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Systematics and biogeography of flying squirrels in the Eastern and the Western Trans-Himalayas

University of Florida Institutional Repository

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SYSTEMATICS AND BIOGEOGRAPHY OF FLYING SQUIRRELS IN THE EASTERN AND THE WESTERN TRANS-HIMALAYAS By FAHONG YU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2002

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Copyright 2002 by FAHONG YU

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iii ACKNOWLEDGMENTS I am most grateful to my committee chair, Dr. Charles Woods, for his continuous guidance, advice, and support throughout my graduate education. Certainly without his support and guidance, I would not have been able to carry out this research. Charles has had the most influence on the direction of my dissertation. He has spent all of his own time to teach me new techniques and to honestly discuss my results. I especially thank Dr. Brian McNab, cochairman of my supervisory committee, for his constant help dealing with all kinds of paperwork and providing me good suggestions during my graduate study. I am extremely thankful to Dr. William Kilpatrick for his creative and unique perspective on issues concerning many aspects of my research. I also appreciate the help and assistance of the other committee members, Dr. Ronald Wolff, Dr. Melvin Sunquist, and Dr. Michael Miyamoto, who were willing to give me their time and offer sound guidance. My sincere appreciation goes to Professor Yingxiang Wang and Professor Yaping Zhang in Kunming Institute of Zoology, China, for allowing me to access the collections and to use equipment in their labs. Also, they provided valuable comments and suggestions throughout the process of developing my dissertation and helped me to significantly improve the quality of the work. I would like to thank Mr. Tian Ming and Dr. Junfen Pang for their interest in my project and for their help when I worked at Kunming. I am grateful for the generosity of Ms. Ginger Clark and Mr. Joel Ernst for allowing me to use the equipment and supplies and to access the computer programs in

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iv laboratory. They helped me solve the contamination problems in my molecular experiments. I greatly appreciated the generosity of the collection managers of Mammal Range of Florida Museum Natural History, Ms. Laurie Wilkins and Candace McCaffery, for their unselfish help and for allowing me to continue accessing the equipment and specimens. My research was primarily supported by Charles Woods (National Fish and Wildlife Foundation); the director foundation of Kunming Institute of Zoology and the director foundation of the Laboratory of Cellular and Molecular Evolution of Kunming Institute of Zoology, the Chinese Academy of Sciences; the visiting scholarship of the American Museum of Natural History, New York. The Department of Zoology provided partial funding of my project. My research project would not have been possible without the support and permissions from the Nature Reserves in Lunchun, Gongshan, Xishuangbaina of Yunnan. Field work by Dr. Shunqing Lu, Mr. Lin, Mr. He, and Mr. Zhang was instrumental in my success. I also wish to thank the American Museum of Natural History, New York; Florida Museum of Natural History, Gainesville; National Museum of Natural History, Washington DC; Chinese Institute of Zoology, Beijing; Kunming Institute of Zoology, Kunming; and Northwestern Institute of Biology, Qinghai, for their permission to examine specimens and to collect tissues. I also owe many thanks to Ms. Karen for her friendly help and service. I would also like to express my appreciation of support and help provided by Professor Feng, Dr. Hongshan Wang, Mr. Lin, and Ms. Linda Gordon, and other colleagues, faculty, graduate students of the Department of Zoology, University of Florida.

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v My wholehearted gratitude goes to my parents, my wife, and my brother for their unconditional love, encouragement, and financial support throughout my six years of study.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS..................................................................................................iii LIST OF TABLES.............................................................................................................ix LIST OF FIGURES...........................................................................................................xii ABSTRACT....................................................................................................................xvi i CHAPTERS 1 INTRODUCTION...........................................................................................................1 1.1 Evolution of Flying Squirrels....................................................................................2 1.2 Evolution of Chinese Flying Squirrels......................................................................6 1.3 Objectives of This Study.........................................................................................10 2 METHODS AND TECHNIQUES OF PHYLOGENETIC STUDY............................13 2.1 Molecular Study......................................................................................................14 2.1.1 Mitochondrial Cytochrome b Gene...............................................................14 2.1.2 Phylogenetic Analysis...................................................................................16 2.1.2.1 Parsimony method...............................................................................17 2.1.2.2 Likelihood method..............................................................................17 2.1.2.3 Distance method..................................................................................18 2.2 Morphometric Study...............................................................................................19 3 PHYLOGENY AND BIOGEOGRAPHY OF EUPETAURUS ....................................22 3.1 Introduction.............................................................................................................22 3.2 Materials and Methods............................................................................................25 3.2.1 Samples.........................................................................................................25 3.2.2 Methods.........................................................................................................27 3.2.2.1 Mitochondrial DNA isolation..............................................................27 3.2.2.2 PCR amplification...............................................................................27 3.2.2.3 Sequence analysis................................................................................28 3.3 Results.................................................................................................................... .30 3.3.1 Phylogenetic Relationship of Eupetaurus between the Eastern and the Western Trans-Himalayas......................................................................................30

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vii 3.3.2 Phylogenetic Analysis between Eupetaurus and Petaurista .........................31 3.4 Discussion...............................................................................................................33 3.4.1 Phylogenetic Status of the Population of Eupetaurus in the Eastern TransHimalayas...............................................................................................................33 3.4.2 Phylogenetic Relationship between Eupetaurus and Petaurista ...................35 3.5 Summary.................................................................................................................39 4 PHYLOGENY OF GIANT FLYING SQUIRREL ( PETAURISTA ) IN SW CHINA AND PAKISTAN: IMPLICATIONS FOR DEVELOPMENT OF MOLECULAR AND MORPHOLOGICAL ANALYSIS.....................................................................49 4.1 Introduction.............................................................................................................49 4.2 Materials and Methods............................................................................................54 4.2.1 Specimens in Morphometric Study...............................................................54 4.2.2 Species for Molecular Analysis.....................................................................56 4.2.3 Morphometric Analysis.................................................................................58 4.2.4 Molecular Analysis........................................................................................59 4.3 Results.................................................................................................................... .60 4.3.1 Phylogenetic Relationships of Chinese P. philippensis ................................60 4.3.1.1 Morphological data.............................................................................60 4.3.1.2 Molecular data.....................................................................................63 4.3.2 Phylogenetic Relationship between P xanthotis and P leucogenys .............65 4.3.3 Phylogenetic Relationship of P. petaurista ...................................................66 4.3.4 Phylogenetic Relationships of Chinese Petaurista ........................................70 4.4 Discussion...............................................................................................................76 4.4.1 Phylogeny of the Trans-Himalayan P. petaurista ( albiventer ).....................76 4.4.2 Taxonomic Status of P. philippensis P. yunanensis and P. hainana ..........80 4.4.3 Phylogenetic Relationship between P. xanthotis and P. leucogenys .............83 4.4.4 Systematics of Chinese Petaurista ................................................................85 4.5 Summary.................................................................................................................89 5 PHYLOGENY OF EOGLAUCOMYS AND HYLOPETES IN THE EASTERN AND THE WESTERN TRANS-HIMALAYAS AS INFERRED FROM MOLECULAR AND MORPHOMETRIC STUDY............................................................................114 5.1 Introduction...........................................................................................................114 5.2 Materials and Methods..........................................................................................117 5.2.1 Materials......................................................................................................117 5.2.2 Morphometric Analysis...............................................................................119 5.2.3 Biochemical Study......................................................................................120 5.3 Results...................................................................................................................1 21 5.3.1 Comparison between Eoglaucomys and the Chinese Hylopetes .................121 5.3.1.1 Morphological data...........................................................................121 5.3.1.2 Molecular data...................................................................................123 5.3.2 Phylogenetic Relationships of Hylopetes ....................................................124

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viii 5.4 Discussion.............................................................................................................128 5.4.1 Taxonomic Status of Eoglaucomys .............................................................128 5.4.2 Phylogenetic Status of H. electilis ...............................................................130 5.4.3 Phylogenetics and Biogeography of Eoglaucomys and Hylopetes ..............132 5.5 Conclusion.............................................................................................................135 6 PYLOGENY OF FLYING SQUIRRELS IN THE TRANS-HIMALAYAS AND OTHER PARTS OF CHINA......................................................................................152 6.1 Introduction...........................................................................................................152 6.2 Materials and Methods..........................................................................................155 6.2.1 Specimens....................................................................................................155 6.2.2 Methods of Phylogenetic Analyses.............................................................158 6.3 Results...................................................................................................................1 58 6.3.1 Comparative Study of the Eastern and the Western Trans-Himalayan Flying Squirrels................................................................................................................158 6.3.2 Phylogenetic Relationships among Flying Squirrels in China....................161 6.4 Discussion.............................................................................................................165 6.4.1 Phylogeny of the Trans-Himalayan Flying Squirrels..................................165 6.4.2 Systematics of Chinese Flying Squirrels.....................................................170 6.5 Summary...............................................................................................................176 7 SUMMARY AND FUTURE WORK.........................................................................192 7.1 Summary...............................................................................................................192 7.2 Future Work..........................................................................................................193 APPENDIX GEOLOGICAL EPOCHS...........................................................................................196 LIST OF REFERENCES................................................................................................197 BIOGRAPHICAL SKETCH..........................................................................................210

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ix LIST OF TABLES Table page 1.1 McKenna’s classification of flying squirrels in Petauristinae.......................................3 1.2 Mein’s classification, determined from dental characters.............................................4 1.3 The latest estimate of flying squirrels in Pteromystinae ...............................................4 1.4 Comparison of different classifications of flying squirrels...........................................5 1.5 Flying squirrels and their distributions.........................................................................6 1.6 Chinese flying squirrels.................................................................................................8 3.1 Thirteen historical and 2 recent specimens of Eupetaurus .........................................24 3.2 Specimens of Eupetaurus and other flying squirrels examined in this study*...........26 3.3 Cycling program of PCR amplification......................................................................28 3.4 Percentage differences of Eupetaurus and Petaurista based on the pairwise comparisons of cytochrome b gene (390 bp)........................................................31 3.5 Transversional substitutions at the third codon positions of cytochrome b gene between Eupetaurus and Petaurista (based on 390 bp)........................................32 3.6 Estimated divergent times among Eupetaurus and Petaurista based on a rate of divergence for the third codon positions of mammalian cytochrome b gene of ca. 0.5% *106 years.....................................................................................................33 4.1 Chinese Petaurista forms............................................................................................50 4.2 Forms of Petaurista .....................................................................................................52 4.3 Major forms of P. philippensis ....................................................................................52 4.4 Species and localities of Petaurista populations examined in morphometric analysis55 4.5 Variables in morphometric study................................................................................56 4.6 Samples of Petaurista examined in molecular study..................................................57

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x 4.7 Sequence data of Petaurista and Pteromys used in this study....................................58 4.8 Discriminant function analysis of five P philippensis forms.....................................62 4.9 Principal components analysis of five P philippensis forms......................................63 4.10 Pairwise comparison based on the partial sequences (409 bp) of cytochrome b gene between five P philippensis forms. Data below the diagonal are the numbers of nucleotide substitutions, transitions vs. transversions. Data above the diagonal represent the genetic differences between samples. The samples were defined in Table 4.6 and 4.7...................................................................................................64 4.11 Percentage of genetic differences between P xanthotis and other giant flying squirrels based on pairwise comparison of the partial cytochrome b sequences (409 bp). See Table 4.6 and Table 4.7 for sample information............................65 4.12 Discriminant function analysis of P. petaurista ........................................................67 4.13 Principal components analysis of P petaurista ........................................................68 4.14 Percentage of differences and the numbers of transversional and transitional substitutions between P. petaurista ( albiventer ) populations based on pairwise comparison of the partial sequence (375 bp) of cytochrome b gene.....................69 4.15 Discriminant function analysis of Chinese Petaurista ..............................................71 4.16 Principal components analysis of Chinese Petaurista ..............................................72 4.17 Pairwise comparison of Chinese Petaurista based on the partial sequences (380 bp) of cytochrome b gene. Data above the diagonal were the percentage of genetic differences between samples, and data below the diagonal were the numbers of transitions vs. transversions between samples......................................................74 4.18 Transversional substitutions at the third codon positions of the partial sequences (375 bp) of cytochrome b gene in Petaurista. .......................................................75 4.19 The estimated divergence time between species based on a divergence rate for the third codon positions of mammalian cytochrome b gene of ca. 0.5% *106 years75 5.1 Species of Hylopetes and Eoglaucomys ....................................................................116 5.2 Species and localities of Hylopetes and Eoglaucomys examined in morphometric analysis................................................................................................................118 5.3 Samples of Eoglaucomys and Hylopetes examined in molecular study...................119 5.4 Discriminant function analysis between the Chinese Hylopetes and Eoglaucomys .122 5.5 Principal components analysis between Chinese Hylopetes and Eoglaucomys ........123

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xi 5.6 Pairwise comparison of cytochrome b nucleotide sequences (400 bp) between Chinese Hylopetes and Eoglaucomys ..................................................................124 5.7 Pairwise comparison of Hylopetes and Eoglaucomsy based on the partial cytochrome b sequences (375 bp)...........................................................................................126 5.8 Transversional substitution rates at the third codon positions of the partial sequences (375 bp) of cytochrome b gene between species.................................................127 5.9 Estimated divergence time between species using the rate of the transversional substitutions at the third codon positions of mammalian cytochrome b gene of ca. 0.5% *106 years...................................................................................................127 6.1 Classification of the extant flying squirrels determined from 44 dental characters..153 6.2 Chinese flying squirrels other than Petaurista ..........................................................153 6.3 Species and localities of flying squirrels used in morphometric analysis.................156 6.4 Samples of flying squirrels used in molecular study.................................................157 6.5 Discriminant function analysis on the eastern and the western trans-Himalayan flying squirrels.....................................................................................................159 6.6 Principal components analysis of trans-Himalayan flying squirrels on the first three factors..................................................................................................................161 6.7 Pairwise comparison based on the partial cytochrome b sequences (375 bp) between species. See Table 6.4 for the sample abbreviations..........................................162 6.8 Pairwise comparison of the transversional substitutions at the third codon positions of the partial cytochrome b sequences (366 bp) between samples. Data below the diagonal are the numbers of transversions at the third codon positions. Data above the diagonal represent the transversional percentage difference between samples. Table 6.4 shows the information of samples........................................163 6.9 Estimated divergence time between samples based on the rate of the transversional substitutions at the third codon of cytochrome b sequences proposed by Irwine et al. (1991). See Table 6.4 for sample abbreviations............................................164 6.10 Multivariate analyses of Chinese flying squirrels...................................................165 6.11 Key to the genera of Chinese flying squirrels.........................................................176

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xii LIST OF FIGURES Figure page 3.1 Historical records of Eupetaurus specimens in the world. The numbers in the map stand for the collecting localities of specimens, which are corresponding to the numbers of Table 3.1.............................................................................................40 3.2 Phylogenetic tree of Eupetaurus reconstructed by the maximum parsimony (MP) method. Numbers above branches indicate the bootstrap values (%). Sample abbreviations are coded in Table 3.2.....................................................................41 3.3 Phylogenetic tree of Eupetaurus reconstructed by the UPGMA method. Sample abbreviations are coded in Table 3.2.....................................................................42 3.4 Phylogenetic tree of Eupetaurus and Petaurita constructed with UPGMA method. Sample abbreviations are defined in Table 3.2.....................................................43 3.5 Phylogenetic relationships of Eupetaurus Petaurita, and G volans constructed with the parsimony maximum (MP) method. Numbers above branches indicate the bootstrap values (%). Sample abbreviations are defined in Table 3.2.................44 3.6 Phylogenetic relationships of Eupetaurus Petaurita, and G volans constructed with the neighbor-joining (NJ) method. Scales in the tree represent branch length in terms of nucleotide substitutions per site. Sample abbreviations are coded in Table 3.2................................................................................................................45 3.7 Eupetaurus cinereus in Pakistan and SW China.........................................................46 3.8 Ventral views of the skulls of E. cinereus, P. petaurista and P. xanthotis ................47 3.9 E. cinereus P. petaurista and P. xanthotis ................................................................48 4.1 Chinese giant flying squirrels ( Petaurista ).................................................................90 4.2 Plot of five P philippensis forms onto discriminant function 1 (CAN I) and function 2 (CAN II).............................................................................................................91 4.3 Plot of five P philippensis forms onto discriminant function 1 (CAN I) and function 3 (CAN III)............................................................................................................92

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xiii 4.4 Principal components analysis of five P philippensis forms onto factor 1 (PRIN I) and factor 2 (PRIN II)...........................................................................................93 4.5 Principal components analysis of five P philippensis forms onto factor 1 (PRIN I) and factor 3 (PRIN III)..........................................................................................94 4.6 Phylogenetic relationships of P philippensis forms based on the cytochrome b gene using maximum parsimony method (MP). Numbers above branches indicate the bootstrap values (%). Sample abbreviations are defined in Table 4.6 and Table 4.7..........................................................................................................................95 4.7 Phylogenetic relationships of P philippensis forms based on the cytochrome b gene using neighbor-joining method (NJ). Scales in the tree represent branch length in terms of nucleotide substitutions per site. See sample abbreviations in Table 4.6 and Table 4.7.........................................................................................................96 4.8 Phylogenetic tree of P. xanthotis and other giant flying squirrels constructed using maximum parsimony method (MP). Numbers above branches indicate the bootstrap values (%). Sample abbreviations are defined in Table 4.6 and Table 4.7..........................................................................................................................97 4.9 Phylogenetic tree of P. xanthotis and other giant flying squirrels constructed using neighbor -joining method (NJ). Scales in the tree represent branch length in terms of nucleotide substitutions per site. See sample codes in Table 4.6 and Table 4.7................................................................................................................98 4.10 Plot of P petaurista populations of discriminant function analysis onto the first and the second discrimiant function (CAN I to CAN II).............................................99 4.11 Plot of P petaurista populations of discriminant function analysis onto the first and the third discrimiant function (CAN I to CAN III).............................................100 4.12 Principal components analysis of P petaurista populations onto factor 1 and factor 2 (PRIN I to PRIN II).............................................................................................101 4.13 Principal components analysis of P petaurista populations onto factor 1 and factor 3 (PRIN I to PRIN III)............................................................................................102 4.14 Phylogenetic relationships within the populations of P. petaurista reconstructed by maximum parsimony (MP) method with Pteromys volans as the outgroup. Numbers above branches are the bootstrap values (%). The abbreviations of taxa are defined in Table 4.6 and Table 4.7................................................................103 4.15 Phylogenetic relationships within the populations of P. petaurista reconstructed by neighbor-joining (NJ) method with Pteromys volans as the outgroup. Scales in the tree represent branch length in terms of nucleotide substitutions per site. The abbreviations of taxa are defined in Table 4.6 and Table 4.7.............................104

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xiv 4.16 Discriminant function analysis of Chinese Petaurista on discriminant function 1 (CAN I) and function 2 (CAN II)........................................................................105 4.17 Discriminant function analysis of Chinese Petaurista on discriminant function 1 (CAN I) to function 3 (CAN III).........................................................................106 4.18 Principal components analysis of Chinese Petaurista on factor 1 (PRIN I) and factor 2 (PRIN II)..........................................................................................................107 4.19 Principal components analysis of Chinese Petaurista on factor 1 (PRIN I) and factor 3 (PRIN III).........................................................................................................108 4.20 Phylogenetic topology of Petaurista based on the maximum parsimony method. The number of bootstrap value (%) is given above each branch. The abbreviations of taxa are defined in Table 4.6 and Table 4.7.............................109 4.21 Phylogenetic topology of Petaurista based on the neighbor-joining method. Scales in the tree represent branch length in terms of nucleotide substitutions per site. The abbreviations of taxa are defined in Table 4.6 and Table 4.7......................110 4.22 Phylogenetic topology of Petaurista based on the UPGMA method. The abbreviations of taxa are defined in Table 4.6 and Table 4.7.............................111 4.23 P. petaurista in Pakistan and W Yunnan, China.....................................................112 4.24 P. philippensis and P. yunanensis in Yunnan, China..............................................113 5.1 Distribution of Chinese Hylopetes and Eoglaucomys ...............................................136 5.2 Discriminant function analysis between Chinese Hylopetes and Eoglaucomys onto function 1 and function 2....................................................................................137 5.3 Discriminant function analysis between Chinese Hylopetes and Eoglaucomys onto function 1 and function 3....................................................................................138 5.4 Principal components analysis of Chinese Hylopetes and Eoglaucomys onto factor 1 and factor 2..........................................................................................................139 5.5 Principal components analysis of Chinese Hylopetes and Eoglaucomys onto factor 1 and factor 3..........................................................................................................140 5.6 Phylogenetic topology of Eoglaucomys and Chinese Hylopetes constructed using the neighbor-joining method based on the partial sequences (400 bp) of cytochrome b gene. Scales in the tree represent branch length in terms of nucleotide substitutions per site. Sample abbreviations are coded in Table 5.3...................141 5.7 Scatter-plot of Hylopetes and Eoglaucomys along the first two discriminant functions..............................................................................................................142

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xv 5.8 Scatter-plot of Hylopetes and Eoglaucomys onto the first and the third discriminant function................................................................................................................143 5.9 Plot of principal components analysis of Hylopetes and Eoglaucomys onto factor 1 and factor 2..........................................................................................................144 5.10 Plot of principal components analysis of Hylopetes and Eoglaucomys onto factor 1 and factor 3..........................................................................................................145 5.11 Phylogenetic tree of Hylopetes and Eoglaucomys generated by MP method on partial sequences (375 bp) of cytochrome b gene. Numbers above branches are the bootstrap values (%). Sample abbreviations are coded in Table 5.3.............146 5.12 Phylogenetic tree of Hylopetes and Eoglaucomys generated by NJ method on partial sequences (375 bp) of cytochrome b gene. Scales in the tree represent branch length in terms of nucleotide substitutions per site. See sample abbreviations in Table 5.3..............................................................................................................147 5.13 Eoglaucomys fimbriatus and Hylopetes alboniger ..................................................148 5.14 Hylopetes electilis and Hylopetes phayrei ..............................................................149 5.15 Hylopetes electilis and Hylopetes nigripes ..............................................................150 5.16 Hylopetes lepidus and Hylopetes spadiceus ............................................................151 6. 1 Phylogenetic reconstruction of flying squirrels, tree squirrels, and fossil squirrels based on 44 dental characteristics.......................................................................178 6.2 Chinese flying squirrels.............................................................................................179 6.3 Trans-Himalayan flying squirrels..............................................................................180 6.4 Scatter-plot of discriminant function analysis of the trans-Himalayan flying squirrels onto the first two functions (CAN I and CAN II)...............................................181 6.5 Scatter-plot of discriminant function analysis of the trans-Himalayan flying squirrels onto function 1 and function 3 (CAN I and CAN III).........................................182 6.6 Principal components analysis of the trans-Himalayan flying squirrels onto the first two factors (PRIN I and PRIN II).......................................................................183 6.7 Principal components analysis of the trans-Himalayan flying squirrels onto the first and the third factors (PRIN I and PRIN III)........................................................184 6.8 Phylogenetic relationships of all Chinese flying squirrels constructed using neighbor-joining (NJ) method. Scales in the tree represent branch length in terms of nucleotide substitutions per site. Sample abbreviations are defined in Table 6.4..............................................................................................................185

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xvi 6.9 Phylogenetic relationships of all Chinese flying squirrels constructed via the maximum parsimony method using heuristic search algorithm. Scales in the tree represent branch length in terms of nucleotide substitutions per site. Sample abbreviations are defined in Table 6.4................................................................186 6.10 Plot of Chinese flying squirrels based on discriminant function analysis onto the function 1 and function 2 (CAN I and CAN II)..................................................187 6.11 Plot of Chinese flying squirrels based on principal components analysis onto the factor 1 and factor 2 (PRIN I and PRIN II).........................................................188 6.12 Trogopterus xanthipes and Pteromys volans ..........................................................189 6.13 Eoglaucomys Hylopetes and Glaucomys ...............................................................190 6.14 Aeretes mlanopterus and Belomys personii ............................................................191

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xvii Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SYSTEMATICS AND BIOGEOGRAPHY OF FLYING SQUIRRELS IN THE EASTERN AND THE WESTERN TRANS-HIMALAYAS By Fahong Yu December 2002 Chair: Charles A. Woods Cochair: Brian K. McNab Major Department: Zoology The areas of the Himalayas where high mountain ranges meet the lowlands of Asia in a series of deep, narrow, and often xeric gorges are described as the “transHimalayas,” including the eastern extreme (SW China, Burma) and the western extreme (Pakistan and Afghanistan). The systematics and biogeography of many flying squirrels in this region, however, are poorly understood and remain uncertain. This is especially true for Chinese flying squirrels. Analyses of the partial sequences of mitochondrial cytochrome b gene and morphological data were performed for investigating the phylogenetic relationships of forms or populations of Eupetaurus Petaurista and Hylopetes ( Eoglaucomys ) along the trans-Himalayas. First, the molecular data revealed that the two specimens in SW China are Eupetaurus which differs significantly from the population in Pakistan, suggesting two distinct species. They diverged at the end of the Miocene. The glaciations and the uplift

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xviii of the Himalayas during the Pliocene-Pleistocene period are the major factors that affected the present distribution of Eupetaurus Second, morphological and molecular data suggested that the population of P. petaurista in Pakistan apparently differentiated from the population in W Yunnan of China. P. yunanensis is distinctive from P philippensis and is a valid species. There is no basis for retaining P. hainana as a recognizable species, however. P. xanthotis is a valid Chinese endemic species and has a close phylogenetic relationship with P. leucogenys in Japan and China. Third the comparative study of Eoglaucomys and Hylopetes in the eastern and the western trans-Himalayas showed that Eoglaucomys is morphologically and genetically distinct from Hylopetes and is a valid genus. H. electilis on the island of Hainan, China, is a valid species of Hylopetes although it shares similar morphological characters with H. phayrei in skull. Last, all Chinese flying squirrels can be divided into five groups: 1) Petaurista ; 2) Pteromys ; 3) Eupetaurus ; 4) Hylopetes and Petinomys ; and 5) Trogopterus and Belomys The morphological and molecular data of this study support that the current distributions of Chinese flying squirrels owe much to both major climatic changes in the late Pleistocene and the physical barriers to migration.

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1 CHAPTER 1 INTRODUCTION The areas of the Himalayas where high mountain ranges meet the lowlands of Asia in a series of deep, narrow, and often xeric gorges are described as the “transHimalayas.” Its eastern and western extremes are considered as the eastern and the western trans-Himalayas. The former includes southwestern Yunnan, eastern Tibet, southern Yunnan, northern Burma and India, while the latter consists of northwestern India, Pakistan, and Afghanistan. They form, in effect, the left and right sides of an open book, with the Tibet Plateau as the center. The eastern trans-Himalayas consists of several distinct topographic regions defined by drainage patterns associated with the parallel mountain chains. These deep gorges have only eastern and southern outlets. The collections of plants and animals made by Pere Armand David, a French missionary-naturalist, were made in the eastern trans-Himalayas, an area also known as the western Chinese highland (Allen, 1940). In this great area the forests are largely of fir and spruce with hardwoods at middle elevations. The high ridges (2,800 to 3,000m) are characterized by thickets of small bamboo and rhododendron. These old north-south orientated ranges are isolated by deep ravines and characterized by diversified habitats which afford asylum for many peculiar, often primitive, types of mammals unknown elsewhere in the world today. However, many of these species are now threatened by logging, over-hunting, trapping, fuel collecting, mushroom rearing, and overgrazing. Deforestation is now widespread in this region where critical habitats for many species are becoming fragmental. The plant and

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2 animal species associated with the once broadly interconnected inter-mountain ecosystems are now fragmented into patterns of disjunctive distributions. More work in western and southern China, as well as adjacent Myanmar, and in Assam, Bhutan, Nepal, northern India and Pakistan, to shed more light on species distributions in this area of great geographic complexity and high relief is clearly desirable (Hoffmann, 2001). The systematics, geographical distributions, and conservation status of many species in these rugged and remote regions are poorly understood. Flying squirrels are especially poorly understood because they occur in deep forest habitats and are nocturnal in habits. 1.1 Evolution of Flying Squirrels Since Cuvier (1798) separated flying squirrels (=Volant) from the non-volant squirrels, placing them in the single genus Pteromys several different revisions of the classification and the distribution of flying squirrels have been proposed based on geographical distributions and external structures (Anderson, 1878; Allen, 1940; Ellerman, 1940; Zahler and Woods, 1997), morphological features (Shaub, 1958; McKenna, 1962; Mein, 1970; Johnson-Murray, 1977; Thorington, 1984; Thorington and Heaney, 1981; Thorington and Darrow, 1996; Thorington et al., 1996, 1997, 1998; Thorington and Stafford, 2001), and biochemical and molecular analyses (Arbogast, 1999; Oshida and Masuda, 2000; Oshida et al., 2000a, 2000b, 2001). By studying the skull morphology and color variation of the flying squirrels in western Yunnan, Anderson (1878) put all 29 species of flying squirrels into the same genus, Pteromys of the family Sciuridae. Allen (1940) classified flying squirrels as an independent family, Petauristidae, based on their unique parachute membrane extending from the ankles to the wrists and characteristics of the skull, such as the short, triangular and slightly raised postorbital processes, and the distinct depression between the orbits, a result of the large

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3 crepuscular eyes. Ellerman (1940) comprehensively summarized the early evolutionary history of the flying squirrels in the book The Families and Genera of Living Rodents McKenna (1962) categorized the living petauristine sciurids into five major tribes, Glaucomys Iomys Petinomys Trogopterus and Petaurisa, on the basis of the differences of the dentitions, auditory regions, and bacula, paralleling the classification of the subfamily Sciurinae (Table 1.1). Later on, McKenna et al. (1977) put all flying squirrels in Pteromyinae instead of Petauristinae because of the membrane. Table 1.1 McKenna’s classification of flying squirrels in Petauristinae Group Genus Glaucomys Eoglaucomys Glaucomys Pteromys Petaurillus Iomys Iomys Petinomys Aeromys Petinomys Hylopetes Trogopterus Pteromyscus Belomys Trogopterus Petaurista Aeretes Petaurista Eupetaurus Mein (1970) made comparisons between the dentition of fossil forms and of modern forms of flying squirrels. Based on his study and description of dental characters, he categorized flying squirrels into three different groups (Table 1.2). The distinct anatomical structures of the wrist support the hypothesis that flying squirrels and non-flying squirrels have different phylogenetic histories (Oshida et al., 2000c, 2000d; Thorington and Darrow, 2001). Corbet and Hill (1992) promoted flying squirrels as a separate family, Pteromyidae. More recently, at the mammal meeting in Seattle, Washington, based on the muscular and skeletal features, Thorington (personal comm.,

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4 1998) suggested the possibility that all flying squirrels belong to two groups: Glaucomys like forms and Petaurista -like forms, each consisting of different subgroups (Table 1.3). Table 1.2 Mein’s classification, determined from dental characters Group Genus I Glaucomys, Eoglaucomys, and Iomys II Pteromys Trogopterus Pteromyscus Belomys Aeretes Petaurista and Eupetaurus III Petinomys, Hylopetes, Aeromys Table 1.3 The latest estimate of flying squirrels in Pteromystinae Group name Subgroup and genus Glaucomys Subgroup I: Glaucomys Eoglaucomys Subgroup II: Hylopetes, Petinomys, Petaurillus Iomys Petaurista Subgroup I: Petaurista Aeretes Subgroup II: Trogopterus, Belomys, Pteromyscus Subgroup III: Eupetaurus Subgroup IV: Aeromys Subgroup V: Pteromys All extant flying squirrels whether in Petauristinae, a subfamily of Sciuridae (Nowak, 1991, 1999; Wilson and Reeder, 1992), or in Pteromyidae (Corbet and Hill, 1992) belong to 14 or 15 genera and 37-52 species (Table 1.4) found in both the Old and New World. Of them, one genus, Glaucomys is confined to North American evergreen and deciduous forests, and the others are centered chiefly in the subtropical forests of the oriental region (Table 1.5). With few exceptions, such as Chakraborty (1981) and Thorington et al. (1996), who considered Eoglaucomys as a valid genus distinct from Hylopetes the current classification of flying squirrels at the generic level is widely accepted (Bruijin and Uney, 1989; Corbet and Hill, 1992; Wilson and Reeder, 1992).

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5 The taxonomic controversies of flying squirrels at the species level are mainly within the genera of Hylopetes and Petaurista Table 1.4 Comparison of different classifications of flying squirrels Number of Species Genus Ellerman 1940 Nowak 1991 Wilson and Reeder 1992 Corbet and Hill 1991 Petaurista 11 6 8 8 Aeromys 2 2 2 2 Pteromys 4 2 2 2 Glaucomys 2 2 2 2 Hyloptetes 13 8 10 11 Eoglaucomys 1 synonymy of Hylopetes synonymy of Hylopetes Petinomys 11 7 8 8 Petaurillus 3 3 3 2 Aeretes 1 1 1 Trogopterus 1 1 1 1 Belomys 1 1 1 1 Pteromyscus 1 1 1 1 Iomys 1 1 2 3 Biswanmoyopterus 1 1 1 Eupetaurus 1 1 1 1 Total 52 37 43 44 However, these classifications are mainly based on the shared primitive features (= plesiomorphic characters) and as a result there is great confusion. Many species or forms are frequently referenced as different species, subspecies or synonyms (Allen, 1940; Corbet and Hill, 1991, 1992; Nowak, 1991; Wilson and Reeder, 1992). Despite the high level of taxonomic, ecological, and morphological information available for some species of flying squirrels, the phylogenetic relationships of many taxa remain uncertain. This taxonomic uncertainty is especially true for Chinese flying squirrels.

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6 Table 1.5 Flying squirrels and their distributions Genus Common name Distribution Petaurista Giant flying squirrel Kashmir, northern Indochina, Malay peninsula, Sumatra, Java, Borneo, Japan, Korea, Manchuria, Taiwan Aeromys Large black flying squirrel Malaya, Sumatra, and Borneo Pteromys Old World flying squirrel Coniferous forest zone of Eurasia (Japan, Finland to Korea) Glaucomys New World flying squirrel Canada, W and E USA, Honduras Hyloptetes Arrow-tailed flying squirrel Northern India, Thailand, south China, Indochina, Sumatra, Java, Borneo, and Naruna Islands Eoglaucomys Small Kashmir flying squirrel Afghanistan, Pakistan, Kashmir Petinomys Dwarf flying squirrel Java, Malaya, Sumatra, Borneo, Philippines, S India, Sri Lanka Petaurillus Pygmy flying squirrel Borneo and Malaya Aeretes Groove-toothed flying squirrel NE China and Sichuan, China Trogopterus Complex-toothed flying squirrel China, Himalayas and Indochina Belomys Hairy-footed flying squirrel E Nepal Indochina, Taiwan Pteromyscus Smoky flying squirrel S Thailand Sumatra, Borneo Iomys Horsfield’s flying squirrelMalaya Java, Borneo, Sumatra Biswanmoyo pterus Namdapha flying squirrelNE India, Southeast Asia Eupetaurus Woolly flying squirrel Pakistan, SW China, North India, Sikkim 1.2 Evolution of Chinese Flying Squirrels The flying squirrels distributed in China belong to seven genera and 14 or 15 recognized species (Allen, 1940; Corbet and Hill, 1991, 1992; Wilson and Reeder, 1992; Nowak, 1999). The most comprehensive discussions of Chinese flying squirrels are found in Allen’s (1940) The Mammals of China and Mongolia Ellerman and MorrisonScott’s (1966) Checklist of Palaearctic and Indian Mammals and Corbet and Hill’s

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7 (1992) The Mammals of the Indomalayan Region Chinese flying squirrels belong to four geographical regions: 1) southwestern China (Tibet, Yunnan, and Sichuan); 2) southern China including the island of Hainan; 3) northern China; and 4) central China (Table 1.6). The region favored by most flying squirrels is southwestern China where there are more than 10 species. Southwestern China is the main part of the eastern trans-Himalayas. It comprises a total area of 767,000 km2, stretching from the southeast corner of Tibet through central and northern Yunnan, western Sichuan, and the hills of the eastern Tibet plateau, particularly the Hengduan and Min mountain systems. Elevation in this region varies from below 1,000 m on the valley floors to over 6,000m on the highest snow covered ridges. The topographical features of this region are very complicated. The limestone bedrock forms diverse landforms including karst landscapes, sharp peaks, intermountane basins, rocky gorges, grottos, and underground rivers. This area can be divided into three great steps with increasing altitudes from low hills at 1,200m in the southeast to high mountain peaks at 3,000-4,000m in the northwest. The vegetation is highly diversified and changes progressively from southeast to northwest. The transition of plant communities in this zone is more altitudinal than latitudinal. The three parallel rivers are the Yangtze, Mekong, and Salaween and are separated by snow-capped mountains. Mountains are vertically stratified with distinct vegetation and typically show a complete spectrum from subtropical evergreen broadleaf forests at lower altitudes to deciduous temperate broadleaf forests, mixed broadleaf coniferous forests at middle levels, and coniferous subalpine forests with dense bamboo and rhododendron associated with alpine meadows at higher altitudes. As a result of the influence of the continental monsoon

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8 from the north and the maritime monsoons from the southwest and southeast, winters are generally cool and dry while summers are warm and wet. Table 1.6 Chinese flying squirrels Genus Species Distribution Aeretes A. melanopterus NE Hebei and Sichuan Belomys B. pearsonii Hunan, SW China, Hainan, and Guizhou Trogopterus T. xanthipes Heber, Huber, Yunnan, Sichuan Eupetaurus E. cinereus N Pakistan, Kashmir to Sikkim (India), Yunnan and Tibet H. alboniger Sichuan, Yunnan, and Hainan Hylopetes H. phayrei Fukien and Hainan P. alborufus Sichuan, and S and C China, Taiwan P. elegans Sichuan and Yunnan P. leucogenys Gansu, Sichuan, and Yunnan P. xanthotis Sichuan, Tibet, Gansu, and Yunnan P. petaurista Sichuan, Yunnan and Fukien P. philippensis Taiwan, South China and Hainan Petaurista P. magnificus Tibet Pteromys P. volans North China and West China Southwestern China is the most interesting and remarkable of the Chinese faunal and floral divisions and has the highest level of biodiversity among Chinese provinces. More than half of the country's protected or endangered mammals, including 25 species under the first class list and 29 of the second, and 50% of the country's total flora, including four first class protected species and 60 of the second class, are found here (Mackinnon et al., 1996). This region provides an ideal habitat for flying squirrels and it is possible that the region is the center of the radiation of flying squirrels. However,

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9 because the region is remote and economic conditions are very poor, the local people and government officially (or unofficially) harvest Yunnan pine, firs, and spruce trees at a very high rate. As a consequence of the rapidly disappearing pine and spruce trees upon which flying squirrels feed and which provide important habitats for flying squirrels, most species are threatened. Eupetaurus Petaurista and Hylopetes are the taxa most threatened, and are the genera where most of the species-level taxonomic controversies exist. Eupetaurus was previously thought to occur only in northern Pakistan and to be very rare or even extinct. However, a review of museum specimens suggests a historical distribution that also includes India, Tibet, Sikkim, and SW China. Zahler (1996) and Zahler and Woods (1997) documented the continued existence of E. cinereus in northern Pakistan. Two “skins” (only skins, no skulls) represented in the collection at the Kunming Institute of Zoology (KIZ) of the Chinese Academy of Sciences have been identified as Eupetaurus based on the pelage color and external features (Wang and Yang, 1986; Corbet and Hill, 1991, 1992). I have found no further evidence of its presence or distribution in southwestern China. Comparisons between Eupetaurus cinereus from Pakistan and the “skins” collected in SW China are important in order to understand the radiation and taxonomy of the genus Eupetaurus and to clarify the taxonomic and phylogenetic status of Eupetaurus within flying squirrels. The separation of Eoglaucomys from Hylopetes in the western trans-Himalayas is based on dental differences (Ellerman, 1947, 1963; Chakraborty, 1981; Thorington et al., 1996). Some authors do not accept Eoglaucomys as a valid genus (Wilson and Reeder, 1992; Corbet and Hill, 1992) and consider that H. alboniger and H. phayrei are the

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10 species of Hylopetes distributed in China. I believe that an examination of the validity of Eoglaucomys based on molecular analysis rather than morphometric is critical for the clarification of the taxonomy of Hylopetes group. As part of this analysis I will examine Hylopetes populations distributed in the eastern trans-Himalayas. The validity of H. electilis in Hainan, China, and the phylogenetic relationships among H. electilis H. alboniger and H. phayrei are as well interesting topics to elucidate the phylogenetic relationship of Chinese flying squirrels. Petaurista is a polymorphic genus with considerable variation in pelage coloring. More than 10 species of Petaurista have been recognized (Corbet and Hill, 1991, 1992; Nowak, 1991, 1999; Wilson and Reeder, 1992; Zhang et al. 1997; Wang, 2002). Of various species included within this genus, it is difficult to resolve the numerous intraspecific and interspecific taxonomic and phylogenetic problems. Various authors still express serious doubts concerning the validity of P. xanthotis, P. philipensis P hainana and P. yunanensis (Nowak, 1999; Wilson and Reeder, 1992; Corbet and Hill, 1992, Wang, 2002). To resolve the affinities of these complex taxa, a comprehensive revision to clarify the relationships among these forms is essential because the available morphometric and molecular data are too scanty to throw any light on the problems. 1.3 Objectives of This Study Most recent phylogenetic studies have concentrated on the analysis of molecular data, particularly DNA sequences. But the combination of both molecular and morphological analyses in systematics has been attracting more and more attention from both systematists and evolutionary biologists (Minelli, 1998; Schierwater and Kuhn, 1998; Benton, 1998; Hugot, 1998; Flynn and Nedbal, 1998; Baker et al., 1998; Smith, 1998; Smith and Patton, 1991; Staongnhope et al., 1998; Barome et al., 1998; Ruedi et

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11 al., 1998; Shoshani and McKenna, 1998; Goodman et al., 1998; Huelsnebeck et al., 1996). Despite the increasing use of DNA sequence data, morphometric analysis still remains one of the most useful techniques available to investigate phylogenetic relationships between taxa (Sanderson et al., 1993). Flying squirrels exhibit a high level of geographic variation in morphological characters such as pelage color, cranium morphology and dental structures. Opportunities for studying flying squirrels traditionally have been limited to studying specimens represented in museums and institutes. In part, this is reflected in the continuing problems of flying squirrel classification. Despite the agreements or disagreements on the taxonomic status of different nominate genera and species in the literature, flying squirrels have not been the main subject of any comprehensive systematic revision. The phylogenetic analysis of mtDNA in rodents has centered largely on murids (Ferris et al., 1983; Smith and Patton, 1991). The overall phylogenetic relationship of flying squirrels remains poorly understood because they are nocturnal, elusive, and difficult to capture. No attempt has been made to investigate the systematics of Chinese flying squirrels. There appear to be similar patterns of speciation and distribution of flying squirrels in the eastern and the western trans-Himalayas. A comprehensive and comparative analysis of the phylogeography and systematic relationships of flying squirrels in the eastern and the western trans-Himalayas will be extremely valuable. In this study, molecular analyses of partial sequences of mitochondrial cytochrome b gene and morphometric study of skull data were performed for the flying squirrels distributed in various areas of China and Pakistan. I also discuss the biogeographic history and

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12 phylogenetic relationships among them as well as the taxonomic status of the groups or forms in Eupetaurus Petaurista and Hylopetes ( Eoglaucomys ) from SW China and Pakistan. The objectives of this study are to seek the answers for the following questions: 1. Search for confirmation of the continued presence of the “lost species” -Eupetaurus cinereus in SW China and investigate the taxonomic status of genus Eupetaurus 2. Examine the taxonomic and phylogenetic relationship between Hylopetes and Eoglaucomys and the taxonomic validity of H. electilis. 3. Determine if the Petaurista petaurista (sensu lato) groups in different localities along the eastern and western trans-Himalayas form a single species P. petaurista ( albiventer ), or a complex of species. Confirm the validity of P. xanthotis, P. philipensis P hainana and P. yunanensis and reconstruct the phylogenetic relationships among Petaurista groups distributed in SW China and Pakistan. 4. Investigate what the systematic relationships are among Chinese flying squirrels, including Petaurista Eupetaurus Trogopterus Hylopetes Eoglaucomys Belomys and Pteromys In the remaining chapters, I will attempt to answer the above questions. In Chapter 2, I will briefly discuss the techniques and methods of phylogenetic analysis used in the present study. Chapter 3 is about the phylogeny and zoogeography of Eupetaurus inferred from an analysis of the cytochrome b gene. Chapter 4 will focus on the phylogenetic relationships of Peaturista distributed in SW China and Pakistan based on a molecular analysis and morphometric study. The phylogenetic relationship between/within Hylopetes and Eoglaucomys is discussed in Chapter 5. Chapter 6 is a discussion of the systematics and biogeography of the trans-Himalayan flying squirrels. The results and the tentative future work are summarized in Chapter 7.

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13 CHAPTER 2 METHODS AND TECHNIQUES OF PHYLOGENETIC STUDY There is little doubt that the introduction of molecular techniques has already significantly enhanced the capacity to address fundamental questions in phylogenetic relationships and will make an even greater contribution in future. Theoretically the phylogenetic analysis of molecular level can provide different and well-corroborated estimates of phylogenetic relationships. It is possible to compare and contrast phylogenies based on morphological data with biochemical data such as genome data, globins, or cytochrome b (Benton, 1998). Messenger and McGuire’s (1998) study of cetaceans showed that combined analyses of the morphological and molecular data provide a well-supported phylogenetic estimate consistent with that based on the morphological data alone. But, intraspecific variation is ubiquitous in systematic characters, including morphology, allozymes, and DNA sequences. Sometimes the analysis of one data set (i.e., molecular) provides one highly corroborated phylogeny; whereas analysis of another data set (i.e., morphological) provides a different highly corroborated phylogeny. The phylogenetic inferences based on characters derived from morphology are corroborated by molecular evidence in some mammal groups (Flynn and Ndebal, 1998; Shoshani and McKenna, 1998). However the integration of distinct data sets, such as the molecular data and morphological data in phylogenetic analysis, has caused considerable debate among evolutionary biologists in recent years (William and Ballard, 1996; Wiens, 1998a, 1998b; Wiens and Servedio, 1998).

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14 2.1 Molecular Study The molecular biological revolution of the last several decades has reached into every conceivable corner of biological investigation (McKenzie and Batterham, 1994; Poinar, 1999). Molecular sequences are information-rich and there are many different ways of extracting useful information from them. Molecular phylogenetic analysis has been developed to infer the branching pattern of different taxa from their common ancestor and the sequential dates when such branching events or cladogenic speciation events occurred. The results of DNA sequence data have been successfully used to reconstruct the phylogenetic relationship in rodents, which provides strong support for the monophyly of rodents, a conclusion that has considerable support from morphology (Honeycutt and Adkins, 1993; Luckeet and Hartenberger, 1993; Frye and Hedges, 1995). 2.1.1 Mitochondrial Cytochrome b Gene The molecular methods used to detect genetic variation within and between species have led to exciting advances in studies of historical biogeography. Molecular survey of DNA sequence data is one of the popular techniques used to quantitatively measure genetic variations among taxa (Storfer, 1996). In principle, any part of the genome can be used for DNA studies. Over the past few years, a variety of different observations have challenged some notions of mitochondrial biology, such as the variable rates of mitochondrial DNA (mtDNA) sequence evolution among taxa (Rand, 1994). However mtDNA is by far the most commonly used methodology in phylogenetic analysis and evolutionary biology since it features several advantages that make it the usual choice for population-level questions (Meyer, 1994). For example, mtDNA sequence data can provide the perspective of a maternally inherited marker on patterns and levels of geographic structuring and the analyses of polymorphic restriction sites. On

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15 the basis of the gene sequences of mtDNA, researchers obtained valuable information on evolutionary relationships and divergence times for mammalian subspecies, species, and higher level taxa (Ferris et al., 1983; Brown et al., 1979; Zhang and Ryder, 1993; Avise,1987, 1994; Janke et al., 1994; Miyamoto and Fitch, 1995; Wettstein et al., 1995; Miyamoto, 1996). To extract historical information from molecular data, it is important to understand the dynamic nature of the sequences and know how molecular sequences change over geological time. Variation of evolutionary rates occurs at several levels in DNA sequences: among the sites (e.g., the second position vs. the third position), among the kinds of substitutions (e. g., transition vs. transversion, or silent vs. replacement), and among regions of the molecule (Ferris et al., 1983; Avise, 1994). At the nucleotide level, which is the most fundamental level for any mutation, there are 12 possible changes, with four being transitional changes and eight being transversional changes. In general, recent divergences are related to rapidly evolving changes and older divergences are related to slowly evolving changes (Graybeal, 1993). These patterns hold for cytochrome b gene, with studies at the population and species level using all informative characters (Smith and Patton, 1991; Moritz, 1994). The mitochondrial cytochrome b gene is one valuable molecule for evolutionary relationship reconstruction among populations, species, and higher taxa in animals and has been used extensively in molecular phylogenetic studies. Because it is slow in terms of amino acid substitutions and the rate of evolution for silent substitutions at the third codon positions is similar to that of other mitochondrial genes, the cytochrome b gene that facilitates the alignment of sequences permits comparisons among widely divergent

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16 taxa (Kocher et al., 1989; Irwin et al., 1991). Using the partial or complete sequences of the mitochondrial cytochrome b gene, researchers and scientists successfully determined the interspecific phylogenies in a wide range of mammalian taxa, such as the phylogenetic analysis of relationships in bats and some murids (Wright et al., 1999; Iudica, 2000, Barome et al., 1998), and genetic analysis of intrageneric relationships of some squirrels and flying squirrels (i.e., Petaurista Glaucomys) (Hafner, et al., 1994; Oshida and Obara, 1992; Oshida and Yoshida, 1999; Oshida et al., 1996, 2000a, 2000b, 2001; Arbogast, 1999). 2.1.2 Phylogenetic Analysis As far as phylogeny is concerned, finding the best trees and using the best tree to reconstruct the phylogenetic relationship are main objectives. With the development of the polymerase chain reaction (PCR), it is possible to recover genetic information from even severely degraded tissues, such as hair, old skin, and excrement. The widespread successful application of molecular analysis in animal phylogenetic reconstruction and evolutionary biology can be attributed partly to the discovery of this versatile PCR technique. The use of DNA from museum skins can reconstruct phylogenetic trees among organisms and develop conservation policies for endangered species. A typical phylogenetic analysis involves a bewildering array of decisions, including what type of data to sample (molecular or morphological), what phylogenetic methods to apply (distance, likelihood, and parsimony), whether or not to order or weight characters, and which taxa and characters to include or exclude. The common methods used for molecular phylogenetic reconstruction include distance method (e.g., the unweighted pair-group method with arithmetic mean (UPGMA), the neighbor-joining, or

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17 the more complicated Fitch-Margoliash method), parsimony method, and likelihood method. 2.1.2.1 Parsimony method Parsimony is a character-based analysis. Each character is considered independent of its neighbor, but only informative sites are considered during the calculation. The parsimony method calculates only the order of the branches of the tree and does not give branch-length estimates. Maximum parsimony is the easiest and most practical to implement, and when evolutionary times are short, maximum parsimony, maximum likelihood, and compatibility tend to yield the same estimated phylogeny (Crandall et al., 1994). The accuracy of parsimony depends largely on how polymorphic characters are coded and the sample size (individuals per species), which is usually an important component of phylogenetic accuracy (Wiens and Servedio, 1998). The advantage of this method is that it uses a logical model and the calculations are rapid. However, a major shortcoming of the method is that a large amount of data that are not informative are discarded. 2.1.2.2 Likelihood method In likelihood method every site of sequences is considered and the likelihood of the replacement of a particular nucleotide from pools of nucleotides is calculated. It is based on random similarity rather than on common descent, and increases with increasing divergence between the outgroup and the ingroup taxa (Milinkovitch and Lyons-Weiler, 1998). Likelihood method considers every site including unchanged sites and gives an accurate estimate of branch lengths. The maximum parsimony method can be viewed as an approximation to the maximum likelihood method, which has been used extensively

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18 for parameter estimation in molecular data analysis. The disadvantage is that this method is very time-consuming. 2.1.2.3 Distance method Distance methods calculate the total number of changes, scored according to the type of change, between every pair of sequences in the alignment. The method is based on distance to calculate branch length that visually represents the number of changes between sequences with consideration of unchanged characters and ambiguous alignments. In practice, the choice among all methods is a serious concern because the intraspecific and interspecific variation is so widespread that the application of different methods can give radically different trees for the same data set. Even subtle differences in how polymorphism is treated can have a significant impact on tree topology (Wiens, 1995; Wiens and Servedio, 1998). UPGMA (unweighted pair group method using arithmetic averages) is the most commonly used clustering method, in which the averaging of the distances is based on the total number of taxa in the cluster analysis (Swofford et al., 1996; Swofford, 2000). Like the likelihood method, it generally gives the most accurate results compared to other techniques (Wiens and Servedio, 1998; Rohlf and Wooten, 1988). In this study, the partial mitochondrial cytochrome b genes (315 420 bp) were amplified with PCR technique. Some sequence data are retrieved from the GenBank of NCBI (National Center for Biotechnology Information). Maximum parsimony (MP), neighbor-joining (NJ), and UPGMA methods were used to reconstruct the molecular phylogeny of Chinese flying squirrels to determine whether the specific genetic structures

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19 and geographic patterns are correlative within each group and to reveal their systematic relationships. 2.2 Morphometric Study Most recent phylogenetic studies focus mainly on the analysis of molecular data, but tracing changes in morphological characters is also an important way to evaluate the distribution of the characters on which those taxonomic units are based. The quantitative description, analysis, and interpretation of shape and shape variation in biology are a fundamental area of research. Taxonomic modifications and reinterpretation of morphological characters in the context of the molecular tree requires further scrutiny. Studies of morphology contribute in different ways to the understanding of evolutionary patterns and processes. Ideally a functional morphological study provides information about the interdependency of characters, and also a transformation scheme of characters that is biomechanically feasible (Galis, 1996). Morphometric studies have applied univariate analyses to differentiate morphotypes (Lee and Cheng., 1996), and multivariate analyses were used to produce an overview of the associations between variables and species patterns (Gauch, 1982). The common multivariate analyses include principal components analysis (PCA), discriminant function analysis, and cluster analysis (Manly, 1994). In multivariate analysis, the selection and the number of the characters used are critical since the interpretation of the results is based on them. Sometimes, raw data probably are transformed by logarithm function to reduce the skewness of original data, make their variances homogeneous, and correct the heterogeneity in magnitude of variables. If there are adequate sample sizes, multivariate analyses allow one to make overall tests as well as a proper posterior test of sets of variables.

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20 Discriminant function analysis is to portray the relationships based on the canonical variables, and to develop linear models of variables to maximize the separation of groups. The discriminant functions are extracted from the between-group covariance matrix standardized by the within-group covariance matrix. Mahalanobis distances are used to measure phenetic distances, which are the indices to evaluate the overlap between pairs of populations, and to transform original distances to maximize power of differentiations between individual specimens to describe the relationship among species. Principal components analysis (PCA) is based upon the variance-covariance matrix of the log-transformed variables. It is performed to identify variables that account for maximum variation in data and to produce a smaller number of uncorrelated factors that are linear combinations of original variables. The first axis lies in the direction of the greatest variability between the sample means, and each succeeding axis lies in the direction of the next greatest variability. Factor loadings, describing the relative contribution of each variable to the principal components, are used to compare the morphological structures between samples. The discrepancy between cluster analysis and other analysis becomes understandable and less important when characteristics of various statistical techniques are considered (Sneath and Sokal, 1973). Cluster accurately represents distance between adjacent groups. Euclidean distance between centroids and an unweighted pair-group method using arithmetic average (UPGMA) clustering algorithm is usually applied to generate a phenogram, depicting morphological relationships among taxa. In this study, all related specimens were pooled together for univariate analysis, which is restricted to one-way analysis of variance (ANOVA) and F-test to calculate the

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21 mean, standard deviation (SD), and the significant test among variables for species. The multivariate analyses including discriminant function analysis, principal components analysis and cluster analysis are used to determine how the groups are related when all the characters are considered simultaneously. Because these multivariate methods are unable to deal with missing data, or else they deal with missing information in a rather arbitrary manner (Rohlf and Marcus, 1990), in this study, only those variables that are available in all sampled groups are selected for further analysis.

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22 CHAPTER 3 PHYLOGENY AND BIOGEOGRAPHY OF EUPETAURUS 3.1 Introduction Eupetaurus cinereus the woolly flying squirrel, is one of the most unusual and least known species in the world (Chakraborty and Agrawal, 1977; Zahler, 1996; Zahler and Woods, 1997). Because observations in the wild have been precluded by the rarity of specimens, virtually, nothing is known of its food habits, reproduction, distribution, habitat preference, behavior, anatomy, or systematics. It is considered among the most endangered mammals (IUCN No.: EN 8 A2ce, B1+2cd1) (Baillie and Groombridge, 1996), probably the most threatened of all flying squirrels. Eupetaurus is a crucial genus in the phylogenetic study of flying squirrels. The cheekteeth of E. cinereus are hypsodont and share many characteristics with rodents that have high-crowned teeth with flat surfaces, such as capromyids (hutias) from the West Indies, thryonomyids (cane rats) from Africa, and New World echimyids (spiny rats), rather than other members of Sciuridae (McKenna, 1962). The highly specialized grinding teeth feature many advantages that allow it to live in relatively treeless rocky areas and possibly supplement its diet during the winter months by eating some abrasive material. Since the structure of teeth is so divergent from other flying squirrels, Schaub (1958), and Grasse and Dekeyser (1955) placed Eupetaurus in its own rodent family, 1 EN: the category of threat is endangered; A2ce: Population decline projected in the future; B1+2cd: small distribution and decline because of the severely fragmented population and habitat; C2a: Small population size and continuing decline by fragmented habitat.

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23 Eupetauridae. By comparing the dentition with the giant flying squirrel ( Petaurista xanthotis ), McKenna (1962) proposed that Eupetaurus is a very high-crown flying squirrel and demonstrably a petauristine sciurid on the basis of a large number of characters other than the dentition, and returned it to the sciurid subfamily Petauristinae. Its present taxonomical status is as follows: Order Rodentia Bowdich, 1821 Suborder Sciuromorph Brandt, 1855 Superfamily Sciuroidea Gill, 1872 Family Sciuridae Gray, 1821 Subfamily Petauristinae Simpson, 1945 Genus Eupetaurus Thomas, 1888 Species Eupetaurus cinereus Thomas, 1888 Eupetaurus had been considered to be very rare or even extinct until a live specimen was captured in northern Pakistan in 1994, which confirmed the existence of woolly flying squirrel. Zahler and Woods (1997) summarized are of the available information on the ecology and conservation of Eupetaurus in Pakistan Eupetaurus in Pakistan is limited to the region of the Sai Valley in the central Indus River Valley near Nanga Parbat, the most westerly main massif in the Himalayan Range. It lives in caves of high alpine zones that are characterized as high, cold desert dominated by Artemisia and Juniperus above 2,000 m, and apparently shows quite unique ecological adaptations for surviving in regions that are inhospitable to any other flying squirrels. The present estimate of the number living in Pakistan is between 1,000 and 3,000 (Zahler and Woods, 1997).

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24 Eupetaurus was historically found from Pakistan, to India, Tibet, Sikkim and SW China, based on 13 available museum specimens in London (British Museum of Natural History), Netherlands (Laiden Museum), Bombay (=Mumbai, Bombay Natural History Society), Calcutta (Indian Museum), and China (Kunming Institute of Zoology, China, KIZ), which confirms presence of Eupetaurus from Pakistan to China (Figure 3.1 and Table 3.1). Table 3.1 Thirteen historical and 2 recent specimens of Eupetaurus Locality Museum ID Collector and date Additional information 1 Tibet, China LM: 19524 Anderson, J. 1878 Skin and skull 2 Yunnan, China KIZ: 73372 Wang, Y. X. 1984 Skin only 3 Yunnan, China KIZ: 73921 Wang, Y. X. 1984 Skin only 4 Sikkim IM: 19103 Gill, J. S. Skin only 5 Gilgit, Pakistan IM: 9492 M. Miles, 1887 Skin and skull 6 Gilgit, Pakistan BNHS: 7107 MacPherson, M. A. 1916 Skin only 7 Sai Nalah, Pakistan BNHS: 7108 Lorimer, Lt. C. D. 1924 The collection site is not associated with the specimen. Skin is in very poor shape. 8 Chitral, Pakistan BNHS: 7109 Fulton, H. T. Skin only 9 Sai Valley, Pakistan BNHS: 7110 Maj. L. MacKenzie, 1924 Skin only 10 Astor, Pakistan BMNH: 88.9.29.1 Purchased by Lydekker, R. 1879 Co-type with a skin and fragmentary snout. The location is said to be from the Astor District. 11 Gyantse Bazar, Tibet BMNH: 23.11.10.2 N o recor d Purchased from Tibet 12 Gilgit, Pakistan N o recor d N o recor d Partial skin 13 Chitral, Pakistan N o recor d N o recor d Partial skin 14 Gilgit, Pakistan UF: 26583 Woods, C. 1996 Partial skin 15 Gilgit, Pakistan UF: 28620 Woods, C. 1996 Skin and skull Note: BNHS: Bombay Natural History Society, India; BMNH: British Museum of Natural History, England; IM: Indian Museum in Calcutta, India; LM: Leiden Museum, the Netherlands; KIZ: Kunming Institute of Zoology, China.

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25 However, it is not possible to establish with certainty that all specimens are the same species, since the majority of the specimens available were collected at the beginning of the last century and some specimens are incomplete and lack skulls. The collecting sites of some specimens are not conclusively associated with individual specimens since the data were recorded solely according to the description of dealers. Therefore taxonomic and phylogenetic studies have been hampered by questionable records and the paucity of Eupetaurus specimens in collections. For example, questions still remain concerning the exact collecting site of the two skins from China represented in the KIZ and the specimen from Sikkim. The phylogentic status of Eupetaurus and the phylogenetic relationships between Eupetaurus and other flying squirrels, such as Petaurista are not yet well understood. In this study, Eupetaurus specimens from different localities were compared with new specimens obtained from Pakistan by analyzing the sequence data from mitochondrial cytochrome b gene using parsimony and distance methods. Here I compare the taxonomic status of Eupetaurus populations in Pakistan and SW China, determine how much variety exists within the genus Eupetaurus along its extensive distribution, and reconstruct the phylogenetic relationship between Eupetaurus and Petaurista 3.2 Materials and Methods 3.2.1 Samples Among those 15 known Eupetaurus specimens represented in museums and institutes, some skins were in very poor shape and without the corresponding skulls; some records were not precisely associated with the specimens; and some specimens were only purchased or shipped by the dealers or a third party. Of 10 available skin

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26 samples obtained from museums and institutes, eight specimens have reliable data associated with them. The rest were described mainly based on comments from either the medicinal dealers or the intermediate persons who shipped or purchased the specimens from different locations. Table 3.2 Specimens of Eupetaurus and other flying squirrels examined in this study* Species Code Collecting localityMuseum ID ECL Tibet, China LM: 19524 ECK1 YUNNAN, CHINA KIZ: 73372 ECK2 Yunnan, China KIZ: 73921 ECI Sikkim IM: 19103 ECB1 Gilgit, Pakistan BNHS: 7107 ECB2 Chitral, Pakistan BNHS: 7109 ECB3 Sai Valley, Pakistan BNHS: 7108 ECF1 Gilgit, Pakistan UF: 26583 ECF2 Gilgit, Pakistan UF: 28620 E cinereus ECD Pakistan Sequence provided by Dr. Roth Other flying squirrels used in this study PPF Gilgit, Pakistan UF ID: 28236 P petaurista PPY Yunnan, China KIZ: 353209 P. xanthotis PTK Gansu, China NBI ID: 85063 G. volans GV Tennessee, US Sequence #: AF063066 Note: The abbreviations of the museums and Institutes see Table 3.1. NIB: Northwestern Biological Institute, China In this study, two samples (ECB3 and ECI) from Pakistan and Sikkim (Table 3.1) were not included because of their highly degraded sequences. The samples that are in good condition and recorded by collectors with certainty were used for molecular

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27 analysis. The detailed information of Eupetaurus and other flying squirrels examined is given in Table 3.2. Dr. Louise Roth, who is an associate professor of biology at Duke University, provided the sequence data for the sample ECD from Pakistan. 3.2.2 Methods 3.2.2.1 Mitochondrial DNA isolation Total DNAs of all samples used were extracted from dry skins using the DNeasy tissue kit (QIAGEN Inc., Valencia, CA91355-1106) and the protocol for animal tissue recommended by the manufacturer. Initially, 180 l of buffer ATL and 20 l of proteinase K (20 mg/ml) were added to the 2 ml tube containing the decalcified material. The sample was mixed and placed into a 550C H2O bath for 48 hours. After vortexed for 15 seconds, 200 l of buffer AL was added. Then it was heated at 700C for 10 minutes. After added 200 l of 100% ethanol, the sample was applied to a Dneasy tissue kit-mini column. During the elution step, 100 l of buffer AE was added. After incubated at room temperature for 1 minute, it was centrifuged and stored at -40C for PCR. 3.2.2.2 PCR amplification The following primers were used to amplify the partial nucleotide sequence (315402 bp) of the mitochondrial DNA (mtDNA) with polymerase chain reaction (PCR) at the interdisciplinary center for biotechnology research (ICBR), University of Florida (UF), and Kunming Institute of Zoology, Kunming, China: L14725 5’-CGA AGC TTG ATA TGA AAA ACC ATC GTT G-3’ L14841 5'-AAA AAG CTT CCA TCC AAC ATC TCA GCA TGA TGA AA-3' H15149 5’AAA CTG CAG CCC CTC AGA ATG ATA TTT GTC CTC A-3’.

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28 Primer names correspond to the light (L) and heavy (H) strand and the 3’ endposition of the primers in the human mtDNA sequence (Anderson et al., 1981). All primers were synthesized at either ICBR of UF or Shanghai of China. The 25 l of PCR reaction mixture contained 2 l 10ng of genomic DNA, 2.5 l of each primer (H15149 and L14725 or L14841), 3.0 l 10x PCR buffer (100 mM TrisHCl, pH 8.3 at 250C, 500 mM KCl, 15 mM MgCl2, 0.01% gelatin), 4.0 l of dNTPs (10 mmol), 3.0 l MgCl2 (25 mmol), and 0.2 l Tag (Sigma tag DNA polymerase (Sigma Chemical CO., St Louis, MO 63178)). The cycling program in Table 3.3 was used for PCR amplification. Table 3.3 Cycling program of PCR amplification Step Temperature (0C) Time Cycles Initial denaturing 94 5 minute 1 Denaturing 94 1 minute Annealing 55 1 minute Extension 72 1 minute 5 second 38 Final extension 72 5 minute 1 PCR products were purified with the Qia-quick PCR purification kit protocol (QIAGEN Inc., Valencia, CA91355-1106). Automatic sequencing was performed with an automated DNA sequencer at University of Florida. The variant sites of sequences were rechecked by comparing the four-color electromorph of sequencing data against the computer results. 3.2.2.3 Sequence analysis Cladistic analysis was performed using the phylogenetic analysis using parsimony (PAUP version 4.0) (Swofford, 2000). Both distance and parsimony methods were applied for phylogenetic analysis to infer gene genealogies of mtDNA. The maximum

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29 parsimony (MP) method using the branch and bound search algorithm (Penny and Hendy, 1986) with the 50% majority-rule consensus, the neighbor-joining (NJ) method (Nei, 1987; Saitou and Nei, 1987), and the UPGMA method (Swofford et al., 1996) were used to reconstruct the phylogenetic trees. In NJ method, numbers of nucleotide substitutions per site were estimated for multiple substitutions by the Kimura’s twoparameter method (Kimura, 1980). Branch-and-bound search was performed to ensure that all minimum-length trees were identified (Zhang and Ryder, 1994). If all characters change sufficiently slowly, they may be equally weighted during phylogenetic inference, even though they do not change actually with equal probability (Felsenstein, 1981,1983, Graham et al., 1998). The MP trees were generated by equal weighted parsimony and the bootstrap values (Felsenstein, 1985) were derived from 1000 heuristic replicates. Quantitative pairwise comparisons between all taxa under the two-parameter model of Kimura (1980) were made for the partial cytochrome b gene sequences. The proportions of nucleotide substitutions between all pairs of sequences were calculated, including the percentage of sequence divergence within and between taxa, the ratio between the transitions and transversions, and the transverstional substitutions at the third codon positions between taxa. Relying on dates of divergence estimated from fossil material for a number of mammalian taxa, Irwin et al. (1991) thought that the average rate of sequence divergence at third positions of the cytochromoe-b gene of mammals is about 10% per million years. But the relative rate of molecular evolution in rodents has been estimated to be ca. 1.5-2 times faster than that of other mammalian lineages (Britten, 1986; Dewalt et al., 1993; Li et al., 1990). In this study, the divergence time between taxa was estimated according to the transverstional substitution rate at the third

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30 codon positions of mammalian cytochrome b gene proposed by Irwin et al. (1991), which was 0.5% per million years. Petaurista petaurista ( albiventer ) from Pakistan and NW China, and P. xanthotis from NW China, which had been considered as the close relatives of Eupetaurus were used for phylogenetic analyses. Glaucomys volans from North America was used as the out-group for phylogenetic reconstruction. The DNA sequence data of G. volans were quoted from GeneBank that is submitted by Arbogast (1999). 3.3 Results The specimens of woolly flying squirrels collected around the turn of the century were mostly from the general region of Gilgit in the area of the confluence of the Himalayan, Karakoram, and Hindu Kush mountain ranges in northern Pakistan. Because some samples were collected more than 100 years ago and some are in very poor condition, the recovered sequences were short and fragmental, usually between 300 to 400 base-pair longs. 3.3.1 Phylogenetic Relationship of Eupetaurus between the Eastern and the Western Trans-Himalayas The partial sequences (389 bp) of cytochrome b gene were successfully determined for seven Eupetaurus samples. The genetic differences obtained from the pairwise comparison (Table 3.4) separated the woolly flying squirrels as two distinct groups. The first group was the eastern tans-Himalayan group ( Eupetaurus I) including samples from Tibet (ECL) and Yunnan (ECK1 and ECK2) in China. The second group was the western trans-Himalayan group ( Eupetaurus II) including samples from Chitral (ECB2) and Gilgit (ECB1, ECD, ECF1, and ECF2) in Pakistan. The genetic difference between these two groups was about 12%. The maximum parsimony (MP) and UPGMA

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31 analysis based on all Eupetaurus individuals generated the similar branching in trees, all of which contained the same two major clades (Figure 3.2 and Figure 3.3). In MP analysis, only one most parsimonious phylogenetic tree was produced with high bootstrap value (99 100%). Table 3.4 Percentage differences of Eupetaurus and Petaurista based on the pairwise comparisons of cytochrome b gene (390 bp) ECL ECK 1 ECK 2 ECB 1 ECB 2 ECF 1 ECF 2 ECD PPY PPF PTK ECL 3.8 3.8 8.4 11.36 11.0 11.0 11.0 16.7 15.9 17.0 ECK1 9/6 0 13.4 14.0 13.3 13.3 13.3 17.9 17.2 19.4 ECK2 9/6 0 13.4 14.0 13.3 13.3 13.3 17.9 17.2 19.4 ECB1 25/6 37/13 37/13 3.6 3.8 3.8 3.8 17.1 16.2 17.5 ECB2 32/11 42/16 42/16 7/4 0 0 0 18.1 17.0 18.7 ECF1 32/11 41/11 41/11 8/6 0/1 0 0 16.9 16.7 19.1 ECF2 32/11 41/11 41/11 8/6 0/1 0 0 16.9 16.7 19.1 ECD 32/11 41/11 41/11 8/6 0/1 0 0 16.9 16.7 19.2 PPY 39/26 44/26 44/26 36/30 42/27 41/25 41/25 41/25 5.2 14.0 PPF 38/24 43/24 43/24 33/30 38/27 40/25 40/25 40/25 18/2 12.1 PTK 41/26 48/28 48/28 35/32 40/32 46/29 46/29 46/29 43/12 37/10 Note: Data below the diagonal are the numbers of nucleotide substitutions, transitions vs. transversions. Data above the diagonal represent the genetic differences between samples. 3.3.2 Phylogenetic Analysis between Eupetaurus and Petaurista Although P. petaurista had been thought as the closest relative of Eupetaurus for long time, Mckenna (1962) regarded P. xanthotis as the closest living relative of E. cinereus for their similar dental structures. The genetic discrepancies between Eupetaurus and Petaurista based on the pairwise comparisons of cytochrome b gene were not consistent with Mckenna’s hypothesis (Table 3.4). The genetic difference between P xanthotis and Eupetaurus was 17.0 19.4%, which was higher than that

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32 between P petaurista and Eupetaurus (16.7 18.1%). The phylogenetic tree did not support his conclusion either, in which all samples were clustered into two genetic clades (Figure 3.4). The estimated dates of divergence among mtDNA clades of Eupetaurus and Petaurista were calculated based on their rates of divergence for the third codon positions of cytochrome b gene (Table 3.5 and Table 3.6). The approximate divergent time between the eastern and the western trans-Himalayan Eupetaurus was about 10 million years ago, and approximately 29.2 32.2 million years ago between Eupetaurus and Petaurista Table 3.5 Transversional substitutions at the third codon positions of cytochrome b gene between Eupetaurus and Petaurista (based on 390 bp) Eupetaurus I Eupetaurus II PPF PPY PTK Eupetaurus I 5.4 14.6 14.6 14.6 Eupetaurus II 7 15.4 15.4 16.1 PPF 19 20 1.0 6.1 PPY 19 20 1 5.4 PTK 19 21 8 7 Note: Data below the diagonal are the numbers of transversions at the third codon positions. Data above the diagonal represent the transversional percentage difference between samples. With Glaucomys volans as the outgroup taxon, the phylogenetic relationships among Eupetaurus P. petaurista and P. xanthotis were reconstructed using maximum parsimony and neighbor-joining methods. Three major clades were formed in both MP and NJ trees (Figure 3.5 and Figure 3.6). The first dichotomy isolated Petaurista including PPY, PPF, and PTK from Eupetaurus group. Then all individuals of

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33 Eupetaurus were clustered as two groups: one was the Pakistan Eupetaurus (ECF1, ECF2, ECB1, ECB2, and ECD); another was the Chinese Eupetaurus (ECK1, ECK2, and ECL). Table 3.6 Estimated divergent times among Eupetaurus and Petaurista based on a rate of divergence for the third codon positions of mammalian cytochrome b gene of ca. 0.5% *106 years mtDNA clades Estimated date of divergence (1.0*106 years) P. petaurista ( albiventer ) 29.2 P. xanthotis 29.2 Eupetaurus I vs. Eupetaurus II 10.8 P. petaurista ( albiventer ) 30.8 Eupetaurus II vs. P. xanthotis 32.2 P. petaurista vs. P. xanthotis 10.8 12.2 3.4 Discussion 3.4.1 Phylogenetic Status of the Population of Eupetaurus in the Eastern TransHimalayas The available morphological comparisons and taxonomic studies of Eupetaurus were solely based on the pelage characteristics and the dental forms of the specimens represented in museums and institutes. The two specimens collected in the deep gorge country of SW Yunnan near the Thailand, Burma, and Tibet border by KIZ were recognized as E. cinereus on the basis of the pelage color and the feet (Wang and Yang, 1986; Corbert and Hill, 1992; Zahler and Woods, 1997), which are externally similar to those in Pakistan (Figure 3.7). The molecular data of this study strongly support their identification. These two Chinese Eupetaurus show the similar molecular features with the population of Eupetaurus distributed in Tibet. The genetic difference is 3.8% (Table 3.4), less than the intraspecific cytochrome b differences of squirrels (Oshida and

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34 Yoshida, 1999). This confirms the presence of Eupetaurus in southwestern China, although lack the morphological data from these specimens. Eupetaurus was commonly considered as a monotypic genus, consisting of a single species, E. cinereus However, the genetic distances in NJ tree and the polycotomy in MP tree of the present study suggest that the populations in the eastern and the western trans-Himalayas can be divided into two major mtDNA clades (Figure 3.2 and Figure 3.3). The genetic distance between these two clades was 11.0 – 13.3% (Table 3.4). Since the reported intraspecific cytochrome b differences of squirrels was < 3.0%, and was applied to other flying squirrels, such as Glaucomys (Arbogast, 1999) and Petaurista (Oshida and Yoshida, 1999), the genetic difference between Eupetaurus I and Eupetaurus II can be referred as the interspecific variation. This implies that the Eupetaurus populations in the western (Pakistan) and the eastern (China) transHimalayas might be two distinct species. Using the substitution rate at the third codon positions estimated from the mammalian cytochrome b genes (Irwin et al., 1991), the divergence between the two groups could have occurred approximately 10.8 Myr (million years) ago (Table 3.8), suggesting that the two populations diverged early before the glacial period and the uplift of the Himalayas and Qinghai-Tibet plateau during Pliocene – Pleistocene (see APPENDIX for time scales). It is inferred that the ancestor stock of Eupetaurus originated somewhere along the Himalayas mountain chain. Before the glacial eustacy, the eastern and the western trans-Himalayan Eupetaurus diverged from the ancestral Eupetaurus and separately migrated to their present locations after the closure of the Tethys Sea at the end of Miocene. During the glacial period of the Pliocene, they became

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35 adapted to the cold environments of mountain rallies. During inter-glacial times, they migrated to higher elevations to avoid warmer conditions. The subsequent glaciations and the uplift of the Himalayas and Qinghai-Tibet plateau in Pleistocene (Xu, 1981; Zheng et al., 2000) caused a great change of climate and ecological system along the Himalayas, which consisted of several distinct topographic regions determined by drainage patterns and the parallel mountain chains in both the western and the eastern trans-Himalayan regions, such as the Yangtze river system, Salaween river system, and Mekong river system. These tectonic events led to southwestern China, also possibly northern Pakistan, becoming refuges for some special mammals, such as Eupetaurus It is possible that the present distribution of Eupetaurus in the trans-Himalayas is secondarily related to the tectonic activities of the Cenozoic that caused dramatic changes of environment in the Eurasian continent (Wang, 1984). In Pakistan, the distributions of E. cinereus, P. petaurista, and Eoglaucomys fimbriatus are sympatic, which correspond to different ecological habitats, indicating their different feeding habits. A similar pattern of sympatric distribution of E. cinereus, P. peturista, and Hylopetes alboniger was also found in SW China where the two Eupetaurus skins were collected. But, without sufficient morphological evidence, it is premature to raise these two groups as distinct species, although it is noteworthy that the genetic characteristics between these two groups are corresponding to their geographic distances of sampling localities. 3.4.2 Phylogenetic Relationship between Eupetaurus and Petaurista Petaurista and Eupetaurus have been considered as the closely related species for a long time because of their similar external structures and sympatric distribution (Figure 4.8). Their common ancestor was likely to have originated in the eastern Himalayas and

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36 Indo-China (Zahler and Woods, 1997). According to McKenna (1962), the differentiation of the recent genus Eupetaurus from a Petaurista -like sciurid provided a significant parallel to the derivation of various dentally high-crowned rodents from sciurid and paramyid stock in the early and middle Tertiary (Wood, 1962). The differences between Petaurista and Eupetaurus are primarily the result of the highcrowned teeth (Figure 3.8). Eupetaurus has high-crowned teeth and survives in more restricted areas that appear to meet their unique habitat requirements. Thomas (1888) believed that woolly flying squirrels fed mainly on lichens, mosses, and other plants associated with rocky areas. Local people in Pakistan believe that Eupetaurus feeds on seeds, needles, bark of conifers, spruce buds, and some abrasive materials (Zahler and Woods, 1997). In eastern Tibet and NW Yunnan, forests grow up to an elevation of 3,500-4,500 m, where there is a mixed coniferous and broad-leaved forest that composes predominantly of spruce, fir, and oak. It is the optimum habitat for Eupetaurus The adaptive shift of the feeding mechanism is analogous to the shifts that led to the distinctive morphology of the dentition of beavers, mylagaulids, eutypomyids, and numerous other high-crown rodents (McKenna, 1962). But, the pattern of the dentition in Eupetaurus is unlike that of any other known rodent, either fossil or recent. A good ecomorph of E. cinereus is Plagiodontia aedium from Hispaniola, which also lives in rock crevices and caves, located in rocky areas at high elevation (3,000m). Both forms probably feed upon the similar abrasive materials for their similar modifications to the masticator apparatus (Woods and Howland, 1979).

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37 Since Eupetaurus is so distinct from other flying squirrels, it appears that E. cinereus is morphologically convergent with capromyids as well as cane rats and New World spiny rats, rather than with other petauristines. The unique dental form of Eupetaurus might be due to isolation in marginal habitats or the strong competitve pressure from P. petaurista and Eoglaucomys fimbriatus or H. alboniger The present molecular findings indicate that Eupetaurus had diverged from Petaurista before the Oligocene-Miocene radiation of giant flying squirrels in Europe (Oshida, et al., 2000a), and that Eupetaurus is a specialized species that genetically differs from Petaurista (Figure 3.5 and Figure 3.6). Petaurista xanthotis and P. petaurista were considered as the closest living relative of Eupetaurus (Mckenna, 1962; Zahler and Woods, 1996) (Figure 3.9), but the genetic data of the present study reveals that the isolation between the ancestors of Eupetaurus and Petaurista occurred approximately at the end of Oligocene, about 30.8 – 32.2 Myr ago (Table 3.4 and Table 3.5). The phylogenetic reconstruction also demonstrates that Eupetaurus and Petaurista are different clades with significant genetic distances, 15.9 – 19.4% (Table 3.3). Considering their divergence time, Eupetaurus diverged from Petaurista -stock much earlier than the formation of P. xanthotis, which diverged from Petaurista about 11 Myr ago. The present distribution of P petaurista ( albiventer ) in western trans-Himalayas is a recent dispersal. Petaurista xanthotis is a Chinese endemic species and is found living from Tsing Hai and Kansu, southeastward to Hubei and southward to Sichuna and Yunnan, China. The elevation ranges from 2,000m in Gansu to 3,300m in Yunnan where the eastern extremes of the Himalayas and the Tibetan Plateau separate the range of P. xanthotis

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38 from Eupetaurus P. xanthotis has semi-hypsodont molariform teeth, which is significantly different from P. petaurista (Figure 3.8). The principal differences between P. xanthotis and Eupetaurus are the result either of the increased height of crown of its molar teeth of Eupetaurus or of a few minor changes in molar crown pattern acquired by P. xanthotis Mckenna (1962) proposed that the lineage leading to P xanthotis had changed little and E. cinereus had modified the dentition and the masticatory musculature to a considerable degree. The similar dental structure between Eupetaurus and P xanthotis might be the convergent adaptation to the similar feeding resources. According to the paleontological records, by the late Miocene the geography of the trans-Himalayas was similar to that of today (Wang, 1984). The presence of the Himalayas helped cause the diversification of climate, and it became an important regulator of the Asian environment. There were either three or four major glacial periods in Europe and Asia, separated by warmer, interglacial periods in Pliocene-Pleistocene. Eupetaurus migrated to its current geographical distribution in the early Miocene when the radiation of Petaurista occurred in the Eurasian continent (Oshida et al., 2000a). After diverging from the ancestral Petaurista in the middle Miocene, P. xanthotis was restricted to the southern parts of the Eurasian continent. During glacial spisodes, it adapted to cold environments and high elevations, and its feeding habits became specialized. With the retreating of glaciers and the uprising of the Himalayas, it subsequently expanded into northward, where it inhabited in the temperate forest. The present distribution of P xanthotis is mainly due to the geographic events of the Pliocene-Pleistocene. The molecular data here indicate that the phylogenetic relationship

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39 between Eupetaurus and P. xanthotis or P. petaurista is not as close as indicated from morphology. 3.5 Summary In this study, the partial cytochrome b gene sequences (390 bp) of Eupetaurus were analyzed to elucidate the phylogenetic status of Eupetaurus in the eastern and the western trans-Himalayas. I also discussed the phylogenetic relationship between Eupetaurus and Petaurista The phylogenetic trees were reconstructed using neighboring-joining and maximum parsimony methods. The following results were concluded in the present study: 1. The two specimens that were collected in northwestern Yunnan, China, are Eupetaurus 2. The Eupetaurus populations in the eastern and the western trans-Himalayas are significantly different (>13%). The genetic characters between these two populations are corresponding to their geographic distribution. They are two distinct species. 3. The divergence time of the two Eupetaurus populations was at the end of Miocene. The glacial period and the uplift of the Himalayas and Qinghai-Tibet plateau during the Pliocene-Pleistocene period are the major factors that secondarily affected on the present distribution of Eupetaurus in trans-Himalayas. 4. There is not a very close phylogenetic relationship between Eupetaurus and P xanthotis The similar dental characters might be of the convergent adaptation to the similar food resource.

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40 Figure 3.1 Historical records of Eupetaurus specimens in the world. The numbers in the map stand for the collecting localities of specimens, which are corresponding to the numbers of Table 3.1.

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41 Figure 3.2 Phylogenetic tree of Eupetaurus reconstructed by the maximum parsimony (MP) method. Numbers above branches indicate the bootstrap values (%). Sample abbreviations are coded in Table 3.2.

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42 Figure 3.3 Phylogenetic tree of Eupetaurus reconstructed by the UPGMA method. Sample abbreviations are coded in Table 3.2.

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43 Figure 3.4 Phylogenetic tree of Eupetaurus and Petaurita constructed with UPGMA method. Sample abbreviations are defined in Table 3.2.

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44 Figure 3.5 Phylogenetic relationships of Eupetaurus Petaurita, and G volans constructed with the parsimony maximum (MP) method. Numbers above branches indicate the bootstrap values (%). Sample abbreviations are defined in Table 3.2.

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45 Figure 3.6 Phylogenetic relationships of Eupetaurus Petaurita, and G volans constructed with the neighbor-joining (NJ) method. Scales in the tree represent branch length in terms of nucleotide substitutions per site. Sample abbreviations are coded in Table 3.2.

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46 Figure 3.7 Eupetaurus cinereus in Pakistan and SW China

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47 Figure 3.8 Ventral views of the skulls of E. cinereus, P. petaurista and P. xanthotis

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48 Figure 3.9 E. cinereus P. petaurista and P. xanthotis

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49 CHAPTER 4 PHYLOGENY OF GIANT FLYING SQUIRREL ( PETAURISTA ) IN SW CHINA AND PAKISTAN: IMPLICATIONS FOR DEVELOPMENT OF MOLECULAR AND MORPHOLOGICAL ANALYSIS 4.1 Introduction Giant flying squirrel, Petaurista contains many recognized distinct species occupying different habitats. They inhabit various kinds of forests either in lowlands or in mountains up to 4,000 meters in elevation from Pakistan, Kashmir to China, northern Indochina, the Malayan Peninsula, Sumatra, Java, Borneo, Japan, Korea, and Manchuria. Several taxonomic revisions have been proposed on the basis of dental and cranial characteristics and external structures (Allen, 1940; Ellerman, 1940; Corbet and Hill, 1991, 1992; Nowak, 1991; Wilson and Reeder, 1992). More than 18 forms in Petaurista have been described as valid species, but some of them are only referenced with very few specimens; some are based solely on skins with no corresponding skulls (Allen, 1940; Ellerman, 1940); and some actually are the synonyms or subspecies of other valid species (Corbet and Hill, 1992). The populations of Petuarista that are distributed in China and occupy different habitats are recognized as ten distinct species by recent authorities (Corbet and Hill, 1992; Zhang et al., 1997) (Table 4.1 and Table 4.2). Figure 4.1 depicted the geographic distributions of Chinese Petaurista In Petaurista the species P. elegans, P. petaurista, P. alborufus, P. magnificus, P. philippensis, and P. leucogeny are commonly accepted as valid species, with each intricately divided into various forms or subspecies (Corbet and Hill, 1991, 1992; Nowak, 1991, 1999; Wilson and Reeder, 1992; Zhang et al., 1977; Wang, 2002).

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50 However, various species with significantly geographical variations are included within this genus, the taxonomy and the intraand the inter-specific phylogenetic relationships are confusing and inconclusive, especially the giant flying squirrels that are distributed in the eastern and western trans-Himalayas. Table 4.1 Chinese Petaurista forms Species Subspecies Distribution Habitat P. p. grandis Taiwan P. p. miloni Guangxi P. p. rufipes Fujian, Guangdong P. p. rubicundus Sichuan, Gansu P. petaurista P. p. nigra W Yunnan Tropical and subtropical forest P. a. alborufus Shanxi, W Sichuan P. a. lena Taiwan P. a. castaneus Hubei, Sichuan, Guizhou P. alborufus P. a. ochraspis Yunnan, Guangxi Tropical and subtropical forest P. e. clarkei Yunnan, Guizhou P. elegans P. e. gorkhali S Xizang Forest P. yunanensis Yunnan, Guangxi, Tibet Tropical forest P. hainana Hainan Tropical forest P. pectoralis Taiwan Tropical forest P. xanthotis Gansu, Qinghai, Yunnan, Xizang Mountain coniferous P. philippensis Yunnan, Guizhou Forest P. magnificus Xizang Mountain forest P. marica Yunnan, Guangxi Tropical forest NOTE: THE ASSIGNMENTS ARE DESCRIBED BY ZHANG ET AL. (1997)

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51 Ellerman (1940), Corbet and Hill (1992), and Wang (2002) had comprehensively studied Chinese Petaurista based on the morphological and external characteristics, but there is not sufficient evidence from either morphometric study or molecular analysis to ascertain conclusively these specific conclusions. The major problems of Chinese Petaurista are about the conspecific relations within P. petaurista (sensu stricto), the taxonomic statuses of P. philippensis, P. xanthotis, P. hainana and P. yunanensis and the phylogenetic relationships of Petaurista at the specific level. Petaurista petaurista is a polymorphic species with considerable variation in pelage coloring (Allen, 1940). This species mainly occurs in plantations as well as in forest in southern China. Its broad distribution is beyond China including northern India, Bhutan, Nepal, Pakistan, northern Afghanistan, and the Greater Sunda Islands. A dozen of nominal subspecies have been described (Allen, 1940; Ellerman, 1940; Ellerman and Morrison-Scott, 1966). Corbet and Hill (1992) put all Petaurista populations into seven major forms (Table 4.2). However, from our comparative study of Eupetaurus (Chapter 3) and observations of some other rodents distributed in the western and eastern trans-Himalayas, it seems extreme to allocate the populations of Petaurista in Pakistan and W Yunnan to the same form, P. p. albiventer Further study needs to be done to clarify the inter-group relations for their various geographical distributions. With few exceptions (Nowak, 1991), P. philippensis and P. xanthotis have been merited as valid species based on their distinct external and dental structures (Corbet and Hill, 1991, 1992; Wilson and Reeder, 1992). P. philippensis is a polymorphic species with extensive geographical variations and used to be included in P. petaurista

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52 (Ellerman, 1940; Ellerman and Morrison-Scott, 1966). Because this species is clearly separable from P. petaurista by the external structures, Corbet and Hill (1991, 1992) ranked P philippensis as a valid species and classified its populations as seven major forms (Table 4.3). All populations distributed in China including the island of Hainan were recognized as P. philippensis yunanensis Without further evidence, much remains to be done to clarify their taxonomic relationships. Some forms included by Corbet and Hill (1992) may warrant separate specific rank. Table 4.2 Forms of Petaurista Form (subspecies) Distribution P. p. albiventer P. p. petaurista P. p. marchio P. p. batuana P. p. terutaus P. p. taylori P. p. candidula W Pakistan, W Yunnan, China Java Borneo and Malayan Peninsula W Sumatra S Thailand S Burma and W Thailand Burma and Thailand Table 4.3 Major forms of P. philippensis Subspecies Distribution P. p. philippensis P. p. cineraceus P. p. mergulus P. p. lylei P. p. annamensis P. p. yunnanensis P. p. grandis Peninsular India and Sri Lanka W Burma Mergui Is, Burma E Burma, Thailand Vietnam, S China SW CHINA, HAINAN, N ASSAM Taiwan Petaurista xanthotis is a Chinese endemic species with an extensive range, from the spruce forests of northwestern Kansu southward in the highlands of western China to the Likiang region. P. xanthotis was considered as a form of Japanese giant flying

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53 squirrel, P. leucogenys by Ellerman and Morrison-Scott (1966), but it was elevated as a distinct species for its much complex cheek-teeth (Corbet and Hill, 1991, 1992, Nowak, 1999). Except for a morphological illustration of teeth by McKenna (1962), almost nothing is known about this species. Petaurista yunanensis and P. hainana are generally treated either as the subspecies (Corbet and Hill, 1992) or as the synonyms (Wilson and Reeder, 1992) of P. philippensis based on the dental structures and pelage coloration, although both were merited as two valid species of Petaurista early by Anderson (1878) and Allen (1940). The former occurs from extreme southwestern Yunnan probably into Burma and Indochina, and the latter is only distributed in Hamfong, Hainan, China (Figure 4.1). Very little seems to be known as to their taxonomic statuses and the interor intraspecific relationships with P. philippensis A comprehensive study on the basis of morphometric and molecular analysis is much needed. Mitochondrial DNA (mtDNA) is one of those valuable molecules for evolutionary relationship reconstruction among populations, subspecies, and species. Using the polymerase chain reaction (PCR), it is possible to recover genetic information from severely degraded tissues. With the mtDNA information it is able to infer the interand intra-specific relationships, to investigate the genetic differentiation, and to reconstruct the phylogenetic topology of the controversial taxa. Although some molecular data and geographical and morphological variations involving morphology, myology, and karyology have been intensively used for studying the species, subspecies or forms of Petaurista (Cuvier, 1856; Bryant, 1945; Harrison, 1960; Johnson-Murray, 1977; Throington and Heaney, 1981; Oshida et al., 1992; Oshida et al., 1996; Oshida

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54 and Masuda, 2000; Oshida et al., 2000a, 2000b), it is too scanty to throw any light on the problems. There are still arguments on the validity of a number of species or forms in Petaurista and very little is known on the phylogenetic relationships within the genus Petaurista In attempting to resolve the affinities of the complex taxa, a comprehensive study based on both morphological data including 14 measurements of skull and molecular data using partial sequences (375 – 400 bp) of mitochondrial cytochrome b gene were conducted in this study. The objectives were to answer the following questions: 1. Do the populations of P. petaurista ( albiventer ) along the eastern and the western trans-Himalayas form a single species -Petaurista albiventer or a complex of species? 2. Are P. philippensis P. xanthotis P. hainana and P. yunanensis valid species, subspecies, or synonyms ? 3. What are the phylogenetic relationships among the populations of Chinese Petaurista and the populations of Petaurista in Pakistan and SE Asia? 4.2 Materials and Methods 4.2.1 Specimens in Morphometric Study A total of 193 recent specimens of giant flying squirrels ( Petaurista ) were used in morphometric analysis (Table 4.4). Specimens were conventional museum specimens preserved as skulls, skeletons, and fluid. These specimens are deposited in the following collections: American Museum of Natural History, New York (AMNH), Florida Museum of Natural History, University of Florida, Gainesville (FLMNH), National Museum of Natural History, Smithsonian Institution, Washington, DC (USNM), Chinese Institute of Zoology, Beijing (China, BIZ), Northwestern Institute of Biology, Qinhai (China, NIB), and Kunming Institute of Zoology, Kunming (China, KIZ).

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55 Fourteen cranial measurements were taken with digital caliper to the nearest 0.01 mm. The definitions and abbreviations of measurements were given in Table 4.5. Table 4.4 Species and localities of Petaurista populations examined in morphometric analysis Species Subspecies Specimens Locality Museum P p. albivente r 10 (5F, 5M) NW Yunnan, China K IZ, AMNH P p. albivente r 31 (14F, 7M) NWFP, Pakistan, Kashmir K IZ, U SMNH P p. candidula 4 (2F, 2M) Lakhuni, India, Chiengmail, Thailand A MNH P p. melanotus 24 (12F, 12 M) E Sumatra, Borneo, Malayan Peninsula U SMNH, A MNH P p. petaurista 10 (6F, 4 M) Java A MNH P petaurista P p. batuana 11 (6F, 5M) W Sumatra A MNH P philippensis 9 (3F, 6 M) Yunnan, Guangxi, China K IZ, BIZ, A MNH P p. lyei 10 (6F, 4M) Indochina, Thailand U SMNH P philippensis P p. grandis 14 (3F, 11U) Taipei, Taiwan A MNH, F LMNH P. yunanensis 15 (5F, 3M) Burma, Yunnan, China B IZ, AMNH P. hainana 9 (5F, 4M) Namfong, Hainan A MNH P. elegans 12 (6F, 6 M) Yunnan, Guangxi, China K IZ P. alborufus 29 (5M, 24U)Taiwan, Yunnan, Burma K IZ, AMNH P. xanthotis 5 (3F, 2 M) Yunnan, Qinhai, China K IZ, NIB Total 193 F = Female, M = Male, U=Unknown sex Note: Assignments of species and subspecies were based upon the classification of Corbet and Hill (1992) and Zhang et al. (1997).

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56 Table 4.5 Variables in morphometric study Variable Definition CRANL Cranial length from the tip of the occipital protuberance to the alveolar BCASEL Braincase length from the tip of the occipital protuberance to orbit CRANW Cranial width, distance between the points on the left and righ t superametal crests above the external acousticmeatus BPORW Width between the left and right zygomatic arch POCL Postorbital constriction, the least distance across the top to skull posterior to the postorbital process PGA Distance from the base of the anterior surface of the postglenoid process to the M3 NAL Nasal length TBL Tympanic bullae length DSL Diastema length between the second incisor and the first premolar MTRL Length of the maxillary tooth row MTRW Width of the maxillary tooth row at M2 LMDL Maximum mandible length LMDH Maximum height of low jaw LMTL Length of the maxillary tooth row of lower jaw 4.2.2 Species for Molecular Analysis Species of giant flying squirrels ( Petaurista ) used in molecular analysis were listed in Table 4.6. All skin tissues were collected from AMNH, USNM, BIZ, NIB, FLMNH, and KIZ. Fresh tissues were either obtained from the Cellbank of Kunming Institute of Zoology, the Chinese Academy of Sciences, or collected from the locality in where the species is distributed. To reconstruct the phylogenetic relationships, the

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57 sequence data of P. petaurista and Pteromys volans were quoted from the GenBank of NCBI. The detailed information was given in Table 4.7. Table 4.6 Samples of Petaurista examined in molecular study Species Code Museum ID Collecting locality PPF1 FLMNH: 28236 Pakistan PPF2 USMNH: 353209Pakistan PPY1 KIZ: 640229 Yunnan, China P. p. albiventer (PPF and PPY) PPY2 KIZ: 73382 Yunnan, China PTK1 QIZ: 85063 Gansu, China P. xanthotis (PTK) PTK2 QIZ: 984 Gansu, China PPH1 KIZ: Fresh tissue Pianma,Yunnan, China PPH2 KIZ: 620028 Yunnan, China PPH3 KIZ: 74540 Yunnan, China P. philippensis (PPH) PPH4 KIZ Yunnan, China PYK1 KIZ: Fresh tissue W Yunnan, China PYK2 KIZ: Fresh tissue Gongshan, Yunnan, China PYK3 KIZ: Fresh tissue Gongshan, Yunnan, China PYK4 KIZ: 73270 Gongshan, Yunnan, China P. yunanensis (PYK) PYK5 KIZ: Gongshan, Yunnan, China PHK1 KIZ: 22686 Hainan, China P. hainana (PHK) PHK2 KIZ: 259442 Namfong, Hainan, China PAK1 KIZ: 006679 Sichuan, China PAK2 KIZ: 43178 Likiang, China PAK3 KIZ: 73369 Yunnan, China P. alborufus (PAK) PAK4 KIZ: 64042 Luodian, Guizhou, China PEK1 KIZ: 84354 Mile, Yunnan, China PEK2 KIZ: 73375 Lijiang, Yunnan, China P. elegans (PEK) PEK3 KIZ: 73369 Bijiang, Yunnan, China

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58 Table 4.7 Sequence data of Petaurista and Pteromys used in this study Species Code Locality Accession No P. p. petaurista** PPB Borneo, E Malaysia AF063067 PPM1 Laos AB023908 P. p. melanotus PPM2 S China AB023909 P. philippensis grandis PPG Natou, Taiwan AB023907 PLL1 Japan AB023903 P. leucogenys leucogenys PLL2 Japan AB023904 PLN1 Japan AB023905 P. leucogenys nikkonis PLN12Japan AB023906 PAC1 S China AB023898 PAC2 S China AB023899 P. alborufus castaneus PAC3 S China AB023900 PAL1 Nantou, Taiwan AB023901 P. alborufus lena PAL2 Hualien, Taiwan AB023902 Pteromys volan PVO Japan AB023910 Note: Most sequence data were quoted from GenBank of NCBI, which were published by Oshida et al. (2000a). ** The sequence was quoted from GenBank of NCBI, which was pu b by Arbogast (1999). 4.2.3 Morphometric Analysis Statistical analyses in morphometric study were performed using the SAS (SAS Institute, 1982) or Statmost program (StatMost, 1995). The associations of cranial characters and species were assessed by discriminant function analysis and principal component analysis (PCA).

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59 Discriminant function analysis carried out a multiple discriminant analysis in a stepwise manner, selecting the variable entered by finding the variable with the greatest F-value. This method was used to determine which of two or more groups a given individual should be assigned to and placed them with the group to which they were nearest on the discriminant functions. The relationships between groups onto the first three discriminant functions were plotted. Principal components analysis was performed to identify variables that account for maximum variation in data set and to accurately represent distances between major groups, in assessing the specific relationships among and between pooled individuals. The loadings or the eigenvector scores describing the relative significance of each variable to principal components were used to compare the morphological similarity and difference of skull. The projections of the row-points on the first three factors were depicted to describe the morphological relationships among samples examined. 4.2.4 Molecular Analysis Because some samples were collected more than 50 years ago, the recovered sequences were usually between 300 to 400 base-pair longs. The following primers were used to amplify the partial nucleotide sequence (300 425 bp) of mitochondrial DNA (mtDNA) with polymerase chain reaction (PCR): L14725 5’CGA AGC TTG ATA TGA AAA ACC ATC GTT G -3’ L14841 5'AAA AAG CTT CCA TCC AAC ATC TCA GCA TGA TGA AA -3' H15149 5’AAA CTG CAG CCC CTC AGA ATG ATA TTT GTC CTCA -3’. ICBR and KIZ synthesized all primers. The techniques and protocols used for DNA isolation, PCR amplification and purification, sequencing analysis, and phylogenetic reconstruction were the same as those in Chapter 3. Most molecular work was done at

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60 the interdisciplinary center for biotechnology research (ICBR), University of Florida, and Kunming Institute of Zoology (KIZ), the Chinese Academy of Sciences, China. Some PCR products were sequenced at the University of Vermont, US. One sample of Pteromys volans from NE China was used as the outgroup taxon for constructing phylogenetic trees. 4.3 Results All measurements of skull were taken by author at museums and institutes in China and US, thereby avoiding the statistical complications and other associated problems of inter-observer error in a multivariate data analysis. A percent difference analysis was performed on 15 randomly chosen individuals to obtain an estimate of the amount of intra-observer error involved in measuring the specimens. In molecular study, because the recovered sequences of some old skins were highly degraded, the sequence sizes of individuals used for building phylogenetic trees were different. 4.3.1 Phylogenetic Relationships of Chinese P. philippensis Following the taxonomic assignments of Corbet and Hill (1992), five forms of P philippensis including 57 specimens of P p philippensis (SW China), P p lyei (Thailand), P p grandis (Taiwan), P yunanensis and P hainana were selected in morphormetric analysis. The molecular analyses were based on the partial sequences (409 bp) of cytochrome b gene. One individual of P petaurista from Borneo was used as an out-group. 4.3.1.1 Morphological data Multivariate analyses revealed highly significant differences in five forms of P philippensis The results of discriminant analysis were given in Table 4.8. The first three axes accounted for 97% of the total variance. The forms in Thailand ( P p lyei ),

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61 Taiwan ( P p grandis ), and Hainan (China, P hainana ) were distinguished as distinct groups without overlapping with other forms on discriminant function 1 (CAN I) (Figure 4.2). The populations from Yunnan, China, including P philippensis and P yunanensis shared similar morphological structures in skull and were clustered together. All characters except for the variable BCASEL (braincase length), PORCL (postorbital constriction), and LMTL (length of the maxillary tooth row of lower jaw) gave high contributions to CAN I. Inspection of the plot based on the discriminant function 1 and 3 revealed that P p grandis and P hainana were still separated as distinct groups (Figure 4.3). BCASEL and MTRWL (Width of the maxillary tooth row) were the variables having the highest canonical scores on CAN II and CAN III, respectively. The PCA results in the first three factors and the eigenvector scores of variables were presented in Table 4.9. Along the first principal component (PRIN I) that accounted for 80% of the original total sample variance, all eigenvector coefficients of variables were positive. The primary separation of taxa along this axis was among P p lylei P p grandis and P hainana The population of P philippensis distributed in Yunnan was overlapped with P yunanensis (Figure 4.4). The morphological variables mainly responsible for this segregation were CRANL and LMDL. In the scatter-plot of PRIN I against PRIN III, P p grandis and P hainana were separated as distinct groups once again, but P p lylei P yunanensis and P philippensis in Yunnan shared similar cranial structures and were greatly overlapped (Figure 4.5). The variable BCASEL and LMDL were strongly correlated with PRIN II and PRIN III, respectively. The results of PCA were consistent with those of disrciminant function analysis.

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62 Table 4.8 Discriminant function analysis of five P philippensis forms CAN Eigenvalue Proportion Cumulative I 15.94 0.72 0.72 II 4.37 0.20 0.92 III 1.14 0.05 0.97 Canonical score Variable CAN I CAN II CAN III CRANL 0.95 0.02 -0.11 BCASEL 0.54 0.59 0.03 CRANW 0.94 0.05 -0.17 BPORW 0.94 -0.05 0.03 PORCL 0.66 -0.08 -0.19 PGA 0.94 0.12 0.02 NAL 0.78 0.03 -0.07 TBL 0.70 0.40 0.07 DSL 0.85 -0.09 -0.01 MTRL 0.84 -0.18 0.18 MTRW 0.76 -0.22 0.31 LMDL 0.94 0.08 0.20 LMDH 0.84 -0.03 0.30 LMTL 0.65 -0.03 -0.02

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63 Table 4.9 Principal components analysis of five P philippensis forms PRIN Eigenvalue Proportion Cumulative I 87.96 0.80 0.80 II 9.14 0.08 0.89 III 3.24 0.03 0.92 Eigenvector score Variables PRIN I PRIN II PRIN II CRANL 0.57 -0.06 -0.51 BCASEL 0.23 0.24 0.13 CRANW 0.26 -0.01 -0.13 BPORW 0.38 -0.10 -0.17 PORCL 0.08 -0.14 -0.05 PGA 0.24 -0.02 -0.05 NAL 0.17 -0.02 -0.22 TBL 0.10 0.00 0.03 DSL 0.13 -0.07 -0.06 MTRL 0.10 -0.04 0.09 MTRW 0.10 -0.02 0.01 LMDL 0.48 -0.26 0.75 LMDH 0.17 0.07 0.15 LMTL 0.11 0.00 0.14 4.3.1.2 Molecular data Totally 12 samples of five forms of P philippensis were examined to reconstruct the phylogenetic relationships. P petaurista from southeastern Asia was used as an outgroup. Table 4.10 showed the sequences differences that were corrected by Kimura’s

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64 two-parameter model (1980), and the numbers of transversions and transitions obtained from pairwise comparison between samples. Table 4.10 Pairwise comparison based on the partial sequences (409 bp) of cytochrome b gene between five P philippensis forms. Data below the diagonal are the numbers of nucleotide substitutions, transitions vs. transversions. Data above the diagonal represent the genetic differences between samples. The samples were defined in Table 4.6 and 4.7. PPH1 PPH2 PPH3 PPH4 PHK1 PHK 2 PYK1 PYK2 PYK3 PYK4 PYK5 PPG PPB PPH1 1.3 1.7 1.5 5.6 4.7 8.3 8.6 8.3 8.3 8.3 12.5 5.7 PPH2 4/1 1.0 1.2 5.9 3.9 9.3 9.4 9.0 9.0 9.0 11.4 6.4 PPH3 5/1 4/0 0 7.1 5.1 9.7 9.7 9.3 9.3 9.3 11.7 6.8 PPH4 4/0 4/0 0 6.6 4.8 9.4 9.4 9.1 9.1 9.1 11.3 6.8 PHK1 21/1 20/2 25/2 25/2 2.2 10.09.6 9.9 10.0 10.0 12.9 8.0 PHK2 19/0 15/0 20/0 19/0 5/1 9.4 8.6 9.0 9.0 9.0 12.9 6.3 PYK1 31/3 33/4 35/4 35/3 35/4 35/3 0.5 0.5 0.5 0.5 13.8 8.6 PYK2 31/4 34/4 35/4 35/3 33/6 35/3 1/1 0.2 0.3 0.2 14.1 8.9 PYK3 30/4 32/4 33/4 33/3 34/6 33/3 1/1 1/0 0 0 13.8 8.3 PYK4 30/4 32/4 33/4 33/3 34/6 33/3 1/1 1/0 0 0 13.8 8.6 PYK5 30/4 32/4 33/4 33/3 34/6 33/3 1/1 1/0 0 0 13.8 8.3 PPG 42/8 37/7 37/8 37/8 43/8 43/8 46/9 46/10 45/10 46/10 45/10 12.1 PPB 23/0 21/2 25/2 27/0 30/2 24/0 32/2 32/2 31/3 30/3 31/3 41/8 The considerable sequence variations existed among all forms. The form from Taiwan ( P p. grandis ) was significantly different from the forms in mainland with high genetic differences (11.3 – 14.1%). The form P yunanensis apparently differed from P philippensis and P hainana with 8.3 – 10% differences in sequence. The genetic difference between P philippensis and P hainana was about 5%. The phylogenetic reconstructions using maximum parsimony (MP) and neighbor-joining distance (NJ)

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65 methods resulted in essentially the same branching patterns. Each of five forms was formed as distinct clade (Figure 4.6 and Figure 4.7). The bootstrap values to support these branching orders were high, ranging from 87% to 100%. 4.3.2 Phylogenetic Relationship between P xanthotis and P leucogenys A partial sequence (409 bp) of cytochrome b gene of P xanthotis was successfully sequenced from two museum specimens. The sequence data of Japanese P leucogenys were retrieved from GenBank of NCBI. Table 4.11 Percentage of genetic differences between P xanthotis and other giant flying squirrels based on pairwise comparison of the partial cytochrome b sequences (409 bp). See Table 4.6 and Table 4.7 for sample information. PTK1 PTK2 PLL1 PLL2 PLN1 PLN2 PYK PPH PHK PPB PTK1 3.8 14.1 12.9 13.2 13.2 14.7 15.5 13.4 14.3 PTK2 11/3 11.7 10.2 9.2 9.2 11.94 11.57 12.7 12.5 PLL1 41/16 34/12 1.3 2.8 2.1 15.4 14.4 15.1 16.0 PLL2 37/15 27/12 4/1 2.6 1.8 14.9 13.6 14.4 15.0 PLN1 37/16 24/12 11/0 9/1 1.0 14.6 13.1 13.5 14.3 PLN2 37/16 24/12 8/0 6/1 3/0 14.3 13.3 14.1 14.3 PYK 46/13 40/8 51/11 47/13 47/11 46/11 8.6 9.6 8.9 PPH 52/10 38/8 49/9 46/8 43/9 44/9 31/4 5.6 5.7 PHK 43/9 41/8 50/11 48/10 43/11 46/11 33/6 21/1 8.1 PPB 47/9 43/7 52/8 50/7 46/8 46/8 32/3 23/0 26/1 Note: Data below the diagonal are the numbers of nucleotide substitutions, transitions vs. transversions. Data above the diagonal represent the genetic differences between samples. For the sake of comparison, P philippensis P yunanensis and P hainana were used to build their phylogenetic relationships with P petaurista distributed in southeastern Asia as an outgroup. Table 4.11 presented the percentage differences that

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66 were corrected by Kimura’s two-parameter model (1980), and the transversional and transitional numbers between samples using pairwise comparison. P xanthotis significantly differentiated from other Petaurista forms for their highly genetic differences, varying from 9.2% to 15.5%. The phylogenetic trees generated with MP and NJ methods were concordant with the results of pairwise comparison. Compared to P philippensis P yunanensis and P hainana there was a closed relationship between P xanthotis and P leucogenys although each of them formed as a distinct clade. (Figure 4.8 and Figure 4.9) The bootstrap value in MP tree for branching was 99%. 4.3.3 Phylogenetic Relationship of P. petaurista The morphological study of P p. albiventer between the eastern and the western trans-Himalayan populations was based on 41 specimens from N Pakistan and W Yunnan, China (Table 4.4). The populations from India, E and W Sumatra, Borneo, Java, and Malayan Peninsula (49 specimens) were included to investigate the morphological relationships among populations of P petaurista. Table 4.12 showed the results of discriminant function analysis on the first three functions, which were outlined graphically in Figure 4.10 and Figure 4.11. The first discriminant function that accounted for 68% of the total variance separated the population in W Yunnan from the rest groups (Figure 4.10). CRANW, BPORW, and LMDL were the major variables contributing to CAN I. The morphological variables contributing to CAN II included LMTL, LMDH, and BCASEL. In the plot of CAN I to CAN III (Figure 4.11), the populations distributed in Yunnan (SW China) and Burma were distinguished as different assemblages. The rest populations were overlapped as a complicated assemblage, although the overlap was little between the populations in Java and in Pakistan. BCASEL was the dominant variable on CAN III.

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67 Table 4.12 Discriminant function analysis of P. petaurista CAN Eigenvalue Proportion Cumulative 20.43 0.68 0.68 5.42 0.18 0.86 I II III 4.16 0.14 1.00 CANONICAL SCORE Variable CAN I CAN II CAN III CRANL 0.76 -0.44 0.38 BCASEL 0.40 -0.57 0.64 CRANW 0.81 -0.03 0.31 BPORW 0.85 -0.21 0.37 PORCL 0.32 0.45 0.25 PGA 0.68 -0.29 0.48 NAL 0.71 -0.54 0.09 TBL 0.02 -0.28 0.14 DSL 0.73 -0.04 0.51 MTRL 0.72 -0.38 0.17 MTRW 0.70 -0.59 0.06 LMDL 0.81 -0.13 -0.26 LMDH 0.68 -0.57 0.06 LMTL 0.64 -0.60 0.07 The results of principal components analysis of P p. albiventer were presented in Table 4.13. The first principal component factor that accounted for 76% of the total variance isolated the W Yunnan and Burma populations from others, forming two distinct clusters (Figure 4.12). The main determinants were CRANL on PRIN I, and

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68 BCASEL and LMDL on PRIN II. All specimens in the plot of the fist principal factor and the third principal factor were clustered as two groups (Figure 4.13). The first group consisted of the populations in Yunnan and Burma, and the second group included the remaining populations. The main variable contributing to the observed association was LMDL. Table 4.13 Principal components analysis of P petaurista PRIN Eigenvalue Proportion Cumulative I 50.07 0.76 0.76 II 6.52 0.10 0.85 III 3.46 0.05 0.91 Eigenvector score Variables PRIN I PRIN II PRIN III CRANL 0.63 0.17 0.04 BCASEL 0.20 0.75 -0.17 CRANW 0.24 -0.16 0.37 BPORW 0.37 -0.16 0.38 PORCL 0.04 -0.06 0.38 PGA 0.24 0.15 0.15 NAL 0.24 -0.03 -0.22 TBL 0.03 0.09 -0.06 DSL 0.18 -0.02 0.33 MTRL 0.14 -0.03 -0.08 MTRW 0.14 -0.03 -0.12 LMDL 0.27 -0.56 -0.35 LMDH 0.28 -0.09 -0.43 LMTL 0.16 -0.01 -0.17

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69 The molecular study of the populations of P p. albiventer was conducted based on the partial sequences (375 bp) that were obtained from 4 museum skin samples. The sequence data of P p. petaurista from Java and P p. melantous from Malaya Peninsular were used to build the phylogenetic topology. Table 4.14 Percentage of differences and the numbers of transversional and transitional substitutions between P. petaurista ( albiventer ) populations based on pairwise comparison of the partial sequence (375 bp) of cytochrome b gene. PPF1 FPPF2 PPY1 PPY2 PPB PPM1 PPM2 PVO PPF1 0.5 8.4 8.8 8.9 13.4 13.8 17.1 PPF2 2/0 8.3 8.6 8.9 13.3 13.8 17.7 PPY1 29/2 29/2 0.5 6.3 11.6 12.2 16.8 PPY2 32/2 32/2 3/0 6.3 11.6 12.2 16.8 PPB 32/1 32/2 24/0 24/1 11.5 11.5 15.9 PPM1 41/7 41/7 37/7 37/7 34/7 0.5 15.9 PPM2 44/7 44/7 39/8 39/8 34/7 2/0 15.9 PVO 43/19 46/19 37/21 39/20 37/20 37/20 37/20 Note: Data above the diagonal were the percentage of genetic differences between samples and data below the diagonal were the numbers of transitions vs. transversions between samples. See Table 4.4 for sample abbreviations. Table 4.14 was the percentage differences and the numbers of transversional and transitional substitutions on the pairwise comparison between samples. The population of P. p. albiventer in Pakistan was apparently different from that in Yunnan with the genetic distance, 8.6 8.9%. The population in Java showed similar genetic characters with the population in Yunnan, China. Both the MP and NJ trees generated the similar branching patterns (Figure 4.14 and Figure 4.15). The populations in Yunnan and in

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70 Pakistan were separated as distinct branches with a high bootstrap value (98%). The population in Malaysia, P. p. melantosus had an early divergence from other groups. 4.3.4 Phylogenetic Relationships of Chinese Petaurista The morphometric analyses were performed on 65 specimens including 5 species, P alborufus P elegans P xanthotis P philippensis and P petaurista ( albiventer ). The results of discriminant function analysis and principal components analysis were displayed in Table 4.15 and Table 4.16, respectively. The first discriminant function (CAN I) accounted for 60% of the original sample variance. Along CAN I, five Petaurista species were assembled as three clusters: P alborufus was one group, P elegans and P xanthotis formed one group, and P philippensis and P p. albiventer formed the third group (Figure 4.16). This separation was attributed to the morphological variable PORCL, DSL, and LMDL on CAN I, and BCASEL on CAN II, which accounted for 32% of the total variance. On the graph plotted on CAN I to CANIII, P alborufus P elegans and P xanthotis were successfully distinguished as distinct groups. The individuals of P philippensis and P p. albiventer were still widely overlapped, showing similar morphological structures (Figure 4.17). The major variables that contributed to CAN III were TBL and MTRW.

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71 Table 4.15 Discriminant function analysis of Chinese Petaurista CAN Eigenvalue Proportion Cumulative I 18.39 0.60 0.60 II 9.67 0.32 0.92 III 1.85 0.06 0.98 Canonical score Variable CAN I CAN II CAN III CRANL 0.54 0.78 0.01 BCASEL -0.16 0.95 0.04 CRANW 0.58 0.74 -0.24 BPORW 0.47 0.83 0.06 PORCL 0.68 0.42 -0.07 PGA 0.55 0.70 -0.06 NAL 0.39 0.71 0.19 TBL 0.24 0.13 0.57 DSL 0.68 0.51 0.01 MTRL 0.42 0.77 0.24 MTRW 0.57 0.58 0.35 LMDL 0.68 0.60 0.14 LMDH 0.39 0.76 0.29 LMTL 0.29 0.55 0.26 In PCA, the first principal factor was the most important, which accounted for 80% of the total variable variance, with the cranial length (CRANL) having the highest eigenvector value (Table 4.16). Along PRIN I, all specimens of Petaurista were identified as three different groups (Figure 4.18), which were consistent with the result

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72 of discriminant function analysis (see Figure 4.16). P alborufus was firstly separated as a group, and the rest four made up other two groups: one group comprising P. elegans and P xanthotis and another group including P philippensis and P. petaurista BCASEL with negative eigenvector score was the dominant variable that contributed to PRIN II. Table 4.16 Principal components analysis of Chinese Petaurista PRIN Eigenvalue Proportion Cumulative I 85.29 0.80 0.80 II 9.63 0.09 0.89 III 2.73 0.03 0.91 Eigenvector score Variables PRIN I PRIN II PRIN II CRANL 0.52 0.13 -0.05 BCASEL 0.37 -0.87 -0.07 CRANW 0.27 0.14 -0.56 BPORW 0.41 0.05 -0.07 PORCL 0.10 0.18 -0.32 PGA 0.22 0.06 -0.19 NAL 0.17 -0.01 0.19 TBL 0.03 0.03 0.17 DSL 0.11 0.15 -0.07 MTRL 0.13 0.00 0.19 MTRW 0.15 0.12 0.21 LMDL 0.38 0.37 0.23 LMDH 0.22 -0.03 0.37 LMTL 0.12 0.00 0.44

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73 When all specimens were plotted onto PRIN I and PRIN III, except for P petaurista and P philippensis that extensively overlapped, P alborufus, P xanthotis and P elegans could be identified as distinct groups, although there existed a few overlaps between each other (Figure 4.19). The most contributions to PRIN III were from the morphological variables CRANW and LMTL. In molecular analyses, the partial sequences (380 bp) were isolated from 8 species (senso lato), including P alborufus P elegans P yunanensis P hainana P philippensis P xanthtis and P petaurista (Zhang et al., 1997; Wang, 2002). The sequence data of P a castaneus P a lena and P l leucogenys (Oshida et al., 2000) were quoted from GenBank of NCBI. Pteromys volans was assigned as an outgroup to reconstruct the phylogeny. Table 4.17 showed the genetic results from pairwise comparisons between samples. P elegans and P xanthotis were the two most diverse species in Petaurista The genetic differences between P elegans and other Petaurista groups were 9.2% 15.0%. P xanthtis showed 12.8% 15.4% differences from other groups. The population of P. alborufus in China (PAC) was apparently different from that in Japan (PAL) with sequence difference at 12.5% level. The phylogenetic reconstructions using UPGMA, MP and NJ methods yielded the similar topological patterns (Figure 4.20, Figure 4.21, and Figure 4.22). P. xanthotis P. elegans, and the population of P. alborufus in Japan formed one group. The population of P. petaurista in Pakistan and P. yunanensis formed another group. And the rest were clustered together as the third group, including the population of P. alborufus in Yunna, P. philippensis the population of P. petaurista in Yunnan, and P. hainana

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74 Table 4.17 Pairwise comparison of Chinese Petaurista based on the partial sequences (380 bp) of cytochrome b gene. Data above the diagonal were the percentage of genetic differences between samples, and data below the diagonal were the numbers of transitions vs. transversions between samples. PAK1 PAK2 PAK3 PAC PHK PPH PPF PYK PPY PEK1 PEK2 PEK3 PAL PTK PVO PAK1 0.5 0.6 0.6 8.5 6.3 9.7 8.4 6.4 13.7 13.1 13.7 12.5 14.4 14.8 PAK2 2/0 0 0 8.2 5.7 9.7 8.4 6.1 13.2 12.8 13.1 12.5 13.8 14.7 PAK3 2/0 0 0 8.2 5.4 9.3 8.4 5.9 13.3 12.8 13.1 12.7 13.7 14.5 PAC 2/0 0 0 8.4 5.9 10.0 8.7 6.0 12.9 12.8 13.1 12.5 14.2 14.7 PHK 30/1 29/1 29/1 30/1 5.6 9.7 9.6 5.3 12.8 12.8 12.9 15.1 13.4 17.0 PPH 24/0 22/0 20/0 22/0 19/1 9.1 8.9 1.8 10.8 9.2 9.9 14.7 15.4 16.4 PPF 35/2 35/2 33/2 36/2 33/3 33/2 6.1 8.3 13.4 13.1 14.0 14.5 12.8 17.0 PYK 28/4 28/4 27/4 28/4 31/5 30/4 21/2 8.9 14.2 14.5 15.0 15.6 14.6 17.6 PPY 24/0 23/0 23/0 22/0 19/1 5/0 30/2 30/4 9.8 9.9 9.9 12.9 14.4 15.8 PEK1 44/8 42/8 42/8 40/8 40/8 33/8 41/10 42/12 30/8 4.5 4.4 16.1 14.1 18.1 PEK2 41/8 40/8 40/8 39/8 38/1026/8 41/8 47/8 29/8 16/1 1.1 13.9 13.5 17.0 PEK3 44/7 41/8 41/8 39/8 37/9 30/7 45/7 49/7 29/8 14/2 4/0 14.5 15.1 17.2 PAL 38/8 38/8 39/9 37/8 46/1047/8 43/11 48/10 44/5 48/13 38/12 42/12 14.1 15.8 PTK 44/10 42/10 41/9 43/10 40/9 48/1038/10 43/12 47/8 41/12 41/9 46/10 38/15 19.3 PVO 33/22 33/22 32/22 33/22 40/2439/2243/20 46/20 38/2343/25 42/21 42/21 38/2050/23 To estimate the divergence time between species and populations, the transversional substitutions at the third codon positions were obtained from pairwise comparison (Table 4.18). The divergence times were calculated using the rate of divergence for the third codon positions of mammalian cytochrome b gene of ca. 0.5% *106 (Table 4.19). P. xanthotis and P. elegans were the earliest species that diverged from Petaurista approximately 11.2 to 13 million years ago. In P. petaurista the divergence time between the population in Pakistan and the population in W Yunnan was about 1.2 – 3.2 million years ago.

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75 Table 4.18 Transversional substitutions at the third codon positions of the partial sequences (375 bp) of cytochrome b gene in Petaurista. PAK PYK PPH PHKPEK PTK PPF PPB PPY PVO PAK 2.4 0 0 4.8 5.6 1.6 0 0 15.4 PYK 3 2.4 2.4 6.5 5.6 0.8 1.6 2.4 13.8 PPH 0 3 0 4.8 5.6 0.8 0 0 15.4 PHK 0 3 0 5.6 5.6 1.6 0 0 15.4 PEK 6 8 6 7 6.5 7.3 5.6 4.8 16.3 PTK 7 7 7 7 8 5.6 6.5 5.6 13.8 PPF 2 1 1 2 9 7 0.8 0.8 13 PPB 0 2 0 0 7 8 1 0 15.4 PPY 0 3 0 0 6 7 1 0 15.4 PVO 19 17 19 19 20 17 16 17 19 Note: Data below the diagonal are the transversional numbers at the third codon positions. Data above the diagonal represent the transversional percentage difference between samples. The sample abbreviations were defined in Table 4.6. Table 4.19 The estimated divergence time between species based on a divergence rate for the third codon positions of mammalian cytochrome b gene of ca. 0.5% *106 years PAK PYK PPH PHK PEK PTK PPF PPB PPY PYK 4.8 PPH 0 4.8 PHK 0 4.8 0 PEK 9.6 13 9.6 11.2 PTK 11.2 11.2 11.2 11.2 13 PPF 3.2 1.6 1.6 3.2 14.611.2 PPB 0 3.2 0 0 11.213 1.6 PPY 0 4.8 0 0 9.6 11.2 1.6 0 PVO 30.8 27.6 30.8 30.8 32.627.6 26 30.8 30.8

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76 4.4 Discussion 4.4.1 Phylogeny of the Trans-Himalayan P. petaurista ( albiventer ) Petaurista petaurista is extensively distributed in southeastern China, Sichuan, Yunnan and Fukien. This species has a broad distribution beyond China including northern India, Bhutan, Nepal, Pakistan, northern Afghanistan, and southeastern Asia. The populations of P. petaurista in Pakistan and W Yunnan, China, are named as the same subspecies, P. p. albiventer (Corbet and Hill, 1992; Zhang et al., 1997) since their similar external and dental structures (Figure 4.23). Wang (2002) elevated the populations of P. petaurista in Pakistan and W Yunnan (China) as a valid species, P. albiventer The present study based on the morphological and molecular analyses reveal that these two populations are significantly different. The principal components analysis indicates that their main differences are in skull size (The eigenvector scores are all positive on the first principal component factor) and the morphological structure of lower jaw, which apparently separate it from the rest populations (Figure 4.10 and Figure 4.12). When compared with other populations, the population in Pakistan shares more cranial characteristics with the populations from SE Asian rather than with P. albiventer in Yunnan, China. This can be inferred that these two populations may occupy different habitats that result in different adaptations. This inference agrees with Ellerman’s (1940) study, which shows that the forms candidula barroni and taylori of P. petaurista in SE Asia are much similar to the Himalayan forms ( P. p. albiventer ). The only species or subspecies of Petaurista distributed in Pakistan is the Himalayan giant flying squirrel, P. ( p. ) albiventer The area of suitable forest where this giant flying squirrel can be found is comparatively limited. In Pakistan, it mainly occurs in Himalayan moist temperate forest, extending in the northwest of Pakistan into Deodar

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77 ( Cedrus deodara ) forest or subtropical pine ( Pinus roxburghii ) zone, elevation from about 1,350 m to upper limit of the tree line at about 3,000 m (Roberts, 1997). P. petaurista is sympatrically distributed in N Pakistan with E. cinereus and Eoglaucomys fimbriatus The competition for food resources among them is unavoidable. Since they have different morphological dental structures, the feeding strategies and food selection are different. The giant flying squirrel selectively feeds upon the young green leaves, the fir and pine cones, the nuts, even the young twigs and tree buds ( Quercus dilatata ), which are partially different from Eupetaurus and Eoglaucomys (Roberts, 1997). Himalayan moist temperate forest commonly consisting of the hill oak ( Quercus dilatata ), horse chestnuts ( Aesculus indica ), and walnuts ( Juglans regia ) supplies this species with sufficient food resource. Since partitioning of microhabitats among competing species is thought to contribute to coexistence among rodent species (Price, 1978), the different habitat selections of these three flying squirrels are a major contributor to this coexistence. The similar sympatric distribution of E. cinereus, P. petaurista, and Hylopetes alboniger are also found in W Yunnan, China. P. p. albiventer in W Yunnan is widely overlapped with H. alboniger from NW Yunnan toward southern Yunnan at different elevations (500m to 3500m) (Zhang et al., 1997). These areas have typically high diversity of plant species that correspond with the mixed dietary habits of these flying squirrels. According to local people, both flying squirrels mainly inhabit in coniferous forest. P. petaurista frequently feeds on walnuts, acorns, and corns, and occasionally descends into farm to feed on corn at dawn. This is a little different from the population of Pakistan, indicating that the Pakistan giant flying squirrel is more adaptable to harsh

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78 or less mesic conditions than the giant flying squirrel in W Yunnan. Their morphological differentiations, such as skin (Figure 4.23), are apparently associated with their different living conditions. The molecular data are partially consistent with the morphological data. The difference in cytochrome b gene between the eastern and the western trans-Himalayan giant flying squirrel ( P petaurista ) is significant (Figure 4.13 and Figure 4.14). The genetic distance is about 8.9%, above the subspecies-level (Table 4.14). Geologic history has been a major factor in understanding evolution of flying squirrels. The geotectonic and paleoclimatic records reveal a series of episodic landscape transformations throughout the past millions of years coincident with changes in taxa and ecological diversity. Based on the rate at the third codon positions of cytochrome b gene (Table 4.18), the Pakistan population of P. petaurista diverged from the Yunnan population about 1.6 million years ago (Table 4.19). The distinct phylogeographic discontinuity between the eastern and the western lineages of P. petaurista suggests a major environmental impediment to gene flow. The possibility that the coincident pattern between these two populations were caused by a shared historical dispersal event is supported by a diverse array of vertebrate taxa that exhibit a similar genetic discontinuity in the trans-Himalayas (Woods, personal communication). The populations of P. petaurista delineated by large phylogenetic gaps are obviously associated with the biogeographic barrier of the great Himalayan chain to gene flow. The climatic changes could lead to an expansion of the Palearctic fauna at the expense of the Oriental fauna, though some generalized species or population of Oriental forms would undoubtedly be able to adapt to the new conditions. The results of the

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79 present study show that the genetic differences among the populations in SW China, SE Asia, and Pakistan are not closely associated with the geographic distances of sampling localities. This implies that P. petaurista rapidly extended into SW China, Pakistan, and SE Asia in a short time during the southward expansion of temperate forests during the glacial stage of Pleistocene. The estimations of the third codon positions of cytochrome b sequences indicate that all populations of P. petaurista diverged from each other during Pleistocene and Holocene. When all populations split from their common ancestor in Pleistocene, one branch migrated to the western side what is now in Pakistan where the climate in northern part and indeed the whole Indus plain, was evidently warmer and more humid in early Pleistocene times (Roberts, 1997). One moved to the eastern side what is now in SW China, and the third branch migrated to SE Asia. During the subsequent glaciation in the late Pleistocene and Holocene that was intervened with shorter periods of warmer moister climatic change, all populations gradually adapted to their present habitats. The similar cranial structures among the populations in SE Asia are apparently the adaptations of similar living conditions. P. petaurista is a polymorphic species and is extensively distributed in various geographical locations. Like many wide-ranging taxa, it is divided into separate species or subspecies by zones of hybridization (Bull, 1991). Because hybrid zones involve closely-related taxa at various stages of speciation, they represent natural settings for study of speciation, gene flow, adaptation, and reinforcement of isolating mechanisms (Baker et al., 1989; Harrison, 1990, 1993; Bendict, 1999). Therefore, the further study of P. petaurista should focus on the populations between SW China and SE Asia, and

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80 between the eastern and the western trans-Himalayas, such as the populations in Burma, India, Thailand, and Laos. 4.4.2 Taxonomic Status of P. philippensis P. yunanensis and P. hainana P. hainana (Hainan giant flying squirrel) in Hainan, P. yunanensis (Yunnan flying squirrel) in Yunnan, and P. grandis (Taiwan giant flying squirrel) in Taiwan were referenced as valid species by Allen (1940) and Ellerman (1940), but Hoffmann et al. (1992) and Nowak (1999) regarded them as the subspecies or synonyms of P. philippensis (gray-backed giant flying squirrel). By checking the collections in Beijing and Yunnan, China, Zhang et al. (1997) and Wang (2002) elevate them as distinct species recently. Taiwan is an island situated on the contnental slope of mainland China and separated by the 150 km strait. It is known to have been connected by a landbridge to mainland China several times during the Pleistocene. In the last warm glacial period (10, 000 to 56, 000 years ago), sea levels decreased by 80 to 150m (Lin, 1966). This indicates that P. grandis migrated to Taiwan from south China during Pleistocene. The findings from both molecular and morphological data in this study support P. p. grandis to be a distinct species (Figure 4.2 and Figure 4.4), which are consistent with Oshida’s et al. (2000a) result and suggest P. grandis to be a valid species. The molecular and morphometric analyses on the remaining populations show some controversial results. The multivariate analyses indicate that P. hainana apparently differs from the rest populations in cranial morphology and P philippensis in Yunnan is morphologically similar to P yunanensis (Figure 4.2 to Figure 4.5). Whereas the molecular data suggest that P. hainana is genetically close to P philippensis with short genetic distance (~ 4 5%). P. yunanensis is evidently distinguishable from P.

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81 hainana and P. philippensis with genetic differences about 10.9% and 8.6%, respectively (Table 4.10). Yunnan flying squirrel P yunanensis is a very handsome maroon flying squirrel with white speckling over the back (Figure 4.24). The type specimen is a skin without a corresponding skull. It was collected from Momein (=Tengyueh), southwestern Yunnan, China, and is represented in the Indian Museum, Calcutta with a catalog number 9486. P yunanensis resembles P. alborufus in size, but is morphologically similar to the population of P. philippensis in Yunnan in the general deep bay coloring (Figure 4.24). Both skulls are large but apparently show no special peculiarities The distribution of P. yunanensis is from the extreme southwestern Yunnan probably into Burma and Indochina, extensively sympatric with P. philippensis in SW China (Zhang et al., 1997). Although at present it is not clear to their feeding habits, their morphological similarity in skull is clearly due to the adaptations to similar habitats. The similar morphological characters and the distinct genetic characters between them suggest that conservative systematic traditions or morphological stasis may be involved. According to the estimated divergence time, P. yunanensis diverged from the stock of P. philippensis about 4.8 million years ago, the early Pliocene. There are known to have been three major periods of successive prolonged glaciation when sea levels sank and huge ice caps developed over all the great mountain massifs during Pliocene, which dramatically affected the climate, biogeographic structures, and flauristic environments along the great Himalayan mountain chain. As a result, the eastern and the western extremes of Himalayas became the optimal shelter of refugees. A quite unique mammalian assemblage including flying squirrels migrates and survives today in the

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82 western side of Yunnan of China. Although the range of P. yunanensis is overlapped with P. philippensis in Yunnan, there is no indication that the two interbreed. The significant differentiation in genetic characters suggest that P. yunanensis is a distinct species, notwithstanding the general similarity in size and body coloration with P. philippensis The type specimen of P. hainana is an adult female skin with the corresponding skull, which was collected from Namfong, the island of Hainan, China, on February 19, 1923, by Clifford H. Pope (Allen, 1940). It is now represented in the collection of American Museum of Natural history. P. hainana is a tropical species that reaches the northern limit of its range in extreme southern China. Allen (1940) predicted that no doubt Hainan giant flying squirrel ( P. hainana ) would be found to show relationship to some forms of the Indo-Chinese mainland. The present study demonstrates that P. hainana is phylogenetically related to P philippensis in Yunnan. P. philippensis is distributed in mountainous coniferous forests at different elevations in W Yunnan; whereas P. hainana is confined to tropical forest on Hainan. Either the feeding habits or the living habitats are significantly different. The morphological difference between them is probably associated with their geographical variations and living conditions. But the close genetic distance between P. hainana and P. philippensis 5 – 7%, is not consistent with their geographic distances of sampling localities. The conflicting placement on the molecular and morphological trees is difficult to reconcile because the phylogenetic hypothesis suggested by the molecular tree requires convergent evolution in morphology and osteology to reflect similarities in these characters among the clades (Austin, 1996). The estimation based on the third codon positions of cytochrome b gene

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83 shows that the divergence of P. hainana from P. philippensis is very recent. The reasonable explanation is that the population of P. hainana rapidly extended to its present distribution in a short time during the southward expansion of temperate forests in southern China during the last glacial stage of Pleistocene because the history of the island of Hainan is about 1 million years. The estimate of the times separating sequences in subspecies within different groups is ranging from 0.94 to 1.52 million years based on a rate estimate of six Sciurus aberti subspecies (Wettstein et al., 1995). Accepting this hypothesis, P. hainana is a subspecies or a synonym of P. philippensis 4.4.3 Phylogenetic Relationship between P. xanthotis and P. leucogenys Petaurista xanthotis is an endemic species in China. It is a large yellowish-gray species with an orange spot behind the ear. Muzzle, forehead, and cheeks shorter-furred than the neck and body, minutely mixed black and white, giving a gray appearance. It has been accepted as a valid species of Petaurista (Corbet and Hill, 1992; Nowak, 1999; Zhang et al., 1997; Wang, 2002). The type specimen of P xanthotis is a mounted skin in the "galerie publique" of the Museum of Natural History at Paris, sent by Pere Armand David from Tibet, near Muping, Sichuan, China (Ellerman, 1940). P. ( Pteromys ) xanthotis was originally named by Milne-Edwards in the belief that it was, if not a distinct species, at least a variety of Pteromys melanopterus (Ellerman, 1940). Buechner (1892) verified that they were not the same animal, but undoubtedly represented P. xanthotis In China, P. xanthotis inhabits from Tsing Hai and Kansu, southeastward to Hubei and southward to Sichuna and Yunnan, China, at different elevations, ranging from 2,000 meters in Gansu to 3,300 meters in Yunnan. The major habitat in NW China is temperate forest. Due to its widely distribution, P xanthotis shows morphological

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84 variations. The population in Yunnan measures slightly less than those from Gansu and Tsing Hai, but the difference is not great and does not warrant their separation as a distinct form. Because of the semi-hypsodont molariform teeth, P. xanthotis had been considered as the closest relative of Eupetaurus (McKenna, 1962). But the molecular data suggest that the similarity of cheekteeth between Eupetaurus and P xanthotis might be the convergent adaptation to the similar food selection (See Chapter 3 for detail). In the historical classification, P xanthotis was included in the Japanese P. leucogenys (Ellerman and Morrison-Scott, 1951). The molecular data in present study reveal a relative close phylogenetic relation between them. But their dental structures are very distinctive, and the cheekteeth of P. xanthotis is more complex than P leucogenys and other Petaurista forms. P. leucogenys inhibits temperate forests in Japan (Nowak, 1999), which is similar to the habitat of P. xanthotis in NW China. According to pelage characteristics, P. leucogenys was classified into three subspecies, P. l. leucogenys, P. l. nikkonis and P. l. oreas (Imaizumi, 1960) Although P. xanthotis is relatively close to P. leucogenys (Figure 4.8 and Figure 4.9), they are indeed genetically different with genetic distances about 9.2 to 14.1% (Table 4.11), suggesting two full species. The islands of Japan are separated from Korean penisula by the Korean Strait, which has a water depth of about 100 m (Kim et al., 1991). During the Pleistocene glacial periods, a landbridge was present between Korea and Japan, and the southern end of the Ryukyu Archipelago was connected with Taiwan in the early Pleistocene (Hikida and Ota, 1997). Based on available fossil records, Oshida et al. (2000c) thought that P. leucogenys migrated from southern China to Japan through the land bridge that was formed around the area of the present East China Sea in the early

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85 middle Pleistocene. If these biogeographic hypotheses are correct, with the estimated divergence time (Table 4.19), P xanthotis diverged from P. leucogenys and other Petaurista forms about 10 to 12 million yeas ago, the middle Miocene, much before the migration of P. leucogenys to Japan. The present distributions of P xanthotis and P. leucogenys are apparently due to the geographic events of the Pliocene-Pleistocene period. During glacial stages in Pleistocene, they moved to southward shifting of temperate forests in both China and Japan. 4.4.4 Systematics of Chinese Petaurista The eastern trans-Himalayan giant flying squirrels at species-level include P. petaurista, P. philippensis, P. yunanensis, P. alborufus, P. elegans, and P. xanthotis. With Pteromys volans as the outgroup, all Petaurista forms are genetically related to each other. The red and white flying squirrel, P. alborufus is distributed in somewhat more northern from Sichuan and Hupei to the mountains of the Likiang Range (Zhang et al., 1997; Wang, 2002) (Figure 4.1). The skull is of nearly maximum size for the genus and is heavily formed with the stout, triangular postorbital processes. The teeth are relatively simple in structure. This species includes three races in mainland China: the typical form of the mountains in southern Muping of Yunnan, with red feet; a more eastern race of Hupei with black feet and little whitish below; and a third form in Yunnan with the pale dorsal area and black feet (Thomas, 1923). P. alborufus is confined to forests of the central and eastern parts of Sichuan. Although P. yunanensis shares similar external structure and pelage coloration with P alborufus the two appear to be distinct species with allopatric distributions. The morphometric analysis on the cranial structure reveals that P. alborufus is significantly different from the rest

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86 Petaurista groups (Figure 4.16 and Figure 4.18). The molecular data are not fully coincident with the morphological inference, however (Figure 4. 20 to Figure 4.22). P. alborufus in mainland is genetically related to P. petaurista, P. philippensis and P. yunanensis Whereas the population of P. alborufus in Taiwan is significantly different from other Petaurista groups, showing an early divergence (Figure 4.21 and Figure 4.22). Oshida’s et al. (2000a) study suggests that the population of P. alborufus in Taiwan could be a distinct species because of its early deviation from P. petaurista and P. philippensis With the geohistorical and biogeographical evidences (Lin, 1966), the population of P alborufus might evolved independently from other P. petaurista species in the late Miocene and migrated to Taiwan island adapting itself to the alpine habitat. If this assumption is reasonable, P. alborufus in mainland might diverge from the lineage of P. petaurista and P. philipensis very recently, during the late Pleistocene or Holocene, and adapt to different living habitat. Petaurista elegans is distributed in Sichuan and Yunnan, China (Hoffmann et al., 1992). Two forms used to be included in this species, P. e. clarikei with ochraceous rufous feet and P. e. punctatus (or marica ) with blackish head. Both forms were also treated as distinct species by some authorities (Allen, 1940; Ellerman, 1940; Zhang et al., 1997; Wang, 2002). The morphological data of this study shows that P. elegans is more similar to P. xanthotis rather than other giant flying squirrels (Figure 4.16 to Figure 4.19), which is consistent with their geographical distributions (Figure 4.1). In Yunnan and eastern Sichuan, the range of P. xanthotis is overlapped with that of P. elegans showing sympatric distribution. But, P. xanthotis is not genetically related to

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87 P. elegans for their highly different genetic characteristics. Both of them diverged from the ancestral stock of Petaurista in the middle Miocene, 11 to 14 million years ago. Paleontological records can usually provide accurate morphological and genetic change information on the amazing diversity of life that existed in the past (Smith, 1998). The available fossil record suggests that there were multiple lineages of flying squirrels during Miocene (James, 1963; Mein, 1970). Several molecular studies have supported the general hypothesis of latest Pleistocene southward depression, followed by postglacial northward expansion of ranges in Palearctic and Nearctic taxa (Cooper et al., 1995). The further support is from the fossil evidence of similar distinctive dentition that was found in an Oligocene example (Bruijn and Uney, 1989). The fossil species, Pteromys lopingensis which was discovered in Loping, Jiangxi, China and described by Young (1947), is considered from Pleistocene and referable to Petaurista or Trogopterus proving that Petaurisa stock had distributed in China before Pleistocene. Integrating the fossil records and the present distributions of these Petaurista forms, their biogeographic history can be interpreted with the following hypothesis. The radiation of Petaurista occurred in the Eurasian continent (Oshida et al., 2000a). After diverged from the ancestral Petaurista in the middle Miocene, both P. xanthotis and P. elegans, maybe the group of P. alborufus in Taiwan, and the ancestral stock of Petaurisa were restricted to the southern parts of the Eurasian continent. During the subsequent three major periods of successive prolonged glaciation, P. xanthotis had adapted to the cold environment and high elevation, and survived with special feeding habit. With the retreating of glacier and the further uprising of the Himalayas, P. xanthotis was expanded northward, where it inhabited temperate forests with mountainous coniferous

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88 habitat; the Taiwanese P. alborufus migrated toward east; and the ancestor of P. petaurista and P. elegans remained in the south. During small upheavals or movements of tectonic plates that created the Himalayan foothill in the late Pliocene or Pleistocene, the other Petaurista forms, such as P. yunanensis, P. petaurista, and the mainland form of P. alborufus explosively diverged. Within the successive periods of glaciation in the late Pleistocene, the intervening shorter periods of warmer, moister climatic changes forced them to dispersal to the present geographical ranges. These multiple dispersal events may have acted to increase haplotypic diversity within these eastern Petaurista groups. On the other hand, phylogenetic analyses using both molecular and morphological information raise some controversial issues particular to the comparison of these data (Patterson, 1988, 1999). In some forms, the genetic results are not concordant with their geographic distances of sampling localities. For example, the population of P. petaurista in Pakistan is close to P. yunanensis rather than the population in Yunnan, which is relatively close to P. philippensis (Figure 4.20 and Figure 4.21). The similar conflict between morphological data and molecular data is also found in other mammals, such as Hugot’s (1998) research on neotropical monkeys, in which the results based on morphological or molecular data were generally conflicting and the phylogeny of the group is debated. Riddle (1996) thinks that populations not clearly delimited by large phylogenetic gaps are genetically connected through ongoing or recent dispersal, and populations delineated by large phylogenetic gaps are usually associated with stable biogeographic barriers to gene flow. It is inferred that the Chinese P. petaurista, P. philippensis, P. yunanensis and P. alborufus rapidly

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89 extended its present distributions in a short time during the last glacial stage of Pleistocene. 4.5 Summary In this chapter, the phylogenetic relationships among Petaurista groups in Pakistan and SW China were investigated. The following results are concluded based on the morphometric and molecular data: 1. The population of P. petaurista in Pakistan is significantly different from the population in W Yunnan of China in morphology and genetics. It is probably a new species, at least a new subspecies. 2. P. yunanensis is genetically different from the population of P philippensis in Yunnan and P. hainana and might be a valid species. P. hainana might be a subspecies or synonym of P philippensis for their closed genetic relationship. The morphological similarity between P. yunanensis and P. philippensis is an adaptation to similar habitat. 3. P. xanthotis is a valid Chinese endemic species and morphological similar to P. elegans but genetically different. P. xanthotis shows a close phylogenetic relationship with P. leucogenys which is distributed in Japan and China. 4. Both P. xanthotis and P. elegans diverged from the ancestral stock of Petaurista in the middle Miocene. The remaining populations of Petaurista rapidly extended its present distribution in a short time during the last glacial stage of Pleistocene. 5. The controversial results from mophometric and molecular analyses in some forms can be interpreted with the Riddle’s (1996) biogeographic theory. Populations not clearly delimited by large phylogenetic gaps are genetically connected through ongoing or recent dispersal and populations delineated by large phylogenetic gaps are usually associated with stable biogeographic barriers to gene flow.

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90 Figure 4.1 Chinese giant flying squirrels ( Petaurista )

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91 -4 -2 0 2 4 6 -8-6-4-20246CAN ICAN II P. hainana P. philippensis in Yunnan P. p. lylei P. p. grandis P. yunanensis Figure 4.2 Plot of five P philippensis forms onto discriminant function 1 (CAN I) and function 2 (CAN II)

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92 -6 -4 -2 0 2 4 -8-6-4-20246 CAN ICAN III P. hainana P. philippensis in Yunnan P. p. lylei P. p. grandis P. yunanensis Figure 4.3 Plot of five P philippensis forms onto discriminant function 1 (CAN I) and function 3 (CAN III)

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93 -8 -6 -4 -2 0 2 4 6 -20-15-10-5051015 PRIN IPRIN II P. hainana P. philippensis in Yunnan P. p. lylei P. p. grandis P. yunanensis Figure 4.4 Principal components analysis of five P philippensis forms onto factor 1 (PRIN I) and factor 2 (PRIN II)

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94 -6 -4 -2 0 2 4 6 -20-15-10-5051015 PRIN IPRIN III P. hainana P. philippensis in Yunnan P. p. lylei P. p. grandis P. yunanensis Figure 4.5 Principal components analysis of five P philippensis forms onto factor 1 (PRIN I) and factor 3 (PRIN III)

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95 Figure 4.6 Phylogenetic relationships of P philippensis forms based on the cytochrome b gene using maximum parsimony method (MP). Numbers above branches indicate the bootstrap values (%). Sample abbreviations are defined in Table 4.6 and Table 4.7.

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96 Figure 4.7 Phylogenetic relationships of P philippensis forms based on the cytochrome b gene using neighbor-joining method (NJ). Scales in the tree represent branch length in terms of nucleotide substitutions per site. See sample abbreviations in Table 4.6 and Table 4.7.

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97 Figure 4.8 Phylogenetic tree of P. xanthotis and other giant flying squirrels constructed using maximum parsimony method (MP). Numbers above branches indicate the bootstrap values (%). Sample abbreviations are defined in Table 4.6 and Table 4.7.

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98 Figure 4.9 Phylogenetic tree of P. xanthotis and other giant flying squirrels constructed using neighbor -joining method (NJ). Scales in the tree represent branch length in terms of nucleotide substitutions per site. See sample codes in Table 4.6 and Table 4.7.

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99 -6 -4 -2 0 2 4 6 -6-4-202468101214CAN ICAN II Population in Yunnan Population in N Pakistan Population in Burma Population in Sumatra Population in Java Population in Borneo Figure 4.10 Plot of P petaurista populations of discriminant function analysis onto the first and the second discrimiant function (CAN I to CAN II)

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100 -4 -2 0 2 4 6 8 10 -5051015 CAN ICAN III Population in Yunnan Population in N Pakistan Population in Burma Population in Sumatra Population in Java Population in Borneo Figure 4.11 Plot of P petaurista populations of discriminant function analysis onto the first and the third discrimiant function (CAN I to CAN III)

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101 -12 -8 -4 0 4 8 -15-10-505101520 PRIN IPRIN II Population in Yunnan Population in N Pakistan Population in Burma Population in Sumatra Population in Java Population in Borneo Figure 4.12 Principal components analysis of P petaurista populations onto factor 1 and factor 2 (PRIN I to PRIN II)

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102 -4 -2 0 2 4 6 -15-10-505101520 PRIN IPRIN III Population in Yunnan Population in N Pakistan Population in Burma Population in Sumatra Population in Java Population in Borneo Figure 4.13 Principal components analysis of P petaurista populations onto factor 1 and factor 3 (PRIN I to PRIN III)

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103 Figure 4.14 Phylogenetic relationships within the populations of P. petaurista reconstructed by maximum parsimony (MP) method with Pteromys volans as the outgroup. Numbers above branches are the bootstrap values (%). The abbreviations of taxa are defined in Table 4.6 and Table 4.7.

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104 Figure 4.15 Phylogenetic relationships within the populations of P. petaurista reconstructed by neighbor-joining (NJ) method with Pteromys volans as the outgroup. Scales in the tree represent branch length in terms of nucleotide substitutions per site. The abbreviations of taxa are defined in Table 4.6 and Table 4.7.

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105 -8 -6 -4 -2 0 2 4 6 -6-4-202468 CAN ICAN II P. alborufus P. elegens P. philippensis P. xanthotis P. petaurista Figure 4.16 Discriminant function analysis of Chinese Petaurista on discriminant function 1 (CAN I) and function 2 (CAN II)

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106 -4 -2 0 2 4 6 8 -6-4-202468CAN ICAN II I P. alborufus P. elegens P. philippensis P. xanthotis P. petaurista Figure 4.17 Discriminant function analysis of Chinese Petaurista on discriminant function 1 (CAN I) to function 3 (CAN III)

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107 -6 -4 -2 0 2 4 6 -20-15-10-505101520PRIN IPRIN II P. alborufus P. elegens P. philippensis P. xanthotis P. petaurista Figure 4.18 Principal components analysis of Chinese Petaurista on factor 1 (PRIN I) and factor 2 (PRIN II)

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108 -6 -4 -2 0 2 4 6 8 -20-15-10-505101520 PRIN IPRIN III P. alborufus P. elegens P. philippensis P. xanthotis P. petaurista Figure 4.19 Principal components analysis of Chinese Petaurista on factor 1 (PRIN I) and factor 3 (PRIN III)

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109 Figure 4.20 Phylogenetic topology of Petaurista based on the maximum parsimony method. The number of bootstrap value (%) is given above each branch. The abbreviations of taxa are defined in Table 4.6 and Table 4.7.

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110 Figure 4.21 Phylogenetic topology of Petaurista based on the neighbor-joining method. Scales in the tree represent branch length in terms of nucleotide substitutions per site. The abbreviations of taxa are defined in Table 4.6 and Table 4.7.

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111 Figure 4.22 Phylogenetic topology of Petaurista based on the UPGMA method. The abbreviations of taxa are defined in Table 4.6 and Table 4.7.

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112 Figure 4.23 P. petaurista in Pakistan and W Yunnan, China

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113 Figure 4.24 P. philippensis and P. yunanensis in Yunnan, China

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114 CHAPTER 5 PHYLOGENY OF EOGLAUCOMYS AND HYLOPETES IN THE EASTERN AND THE WESTERN TRANS-HIMALAYAS AS INFERRED FROM MOLECULAR AND MORPHOMETRIC STUDY 5.1 Introduction The generic status of Hyloptetes (arrow-tailed flying squirrel) and Eoglaucomys (small Kashmir flying squirrel) has been controversial for a long time. Eoglaucomys was maintained as a subgenus of Glaucomys (Thomas, 1908) until Howell (1915) first described it as a distinct genus. The morphological structures of the skull and baculum suggest that Eoglaucomys in the western extreme of the Himalayas could be a different genus from Hylopetes in the eastern Himalayas and SE Asia (Pocock, 1923; JohnsonMurray, 1977; Chakraborty, 1981; Roberts, 1997; Baillie and Groombridge, 1996). The main differences between Hylopetes and Eoglaucomys are related to the dental enamel, the cuspid of the third upper molar, and other dental structures of maxillary teeth. By carefully examining the baculum, footpads, musculature, crania including teeth, and postcranial structures (i.e., ankle and wrist joints) of the specimens from Afghanistan and Pakistan, Thorington et al. (1996) concluded that Eoglaucomys and Hylopetes are distinct clades. In the sixth edition of Walker’s mammals of the world, Nowak (1999) accepted Thorington’s recognition and treated Eoglaucomys as a valid genus. However, Eoglaucomys is still widely referenced as subgenus or synonym of Hylopetes because the morphological differences between them did not seem to be of more than subgeneric importance (Ellerman, 1947; Ellerman and Morrison-Scott, 1966; McLaughlin, 1967,

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115 1984; Honacki et al., 1982; Corbet and Hill, 1991, 1992; Nowak, 1991; Wilson and Reeder, 1992). Eoglaucomys is a monotypic genus with a single species E. fimbriatus the small Kashmir flying squirrel. According to the difference in size between the crown areas of the first molar and the fourth premolar, Chakraborty (1981) elevated the population distributed in northeastern Afghanistan and northern Pakistan as another possible valid species, E. baberi Corbet and Hill (1992) also accepted Hylopetes ( Eoglaucomys ) baberi as a full species. But Thorington et al. (1996) argued that E. fimbriatus and E. baberi are conspecific. By rechecking the type specimen and other specimens in the British Natural History Museum, Roberts (1997) found no evidence of consistent size differentiation between fimbriatus and baberi supports Chakraborty’s (1981) result, and he thought that there is no basis for retaining baberi as a distinct species, even as a recognizable subspecies. Hylopetes is a polymorphic genus and consists of 8 (Nowak, 1991) or 10 (Corbet and Hill, 1992) species (Table 5.1), occurring from the eastern Himalayas to Greater Sunda Island (SE Asia). Except for recognition of Eoglaucomys as a separate genus, the arrangements of Hylopetes proposed by Corbet and Hill (1992) were commonly accepted (Wilson and Reeder, 1992; Nowak, 1999). Three species, H. alboniger H. phayrei, and H. electilis are recognized in China, but the recognition of discrete species of H. electilis that is distributed in the island of Hainan, China, is not justified unanimously. H. electilis is treated as a subspecies of H. phayrei in recent references (Nowak,1999; Wang, 2002). Zhang et al. (1997) even combined H. electilis and the population of H. phayrei in China together as a valid species, Petinomys electilis under the genus Petinomys In Yunnan,

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116 H. alboniger is mainly distributed in western mountainous areas (Figure 5.1) and is sympatric with Petaurista Table 5.1 Species of Hylopetes and Eoglaucomys Nowak, 1991 Corbet and Hill, 1992 Locality H. alboniger H. alboniger Nepal to Indochina H. phayrei H. phayrei Burma, Thailand, Laos H. nigripes H. nigripes Palawan Island, Philippines H. lepidus H. lepidus Malay Peninsular, Sumatra, Borneo H. spadiceus H. spadiceus S Burma, Thailand, Indochina, Sumatra, Java, Borneo H. mindanensis Mindanao H. fimbriatus H. fimbriatus H. baberi Afghanistan, Pakistan, N India H. electilis Hainan, China H. sipora W Sumatra H. bartelsi W Java H. winstoni N Sumatra In the absence of experimental breeding data, the classification of geographically isolated populations as separate species or subspecies must rely on relative differences in morphology, behavior, and biochemical and genetic data (Garner and Ryder, 1996). The variability of mitochondrial DNA sequence is one of the many factors that should be considered in the classification of populations of flying squirrels. To date, most studies of Eoglaucomys and Hylopetes are mainly focused on the morphological, external, and ecological comparisons (McLaughlin, 1984; Honacki et al., 1982; Thorington et al., 1996; Roberts, 1997). The available data from either morphological or molecular study is fragmental and is not sufficient to construct the phylogenetic relationships. To test the

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117 different hypotheses or revisions about the taxonomy and phylogeny of Eoglaucomys and Hylopetes the morphological study with multivariate analysis and biochemical study with DNA sequencing analysis were conducted in this study. Finding the answers to the following questions would be important for reconstructing the phylogenetic relationships of the trans-Himalayan flying squirrels. 1. Is Eoglaucomys a valid genus or a subgenus of Hylopetes ? 2. What is the phylogenetic relationship between Eoglaucomys in the western transHimalayas and the population of Hylopetes in the eastern trans-Himalayas? 3. Is H. electilis a full species or a subspecies of H. phayrei ? 4. What’s the phylogenetic relationship between E. fimbriatus and the populations of Hylopetes ? 5.2 Materials and Methods 5.2.1 Materials In the morphometric study, a total of 117 specimens of Eoglaucomys and Hylopetes were examined (Table 5.2). All specimens were represented in museums and institutes, including American Museum of Natural History, New York (AMNH), Florida Museum of Natural History, University of Florida, Gainesville (FLMNH), National Museum of Natural History, Smithsonian Institution, Washington DC (USNM), Chinese Institute of Zoology, Beijing (China, BIZ), and Kunming Institute of Zoology, Kunming (China, KIZ). Fourteen measurements of skull were used in multivariate analyses, which were defined in Table 4.5 (see Chapter 4).

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118 Table 5.2 Species and localities of Hylopetes and Eoglaucomys examined in morphometric analysis Species Specimen Sex Locality Museum H. alboniger 8 4 F, 4 M Yunnan, ChinaKIZ H. electilis 24 12 F, 12 MHainan, China KIZ, AMNH H. phayrei 19 9 F, 10 M Burma, Java AMNH, USMNH H. lepidus 10 5 F, 5 M W Malaysia USMNH H. spadiceus 9 4 F, 5 M W Malaysia USMNH H. nigripes 13 7 F, 6 M Palawan, Philippines USMNH, AMNH Eoglaucomys fimbriatus 34 20 F, 14 M W Pakistan, Kashmir USMNH Total 107 F = Female M = Male In the molecular study, the partial mitochondrial cytochrome b sequences were used for reconstructing the phylogenetic trees. Except for the sequence data of Phayre’s flying squirrel ( H. phayrei ) and white-bellied flying squirrel ( Petinomys setosus ), which were retrieved from GenBank of NCBI, the sequences of Hylopetes and Eoglaucomys were determined from museum specimens, either hairs or skins. The detailed information of samples used was given in Table 5.3.

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119 Table 5.3 Samples of Eoglaucomys and Hylopetes examined in molecular study Species Code Museum ID Locality HAK1 KIZ: 5654 Menla, Yunnan, China HAK2 KIZ: 74545 Luchun, Yunnan, China HAK3 KIZ: 74546 Luchun, Yunnan, China H. alboniger HAK4 KIZ: 55660 Menhai, Yunnan, China H. electilis HEK KIZ: 29273 Hainan, China EFP1 USMNH: 353237 W Pakistan EFP2 USMNH: 353244 W Pakistan E. fimbriatus EFP3 FLMNH: 26513 Pakistan H. nigripes HNP BIZ: 47792 Philippines HLP1 USMNH: 488619 W Malaya H. lepidus HLP2 USMNH: 488632 Malaya HSP1 USMNH: 481112 W Malaya H. spadiceus HSP2 USMNH: 48495 W Malaya H. phayrei HPT AB030259 Thailand Petinomys setosus PSE AB030260 Indochina peninsula G. volans GV AF063066 Tennessee, US 5.2.2 Morphometric Analysis Morphometric study of Eoglaucomys and Hylopetes was based on 7 species and 14 skull measurements. According to the localities, all specimens of Hylopetes were divided into two groups: the Chinese Hylopetes containing the populations of Hylopetes in the eastern trans-Himalayas, and the southeastern Hylopetes consisting of the populations in SE Asia. I first compared the morphological structures between

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120 Eoglaucomys and the Chinese Hylopetes ; then pooled both Hylopetes groups and Eoglaucomys together to assess the overall morphological relationships. Multivariate analyses including principal components analysis (PCA) and discriminant function analysis were applied to make these comparisons. The technical information of morphometric methods was described in the section 4.2 of Chapter 4. 5.2.3 Biochemical Study The cytochrome b sequences of Eoglaucomys and Hylopetes in different localities determined from museum specimens were analyzed to rebuild the phylogenetic relationships. Two primer pairs, L14725 and H15149, and L14841 and H15149 (see Chapter 3 for detail), were synthesized at ICBR (Interdisciplinary center for biotechnology research, University of Florida, US) and KIZ (Kunming Institute of Zoology, China) for PCR amplification and PCR product sequencing. The techniques and protocols for DNA isolation, PCR amplification, PCR products purification, and DNA sequencing analysis were the same as those described in the section 3.2 of Chapter 3. The sequence data of some samples used for phylogenetic study were quoted from the GenBank of NCBI. The white-bellied flying squirrel ( Petinomys setosus ) from Indochina Peninsula was used as an outgroup for constructing the phylogenetic trees. Its sequence data was retrieved from GenBank of NCBI that is provided by Oshida et al. (2000a). To avoid any confusion, only the specimens or samples that were collected in Pakistan and Kashmir in where Eoglaucomys fimbriauts is distributed were used in both mophometric and molecular studies. G. volans is used as the outgroup to reconstruct the phylogenetic trees.

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121 5.3 Results 5.3.1 Comparison between Eoglaucomys and the Chinese Hylopetes 5.3.1.1 Morphological data The multivariate analyses for comparative study of the Chinese Hylopetes and Eoglaucomys were discriminant function analysis and principal components analysis (PCA). The taxa included H. alboniger H. electilis H. phayrei and Eoglaucomys fimbriatus The results of discriminant function analysis were showed in Table 5.4. Along the first discriminant function (CAN I, 78% of variance), four species were discriminated as three groups. Eoglaucomys fimbriatus and H. alboniger were separated as two distinct groups, and H. electilis was clustered with H. phayrei as one group (Figure 5.2). All variables had equal contributions to this axis. The CAN II was strongly associated with the morphological variable BCASEL. The same grouping pattern was plotted in discriminant function analysis onto function 1 (CAN I) and function 3 (CAN III) (Figure 5.3). The major contributions to CAN III were from TBL and PORCL with a negative score. Table 5.5 presented the results of principal components analysis of the Chinese Hylopetes and Eoglaucomys The first principal component factor (PRIN I), the size factor, that accounted for 98% of the total original variance defined all specimens as three groups: E. fimbriatus and H. alboniger formed two distinct groups; H. electilis and H. phayrei combined as the third group for their extensively overlapping (Figure 5.4). The major contributions were from the variable CRANL and BCASEL. BCASEL also had the highest score on the second principal component factor (PRIN II). On the plot of the principal component factor 1 and factor 3 (PRIN III), E. fimbriatus was isolated from

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122 Chinese Hylopetes and formed an independent group (Figure 5.5). There was a considerable overlap between H. electilis and H. phayrei Along the third principal component factor (PRIN III), the main morphological variable contributing to this association was PGA. Table 5.4 Discriminant function analysis between the Chinese Hylopetes and Eoglaucomys CAN Eigenvalue Proportion Cumulative I 46.93 0.78 0.78 II 12.53 0.21 0.99 III 0.87 1.00 Canonical score Variable CAN I CAN II CAN III CRANL 0.99 0.08 0.01 BCASEL 0.97 0.22 0.03 CRANW 0.99 0.01 0.00 BPORW 0.99 0.00 0.05 PORCL 0.76 -0.03 -0.35 PGA 0.96 0.08 0.05 NAL 0.98 0.12 0.00 TBL 0.92 0.33 0.14 DSL 0.97 0.14 0.03 MTRL 0.98 0.00 0.06 MTRW 0.97 -0.06 0.03 LMDL 0.98 0.05 0.09 LMDH 0.98 0.06 -0.01 LMTL 0.99 -0.08 0.11

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123 Table 5.5 Principal components analysis between Chinese Hylopetes and Eoglaucomys PRIN Eigenvalue Proportion Cumulative I 366.71 0.98 0.98 II 2.56 0.01 0.99 III 1.58 0.00 0.99 Eigenvector score Variables PRIN I PRIN II PRIN II CRANL 0.55 0.01 -0.11 BCASEL 0.44 -0.70 -0.11 CRANW 0.18 0.20 -0.16 BPORW 0.29 0.42 -0.02 PORCL 0.05 0.04 -0.14 PGA 0.23 0.00 0.94 NAL 0.23 -0.10 -0.07 TBL 0.09 -0.28 0.07 DSL 0.13 -0.10 -0.05 MTRL 0.10 0.09 0.06 MTRW 0.11 0.18 0.01 LMDL 0.42 0.33 -0.09 LMDH 0.22 0.08 -0.11 LMTL 0.10 0.16 0.02 5.3.1.2 Molecular data The molecular analyses were conducted on the basis of the partial sequences (400 bp) of cytochrome b gene. Nine samples from 4 species were used for studying the phylogenetic relationships of the Chinese Hylopetes and Eoglaucomys Table 5.6 showed

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124 the genetic differences and the substitutions of transversions and transitions based on the pairwise comparison between samples. The genetic differences between Eoglaucomys and H. alboniger and between Eoglaucomys and H. electilis and H. phayrei were significant, varying from 12.8% to 17.1%. H. electilis was genetically distinct from H. phayrei and H. alboniger with 11.7% and 12% variations in sequence, respectively. Table 5.6 Pairwise comparison of cytochrome b nucleotide sequences (400 bp) between Chinese Hylopetes and Eoglaucomys HAK1 HAK2HAK3 HAK4 EFP1EFP2 EFP3HPT HEK H AK1 1.0 0.8 1.2 13.5 14.6 14.7 10.1 13.1 HAK2 2/1 0.1 1.7 14.6 15.6 15.6 10.8 12.2 HAK3 3/0 2/1 0.5 13.3 14.3 14.5 10.6 12.8 HAK4 5/0 5/1 2/0 12.8 13.8 14 10.7 12.2 EFP1 33/21 32/2530/21 1.1 2.3 16.2 16.1 EFP2 35/23 33/2634/2332/233/1 1.5 17.1 16.8 EFP3 38/21 39/2637/2135/217/2 5/1 16.5 15.7 HPT 39/1 35/2 41/1 41/1 43/2144/23 44/21 11.8 HEK 43/8 32/5 43/8 40/8 40/8 42/21 43/1835/5 Note: Data below the diagonal are the numbers of substitutions of transitions vs. transversions. Data above the diagonal represent the genetic differences between samples The neighbor-joining analyses generated similar tree topology (Figure 5.6). Three distinct clades were formed in both trees: H. alboniger Eoglaucomys and the combination of H. electilis and H. phayrei 5.3.2 Phylogenetic Relationships of Hylopetes To assess the overall morphological relationships, E. fimbriatus and three other Hylopetes including H. nigripes H. lepidus and H. spadiceus from SE Asia, were

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125 included in discriminant function analysis and principal components analysis. Figure 5.7 and Figure 5.8 were the scatter-plots of discriminant function analysis on function 1 to function 2, and to function 3. In both plots, except for H. electilis and H. phayrei that were combined as one group, the rest were distinguished as distinct groups, showing different morphological structures of skulls. The principal components analysis onto the first factor and the second factor plotted similar morphological patterns as those of discriminant function analysis with an exception of H. spadiceus, which shared the similar skull characters with H. electilis and H. phayrei (Figure 5.9). The plot between factor 1 and factor 3 clustered the populations of Hylopetes in China as a complex group (Figure 5.10), and the rest were separated as different groups. The sequence data of H. nigripes H. lepidus and H. spadiceus from SE Asia were isolated from museum skins. Table 5.7 presented the genetic results of pairwise comparison on the partial sequences, 375 bp, of cytochrome b gene. E. fimbriatus apparently differed from all Hylopetes with a high sequence differentiation, 16.2% 17.8%. The genetic variation between H. phayrei and Petinomys setosus was about 3%. Considering Petinomys setosus as an outgroup, the NJ tree and MP tree generated similar branching patterns. All Hylopetes were separated as three clades: H. lepidus and H. spadiceus as one group with a high bootstrap value (89%), H. phayrei as the second group, the rest species, including Eoglaucomys fimbriatus H. nigripes H. alboniger and H. electilis, as the third group (Figure 11 and Figure 12).

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126 Table 5.7 Pairwise comparison of Hylopetes and Eoglaucomsy based on the partial cytochrome b sequences (375 bp) HAK EFP HPTHEK HLP1HLP2 HSP1HSP2 HNP PSE HAK 13.3 10.3 12.3 13.0 13.1 13.915.311.8 12.6 EFP 30/20 16.3 16.2 17.8 17.3 17.817.417.1 18.5 HPT 37/1 40/20 11.4 11.5 11.9 9.3 11.715.1 3.0 HEK 40/5 39/20 33/3 12.0 12.0 10.712.010.3 12.0 HLP1 41/7 44/21 36/6 39/5 0 7.5 8.4 12.6 13.6 HLP2 42/7 44/21 38/6 39/5 0 7.5 8.8 12.7 13.8 HSP1 45/6 43/22 28/4 32/5 20/6 21/6 1.1 9.8 11.4 HSP2 50/6 48/18 36/4 37/4 25/5 27/5 1/2 11.2 13.7 HNP 37/6 46/17 48/4 30/4 41/5 41/5 31/2 36/2 15.8 PSE 41/6 44/25 7/5 32/5 39/11 40/10 29/9 38/8 49/9 Note: Data below the diagonal are the numbers of substitutions of transitions vs. transversions. Data above the diagonal represent the genetic differences between samples The divergence time between Hylopetes and Eoglaucomys was calculated based on the transversional substitution rates at the third codon positions of cytochrome b sequences (375 bp) (Table 5.8). Table 5.9 gave the divergence time between species that were calculated from the percentage of the transversional substitutions at the third codon positions of the sequences. The divergence rate used here is about 0.5% per million years (Irwine et al., 1991). The results indicated that Eoglaucomys diverged from its ancestor stock about 24 to 33 million years ago. The divergence time between the Chinese Hylopetes the SE Asian populations was about 6.4 to 9 million years ago. H. electilis was genetically distinguishable from H. alboniger as early as 8 million years ago.

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127 Table 5.8 Transversional substitution rates at the third codon positions of the partial sequences (375 bp) of cytochrome b gene between species HAK EFP HEK HPT HLP HSP HNP PSE HAK 13.6 4 0.8 3.2 4 4.8 2.4 EFP 17 12 14.4 15.2 12 12 16.8 HEK 5 15 4.8 3.2 3.2 3.2 6.4 HPT 1 18 6 4.8 4 3.2 1.6 HLP 4 19 4 6 3.2 4 6.4 HSP 5 15 4 5 4 0.8 6.4 HNP 6 15 4 4 5 1 5.6 PSE 3 21 8 2 8 8 7 Note: Data below the diagonal are the numbers of transversions at the third codon positions. Data above the diagonal represent the transversional percentage difference between samples. Table 5.9 Estimated divergence time between species using the rate of the transversional substitutions at the third codon positions of mammalian cytochrome b gene of ca. 0.5% *106 years HAK EFP HEK HPT HLP HSP HNP EFP 27.2 HEK 8 24 HPT 1.6 28.8 9.6 HLP 6.4 30.4 6.4 9.6 HSP 8 24 6.4 8 6.4 HNP 9.6 24 6.4 6.4 8 1.6 PSE 4.8 33.6 12.8 3.2 12.8 12.8 11.2

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128 5.4 Discussion 5.4.1 Taxonomic Status of Eoglaucomys The small Kashmir flying squirrel Eoglaucomys is a monotypic genus and is found in northeastern Afghanistan, Kashmir, adjacent parts of northern Pakistan, and India (Niethammer, 1990; Roberts, 1997). It is designated as a near threatened species by IUCN (Baillie and Groombridge, 1996). The single species Eoglaucomys ( Hylopetes ) fimbriatus was originally named as Sciuropterus baberi by Blyth (1874). The type locality is in Northwest India, Punjab, Simla (Hassinger, 1973). Based on the drawing of the specimen from the mountain district of Nijrow, Afghanistan, by Burnes, Blyth (1847) described Sciuropterus baberis as a larger species than S. fimbriatus Ellerman (1940) described fimbriatus as a polytypic species under the genus Eoglaucomys Howell, and baberi was treated as a subspecies of it. Ellerman (1963) elevated baberi as a different race from fimbriatus according to the size of skull and the distribution, and considered baberi as a more western subspecies being found in Afghanistan, Pakistan (Northwest Frontier Province and Kagan Valley), Kashmir, and fimbriatus as an eastern subspecies found in Punjab, Kashmir (Islamabad district, Gilgit) and Himachal Prades By comparing the cranial and dental characters of skulls that were collected around the town of Islamabad (formerly Anatnag) in the main valley of Kashmir -33 o 42’ E, 75o 09’N (Shikargarh, Daksum Chaprot, Gilgit, Ladakh) and Sardalla and Chitral (Ellerman, 1963; Blanford, 1891), Chakraborty (1981) elevated baberi as a distinct species from fimbriatus. But Roberts (1997) argued that unless it can be proved that there is reproductive isolation between these two identical and certainly sympatric populations, there is no basis for retaining baberi even as a recognizable subspecies.

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129 Eoglaucomys fimbriatus is larger than Hylopetes (Figure 5.13). Both the molecular and morphological data of the present study yield the consistent results, supporting Eoglaucomys as a distinct genus. Although the phylogenetic differentiation between Eoglaucomys and H. alboniger is about 13%, the genetic differences between Eoglaucomys and other Hylopetes groups in Thailand ( H. phayrei ) and SE Asia ( H lepidus and H. spadiceus ) have reached as high as about 17% (Table 5.7). This reveals that Eoglaucomys is quite distinctive from all Hylopetes groups. In multivariate analyses, the cranial structures of Eoglaucomys are significantly distinguishable in both discriminant function analysis and principal components analysis (Figure 5.2 to Figure 5.5). The major differentiation is associated with the skull size. The different morphological characters could be viewed as reflections of various adaptations to various ecological niches, which typically occur as a result of competition between species. Competition usually varies among habitats, and habitat selection is a major contributor to coexistence. The coexistence of competing species by partitioning microhabitat is very common among rodent species (Price, 1978; Jorgensen and Demarais, 1999). This phenomenon exists among the trans-Himalayan flying squirrels as well (Roberts, 1997; Wang, 2002). In Pakistan, the small Kashmir flying squirrel ( E. fimbriatus ) is sympatric with the Pakistan giant flying squirrel ( P. petaurista ) and woolly flying squirrel ( Eupetaurus cinereus ) (Hassinger, 1973). The competitions for habitat and utilization of the available food resources are unavoidable. The habitat of Eupetaurus is characterized as high, cold desert dominated by Artemisia and Juniperus above 2,000 m, with many valleys having scattered forests of Pinus at high altitudes (Zahler and Woods, 1997). E fimbriatus is

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130 confined mainly to the Himalayan moist temperate forest that has a mixture of deciduous and coniferous tree species. Compared to P petaurista Eogalucomys is more adaptable to less mesic conditions and to living in drier regions where the forest is predominantly coniferous or pine forest, such as deodar ( Cedrus deodara ), Holly Oak ( Quercus ilex ) (Roberts, 1997), at elevation ranging from 2,000m to the limits of the tree-line. It is also found in spruce forest ( Picea smithiana ), such as in Indus Kohistan regions where Petaurista does not occur. E. fimbriatus feeds briefly upon the leaves and cones of pine, walnut, and barks of some trees (Chakraborty, 1981), which are partially similar to the food items of P. petaurista (Nowak, 1999). The particolored flying squirrel H. alboniger is a small size flying squirrel and distributed in Yunnan, Sichuan, and S China, at altitudes of 1,500 – 3,300m China. It is smaller than Eoglaucomys fimbriatus (Figure 5.13). According to local people, H. alboniger feeds on oak trees and pine trees to which they are probably attracted by the acorns, cones, young shoots, and buds of fir or pine. In this region, other flying squirrels with large size, such as Petaurista and Trogopterus were also observed in the same habitat, even in the same tree. I had heard them actively feeding on such trees at night during my field trips in Luchun of Yunnan, China, in 2000, implying their coexistence but distinctive in feeding preference. Because H alboniger is strictly nocturnal and spend the day curled up asleep in hollow trees or the hidden holes, practically nothing is known about its biological activities. 5.4.2 Phylogenetic Status of H. electilis Phayre’s flying squirrel ( H. phayrei ) (Wang, 2002) and Hainan flying squirrel ( H electilis ) (Corbet and Hill, 1992) are other valid species of Hylopetes in China, occurring in Fujian, and the island of Hainan. Hainan flying squirrel is a rather small species and is

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131 restricted to the island of Hainan, China (Figure 5.14). The type specimen used for species elevation is an adult female skin with the corresponding skull, which was collected by Clifford H Pope from Namfong, Hainan, China, during the central Asiatic expeditions in April 1923 (Ellerman, 1940). It is now represented in the collection of American Museum of Natural History with catalog number No. 58177. The skull shows short rostrum and low, uninflated bullae. The skin is with pale russet back, grading into fuscous on the upper part of the membrane, naked ears, and a distichous tail. The validity of the Hainan flying squirrel has been controversial for a long time; even to date it is still referenced as different taxonomic group (Corbet and Hill, 1992; Zhang et al., 1997; Nowak, 1999; Wang, 2002). H. electilis used to be maintained as a distinct species (Ellerman, 1940; Corbet and Hill, 1991; Nowak, 1991), but is demoted as a subspecies of H. phayrei in recent references (Corbet and Hill, 1992; Wilson and Reeder, 1992; Nowak, 1999; Wang, 2002), or a species of the genus Petinomys (Zhang et al., 1997). The data in this study show that H. electilis is morphologically similar to H. phayrei demonstrating their similar living environments. Both Hainan and Fujian belong to the same Chinese zoological region. A moist, monsoon climate during the summer gives way to cool, dry air in the winter. The habitat is a tropical, subtropical, or evergreen forest, and is within reach of the southwest and south monsoon. The simplest interpretation of the similar morphological structures between H. electilis and H. phayrei might be the adaptations to the similar living conditions. The molecular data presented in this study contrast with the morphological result. The genetic difference either between H. electilis and H. alboniger (12.3%) or between H. electilis and the population of H. phayrei (11.4%) in Thailand contradict the present view of the classification, which

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132 assumes that H. electilis is a subspecies of H. phayrei In fact, the most easily recognized symptom of disagreement between molecular and morphological character information manifests itself in different topologies resulting from separate analysis. The findings in this study are concordant with the recent claims that morphological data have less utility in systematic studies than do molecular data (Baker et al., 1998). The phylogenetic differentiation between H. electilis and both H. phayrei and H. alboniger revealed in this study confirms the species validity of H. electilis which might be related to genetic isolation. The island of Hainan is about one million years old and has two endemic mammal species, the Hainan moonrat and Hainan flying squirrel. The isolation and rejoining between the island and the mainland of China alternatively reduced and facilitated the gene flow of populations as fluctuations of sea level. This is an ideal situation for speciation and many endemic species have evolved. As the island populations regained genetic contact with mainland, some could still interbreed with the mainland forms and were thus still the same genetic species, such as the Hainan giant flying squirrel ( P. hainana ) (See Chapter 4 for detail), but some had diverged enough for reproductive barriers to be erected and were thus new species, like H. electili s here. One of the most serious gaps in the knowledge of Hainan flying squirrel is in the area of ecology, the study of other animals and plants in relation to each other and to its environment. For H. electilis and another flying squirrel Petaurista in the island of Hainan, virtually nothing is known, not even preferred habitat, so any field observation is likely to prove valuable. Much remains to be done in future. 5.4.3 Phylogenetics and Biogeography of Eoglaucomys and Hylopetes Molecular sequences in extant taxa can be used to infer speciation and biogeographic historical distributions produced by range shifts in shallow time (Riddle,

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133 1996), and thus provide a basis for constructing bridges between historical biogeographic, paleoecological, and ecological biogeographic perspectives. Genetic drift, adaptation to varying conditions, and genetic isolation can be expected to quickly evolve new species. H. nigripes, H. lepidus, and H. spadiceus are distributed in SE Asia, which are externally similar to H. electilis and H. phayrei (Figure 5.15 and Figure 5.16). The estimated divergence time inferred from the substitution rate at the third codon positions of cytochrome b gene suggests that Eoglaucomys had an early divergence from other groups, which is about 24 to 33 million years ago, the middle Oligocene. The split time between the populations of Hylopetes was in the late Miocene and the early Pliocene, about 4 to 6 million years ago (Table 5.8). This is consistent with the Oshida’s et al. (2000a) conclusion. The patterns of morphological and molecular characters and species associations in this study correspond with variations of local geological formations in Pakistan, China, and SE Asia (Figure 5.6 to Figure 5.11). The radical geological and climatological changes over relatively short periods of time have had remarkable effects on the evolution of mammals in southeastern Asia. South China is located at the crossroads of southeastern Asia and has been a bypass for animal dispersal from mainland Asia southward into the Indo-Malayan region. Of particular importance for the southeastern fauna were the changes in sea level which accompanied the isolation and rejoining of populations during the last few million years. In southeastern Asia, mammals are overwhelmingly affiliated with the Oriental fauna, with relatively few species of Palearctic affinities. Many species have migrated south from Burma and southern China along the forested mountains of the Thai-Burma border into Malaya, Sumatra, Java, and Borneo, such as gibbon (Lekagul and McNeely, 1988).

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134 Similarly, many species have expanded northward and higher in elevation with warmer conditions. The morphological similarities and the genetic differences within Hylopetes groups concluded from the present study also prove this hypothesis. The genetic similarity between H. alboniger and SE Asiatic groups reveals their close phylogenetic relationship. For example, the early divergence of Eoglaucomys is closely associated with its unique morphological structures and high genetic difference. The similar morphological and genetic characters in the populations of H. lepidus and H. spadiceus are associated with their similar living conditions and recent divergence. The divergence between H. phayrei and Petinomys had occurred in southeastern or south Asia approximately 2 to 3 million years ago (Oshida et al., 2000a), suggesting a late and rapid divergence. Based on the paleontological data, the Miocene fossil remains of Petinomys found in Europe imply a radiation of flying squirrels in Europe during the Oligocene-Miocene period. Accepting this hypothesis, there is a close biogeographical relationship between Eoglaucomys and Hylopetes During the Oligocene-Miocene radiation in Europe, Eoglaucomys and Hylopetes diverged from their common ancestor stock. With the tectonic movements in the Miocene, these two stocks, Eoglaucomys and the ancestor of recent Hylopetes migrated southward independently. Eoglaucomys migrated to the western extreme of the great Himalayan chain before the main uplift of the northwestern Tibetan Plateau that began ca. 4.5 million years ago (Zheng et al., 2000). About 6 million years ago, the late Miocene, the SE Asiatic and the Chinese Hylopetes diverged from their ancestor. During the Pliocene-Pleistocene period, the southeastern Asiatic

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135 branch invaded to Southeast or South Asia along the forested mountains of the ThaiBurma border. However, the ranges of these flying squirrels are still poorly known, with nearly every new collection revealing new distribution limits. The taxonomy and the phylogentic relationships among Hyloeptes are still unsettled, especially at the subspecies level. Further research will undoubtedly lead to changes in details. 5.5 Conclusion This chapter focuses mainly on the phylogenetic relationships between Eoglaucomys and Hylopetes based on the morphometric and molecular analyses. The following results are concluded. 1. Eoglaucomys differs morphologically and genetically from Hylopetes in China and SE Asia. It is reasonable to recognize Eoglaucomys as a valid genus. 2. H. electilis is a possible valid species of Hylopetes based on its genetic characters, although it shares similar morphological characters with H. phayrei in skull features. 3. Eoglaucomys diverged from other Hylopetes populations as early as 24 million years ago, the middle to late Oligocene. All Hylopetes groups including the SE Asiatic and Chinese populations diverged from the ancestor stock of Hylopetes in the early Pliocene, after the European radiation of flying squirrels in Oligocene-Miocene. The migration of Hylopetes to the present geographical distribution is due to the tectonic movements of the Himalayas during the Pliocene-Pleistocene period.

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136 Figure 5.1 Distribution of Chinese Hylopetes and Eoglaucomys

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137 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 -10-8-6-4-20246810CAN ICAN II H. alboniger H. electilis H. phayrei Eoglaucomys fimbriatus Figure 5.2 Discriminant function analysis between Chinese Hylopetes and Eoglaucomys onto function 1 and function 2

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138 -4 -2 0 2 4 -10-50510 CAN ICAN II I H. alboniger H. electilis H. phayrei Eoglaucomys fimbriatus Figure 5.3 Discriminant function analysis between Chinese Hylopetes and Eoglaucomys onto function 1 and function 3

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139 -2 0 2 4 6 8 -30-20-100102030 PRIN IPRIN II H. alboniger H. electilis H. phayrei Eoglaucomys fimbriatus Figure 5.4 Principal components analysis of Chinese Hylopetes and Eoglaucomys onto factor 1 and factor 2

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140 -1.5 -1 -0.5 0 0.5 1 -25-20-15-10-5051015202530 PRIN IPRIN III H. alboniger H. electilis H. phayrei Eoglaucomys fimbriatus Figure 5.5 Principal components analysis of Chinese Hylopetes and Eoglaucomys onto factor 1 and factor 3

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141 Figure 5.6 Phylogenetic topology of Eoglaucomys and Chinese Hylopetes constructed using the neighbor-joining method based on the partial sequences (400 bp) of cytochrome b gene. Scales in the tree represent branch length in terms of nucleotide substitutions per site. Sample abbreviations are coded in Table 5.3.

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142 -15 -12 -9 -6 -3 0 3 6 -15-10-5051015CAN ICAN II H. alboniger H. electilis H. phayrei Eoglaucomys fimbriatus H. nigripes H. lepidus H. spadiceus Figure 5.7 Scatter-plot of Hylopetes and Eoglaucomys along the first two discriminant functions

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143 -6 -4 -2 0 2 4 6 8 -15-10-5051015 CAN ICAN III H. alboniger H. electilis H. phayrei Eoglaucomys fimbriatus H. nigripes H. lepidus H. spadiceus Figure 5.8 Scatter-plot of Hylopetes and Eoglaucomys onto the first and the third discriminant function

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144 -3 -2 -1 0 1 2 3 4 5 6 -30-25-20-15-10-505101520253035PRIN IPRIN II H. alboniger H. electilis H. phayrei Eoglaucomys fimbriatus H. nigripes H. lepidus H. spadiceus Figure 5.9 Plot of principal components analysis of Hylopetes and Eoglaucomys onto factor 1 and factor 2

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145 -3 -2 -1 0 1 2 3 4 -30-20-10010203040PRIN I H. alboniger H. electilis H. phayrei Eoglaucomys fimbriatus H. nigripes H. lepidus H. spadiceus Figure 5.10 Plot of principal components analysis of Hylopetes and Eoglaucomys onto factor 1 and factor 3

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146 Figure 5.11 Phylogenetic tree of Hylopetes and Eoglaucomys generated by MP method on partial sequences (375 bp) of cytochrome b gene. Numbers above branches are the bootstrap values (%). Sample abbreviations are coded in Table 5.3.

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147 Figure 5.12 Phylogenetic tree of Hylopetes and Eoglaucomys generated by NJ method on partial sequences (375 bp) of cytochrome b gene. Scales in the tree represent branch length in terms of nucleotide substitutions per site. See sample abbreviations in Table 5.3.

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148 Figure 5.13 Eoglaucomys fimbriatus and Hylopetes alboniger

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149 Figure 5.14 Hylopetes electilis and Hylopetes phayrei

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150 Figure 5.15 Hylopetes electilis and Hylopetes nigripes

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151 Figure 5.16 Hylopetes lepidus and Hylopetes spadiceus

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152 CHAPTER 6 PYLOGENY OF FLYING SQUIRRELS IN THE TRANS-HIMALAYAS AND OTHER PARTS OF CHINA 6.1 Introduction Flying squirrels are better known by sight to people than most non-volant arboreal and terrestrial squirrels. It has a furred gliding membrane along the sides of body from the arms to the legs, even to the neck and tail in some genera. The membrane consists of sheets of muscles that can be tensed or relaxed. Varying the tension of membranes and the slant of the tail controls the direction of glide (Gupta, 1966). All flying squirrels are similar in postcranial anatomy and have evolved adaptations to the same locomotor problems (Thorington et al., 1997). Gliding has evolved among recent mammals at least six different times (Walker, 1975), but the complex wrist anatomy of flying squirrels provides evidence that gliding evolved only once among sciurids and that flying squirrels are a monophyletic group (Thorington, 1984; Thorington et al., 1998). This is contrast to Black’s (1963) diphyletic and Mein’s (1970) polyphyletic hypotheses. Flying squirrels are nocturnal and are found in both the Old and New World. Fourteen or fifteen forms containing 38 52 species have been given generic rank in recent years. Based on the dental characteristics of the extant and extinct forms, Mein (1970) assembled all flying squirrels into three distinct groups (Table 1.2). However, the phylogeny does not support Mein’s three-group hypothesis; instead, the extant flying squirrels are divided into four groups (Table 6.1). Figure 6.1 is the most recent

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153 phylogenetic reconstruction of flying squirrels, tree squirrels, and fossil squirrels based on 44 dental characteristics (Pappas et al., 2002). Table 6.1 Classification of the extant flying squirrels determined from 44 dental characters Group Genus I Glaucomys, Eoglaucomys, Petaurillus, Iomys, Pteromys Aeretes Petaurista Eupetaurus II Trogopterus Pteromyscus Belomys III Petinomys, Hylopetes IV Aeromys Table 6.2 Chinese flying squirrels other than Petaurista Genera Species Distribution Habitat Trogopterus T. xanthipes NW and SW China, Hubei, Guangxi, Henan Subtropical and warmtemperate forest Belomys B. pearsonii Yunnan, Guizhou, Guangxi, Hainan, Guangdong, Taiwan Tropical and subtropical forest Aeretes A. melanopterus Heiberi, Sichuan, Gansu Mountain forest Pteromys P. volans NE and NW China, Hebei, Henan, Sichuan Forest Eupetaurus E. cinereus Yunnan Subtropical evergreen broadleaf forest Hylopetes H. alboniger Sichuan, Yunnan, Guizhou, Zhejiang, Xizang, Hainan Mountain forest Petinomys P. electilis Hainan, Fujian, Guangxi Tropical and subtropical forest There are seven or eight genera consisting of 14 or 15 recognized species of flying squirrels in China (Corbet and Hill, 1991, 1992; Wilson and Reeder, 1992; Nowak,

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154 1999; Wang, 2002) (Figure 6.2). Table 6.2 shows the distributions and habitats of all Chinese flying squirrels except for Petaurista which was discussed in Chapter 4. Flying squirrels in mainland of China are mainly distributed in four geographical regions: southwestern China (Tibet, Yunnan, and Sichuan); southern China including Hainan island; northern China; and central China (see Table 1.6 for details). According to Pappas’ et al. (2002) classification, the Chinese flying squirrels are categorized into three groups: Pteromys Aeretes Petaurista Eupetaurus as one group, Trogopterus and Belomys as another group, and Petinomys, Hylopetes as the third group. The present taxonomic controversies at the specific level are mainly within the genera Hylopetes and Petaurista The major information about Chinese flying squirrels is from the morphological comparisons and external descriptions, such as Allen’s (1940) The Mammals of China and Mongolia and Corbet and Hill’s (1992) The Mammals of the Indomalayan Region. However, the specimens of flying squirrels, especially the skins and fluid, are not as common in museums or research institutes as the non-flying squirrels, and many of the forms are very little known. The latest taxonomical revisions are either a summary of the previous studies (Nowak, 1999; Zhang et al., 1997), or a pure morphological taxonomy that is based on the previous data and the observations of external structures (hairs, skins, pelage colors) (Wang, 2002). Many species remain nothing known and taxonomically controversial. Thus far, none of revisions in the literature is based on the morphologically quantitative comparisons or molecular analyses. In this chapter, I focus on the comparative studies of the trans-Himalayan flying squirrels using both morphological and molecular techniques. The flying squirrels of the

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155 eastern and the western trans-Himalayas, including Eupetaurus Petaurista Eoglaucomys and Hylopetes are compared (Figure 6.3), and all flying squirrels distributed in China are discussed. The purposes are to seek the answers for the following questions: 1. What are the phylogenetic relationships between the eastern and the western trans-Himalayan flying squirrels? 2. What are the phylogenetic relationships among Chinese flying squirrels? 3. Are the phlyogenetic relationships among Chinese flying squirrels obtained from morphological and molecular studies consistent with their geographical variations? 6.2 Materials and Methods 6.2.1 Specimens In the morphometric study, 188 specimens representing 14 species were examined and 14 variables were measured (Table 6.3). A total of 25 samples including 18 species were used in the molecular study for reconstructing the phylogenetic relationships among the eastern and the western trans-Himalayan flying squirrels (Table 6.4). The tissues used in this study were collected from museums and academic institutes in China and US (See Chapter 3, 4, and 5 for detail). The sequence data of some species were retrieved from GenBank of NCBI that were provided by Oshida et al. (2000a, b, and c) and Arbogast (1999). The fresh tissues of flying squirrels and tree squirrels were either collected from the localities in where the species are distributed, or obtained from the mammal department of Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, China. Two tree squirrels ( Callosciurus erythraeus ) in Kunming, China, were used as the outgroup taxon.

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156 Table 6.3 Species and localities of flying squirrels used in morphometric analysis Species Specimen Sex Locality Museum Aertes melanopterus 3 2 F, 1 M Hebei, China AMNH Belomys pearsoni 4 2 F, 2 M Indochina AMNH, USMNH Trogopterus xanthipes 3 2 F, 1 M Beijing, China BIZ Pteromys volans 11 7 F, 4 M Jinin, China BIZ Petinomys setosus 5 3 F, 2 M Mentawai Island AMNH Petaurista albiventer 9 5 F, 4M Yunnan, China KIZ P. yunanensis 15 7 F, 6 M, 2 U Yunnan, China BIZ, KIZ P. philippensis 9 3 F, 5 M, 1 U Yunnan, China KIZ P. elegans 12 5 F, 5 M, 2 U Yunnan, China KIZ P. alborufus 15 6 F, 8 M, 1 U Hupei, China, Burma AMNH P. albiventer 32 13 M, 19 M N Pakistan USMNH Eoglaucomys fimbriatus 34 20F, 14 M W Pakistan USMNH Hylopetes alboniger 7 4 F, 3 M Yunnan, China KIZ, BIZ H. phayrei 19 9 F, 10 M Mandalaypopa, Burma AMNH Glaucomys volans 10 4 F, 6 M Florida, US UF Total 188 Note: F = Female, M = Male, U = Unknown sex

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157 Table 6.4 Samples of flying squirrels used in molecular study Species Code Museum ID Locality E. cinereus ECK KIZ: 73372 Yunnan, China E. cinereus ECF UF: 26583 Gilgit, Pakistan P. albiventer PPF USMNH: 353209 N Pakistan P. philippensis PPH KIZ: Fresh tissue Pianma,Yunnan, China P. yunanensis PYK KIZ: Fresh tissue Gongshan, Yunnan, China P. hainana PHK KIZ: 22686 Hainan, China P. xanthotis PTK QIZ: 85063 Gansu, China P. alborufus PAK KIZ: 006679 Sichuan, China P. elegans PEK KIZ: 84354 Mile, Yunnan, China H. alboniger HAK KIZ: 74546 Luchun, Yunnan Eo. fimbriatus EFP USMNH: 353237 W Pakistan H. lepidus HLP USMNH: 488619 W Malaya H. spadiceus HSP2 USMNH: 48495 W Malaya Tr. xanthepes TRX1KIZ: 73378 Deqing, Yunnan Tr. xanthepes TRX2KIZ: Cell Bank Yunnan Pteromys. volans PVO KIZ: None NorthEast, China Belomys pearsonii BPE KIZ: 2970 Luchong, Yunnan Callosciurus erythraeus TSK1 KIZ: Fresh tissue Kunniming Callosciurus erythraeus TSK2 KIZ: Fresh tissue Kunniming P. p. petaurista PPB AF063067 Borneo, E Malaysia H. phyrei HPT AB030259 Thailand Petinomys setosus PSE AB030260 Indochina peninsula Pteromys volans PVO AB023910 Japan Glaucomys volans GV AF063066 Tennessee, US

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158 6.2.2 Methods of Phylogenetic Analyses Most specimens used here were selected from the previous chapters. Multivariate analyses, discriminant function analysis and principal components analysis, were applied for the comparative studies between the eastern and the western trans-Himalayan flying squirrels, and among all Chinese flying squirrels. The detailed information of the morphometric techniques was described in Chapter 4. To reduce the sexual dimorphism, the equal male and female specimens of each species were selected in morphometrics. Most samples in molecular study were the same as those used in Chapter 3, Chapter 4, and Chapter 5. In addition, the samples of Trogopterus xanthepes in Yunnan, Pteromy volans in NE China, Belomys pearsoni in Yunnan, and Callosciurus erythraeus in Kunming, Yunnan, were included. The techniques and protocols of DNA isolation, PCR amplification and purification, and DNA sequencing analysis were the same as those described in Section 3.2 of Chapter 3. The neighbor-joining method and maximum parsimony using a heuristic search algorithm with the 50% majority-rule consensus were used to construct the phylogenetic topology of flying squirrels. Two individuals of Callosciurus erythraeus were used as the out-group. 6.3 Results 6.3.1 Comparative Study of the Eastern and the Western Trans-Himalayan Flying Squirrels To investigate the phylogenetic relationships between the flying squirrels in the eastern and the western trans-Himalayas, three genera consisting of 9 species were examined in multivariate analyses.

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159 Table 6.5 Discriminant function analysis on the eastern and the western trans-Himalayan flying squirrels Axis Eigenvalue Proportion Cumulative CAN I 275.49 0.72 0.72 CAN II 73.85 0.19 0.91 CAN III 22.62 0.06 0.97 Canonical score Variable CAN I CAN II CAN III CRANL 0.33 0.83 0.41 BCASEL 0.44 0.75 0.30 CRANW 0.40 0.85 0.32 BPORW 0.39 0.84 0.36 PORCL 0.47 0.79 0.28 PGA 0.36 0.80 0.43 NAL 0.26 0.78 0.42 TBL 0.42 0.70 0.23 DSL 0.14 0.74 0.60 MTRL -0.48 0.82 -0.28 MTRW 0.09 0.90 0.39 LMDL -0.77 0.55 0.30 LMDH 0.86 0.49 0.09 LMTL 0.92 0.37 0.07 The results of discriminant function analysis were presented in Table 6.5. The first two axes accounted almost for 91% of the original specimen variance. When all specimens were plotted onto the first two discriminant functions (CAN I and CAN II), five groups were identified (Figure 6.4). Eoglaucomys Hylopetes, and P. albiventer in

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160 Pakistan were distinguished as distinct groups; P alborufus and P. elegans were combined as one group; and P. albiventer in Yunnan P. yunanensis, P. philippensis were clustered as one group. The measurements of lower jaw were the key factors responsible for these separations along CAN I. The remaining variables contributing to the observed associations were strongly correlated with CAN II. Function 3 (CAN III), accounting for 6% of the total variance, described all trans-Himalayan flying squirrels as four groups (Figure 6.5). The clusters were similar to those in Figure 6.4, except for Eoglaucomys which was merged into the group of P. albiventer in Yunnan P. yunanensis, and P. philippensis DSL was the major variable contributing to CAN III. When all specimens used in discriminant function analysis were included in principal components analysis, a similar clustering pattern was generated. Table 6.6 presented the results of principal components analysis of trans-Himalayan flying squirrels. The first two principal component factors that accounted for 67% and 29% of the total variance, respectively, partitioned all species into four groups. These groups are concordant with the results of discriminant function analysis. Three genera, Petaurista Eoglaucomys and Hylopetes are separated as distinct taxa (Figure 6.6). The populations in Petaurista were divided into two groups: one group containing P. albiventer in Pakistan, P alborufus and P. elegans and another group comprising P. albiventer, P. yunanensis, and P. philippensis CRANL and LMDL were the dominant variables on PRIN I and PRIN II, respectively. The clusters onto PRIN I and PRIN III showed that P. albiventer in Pakistan was morphologically similar to P. albiventer in Yunnan P. yunanensis, and P. philippensis The remaining species represented distinct taxa along PRIN III (Figure 6.7). MTRL was the morphological variable achieving this discrimination.

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161 Table 6.6 Principal components analysis of trans-Himalayan flying squirrels on the first three factors Axis Eigenvalue Proportion Cumulative PRIN I 559.30 0.67 0.67 PRIN II 241.53 0.29 0.97 PRIN III 10.04 0.01 0.98 Eigenvector score Variable PCA I PCA II PCA III CRANL 0.51 0.15 -0.25 BCASEL 0.38 0.02 -0.39 CRANW 0.27 0.05 0.25 BPORW 0.38 0.08 0.23 PORCL 0.13 0.00 0.20 PGA 0.21 0.05 -0.13 NAL 0.17 0.07 -0.21 TBL 0.07 0.00 -0.04 DSL 0.10 0.06 -0.19 MTRL 0.06 0.21 0.68 MTRW 0.12 0.09 0.08 LMDL 0.08 0.82 0.03 LMDH 0.41 -0.35 0.20 LMTL 0.29 -0.34 0.14 6.3.2 Phylogenetic Relationships among Flying Squirrels in China Table 6.7 showed the percentage of genetic differences and the numbers of transversional and transitional substitutions based on pairwise comparison of the cytochrome b sequences (375 bp). When tree squirrel ( Callosciurus erythraeus ) was

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162 used as the outgroup, except for Eupetaurus all trans-Himalayan flying squirrels were branched into five similar groups in both MP tree and NJ tree. All populations of Petaurista Hylopetes and Pteromys were isolated as distinct clades (Figure 6.8 and Figure 6.9). Glaucomys and Eoglaucomys represented a clade. Eupetaurus was either separated as a distinct group in MP tree or combined with Trogopterus and Belomys as a clade in NJ tree. The pairwise comparison of the partial cytochrome b sequences at the third codon positions between samples was given in Table 6.8. Table 6.7 Pairwise comparison based on the partial cytochrome b sequences (375 bp) between species. See Table 6.4 for the sample abbreviations. ECF ECK PPF PPH PYK HAK HPT EFP TRX PVO BLP PSE GV TSK ECF 13.1 16.4 16.1 16.4 15.3 16.4 18 17.2 14 16.4 17.9 18.5 17.2 ECK 37/11 17.7 15.8 18.3 14.4 15.5 17.7 17.7 16.1 16.1 16.7 15.3 19 PPF 38/22 42/23 8.7 5.7 16.9 17.9 18.3 18.0 17.1 18.9 19.5 16.4 20.4 PPH 37/22 35/23 30/2 8.2 13.6 16.5 14.4 17.4 15.9 16.8 18.3 15.5 18.8 PYK 39/21 43/24 20/1 27/3 14.4 18.3 18.9 18.5 16.7 19.1 20.4 17.2 20.4 HAK 40/16 40/13 40/22 30/20 30/13 10.6 14 16.1 13.3 14.7 12.7 13.9 17.2 HPT 43/16 44/11 42/23 39/20 42/24 36/2 16.4 17.8 16.2 16.1 3 13.2 20 EFP 41/25 41/24 48/19 34/19 40/20 32/19 40/19 19.1 17.3 18.8 18.5 14.4 17 TRX 32/31 37/28 41/25 39/25 44/24 40/19 43/21 42/28 17 12.8 19.4 18.3 19.1 PVO 32/19 39/19 42/19 36/21 42/18 30/18 38/17 40/22 39/23 16.7 17.1 14.7 16.4 BLP 32/27 35/23 41/27 33/27 41/28 33/19 34/20 38/30 31/15 35/21 17.3 15.8 19.6 PSE 45/20 44/16 44/27 41/25 46/28 39/6 6/4 43/23 44/26 37/21 36/22 14.4 20.9 GV 49/19 42/14 37/23 36/21 39/24 40/11 37/11 31/22 47/20 32/20 37/20 37/15 17.2 TSK 40/23 42/28 48/27 42/27 49/26 38/25 64/24 39/22 46/24 37/22 47/24 40/23 39/24 Note: Data below the diagonal are the numbers of transitions vs. transversions. Data above the diagonal represent the genetic differences between samples

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163 Table 6.8 Pairwise comparison of the transversional substitutions at the third codon positions of the partial cytochrome b sequences (366 bp) between samples. Data below the diagonal are the numbers of transversions at the third codon positions. Data above the diagonal represent the transversional percentage difference between samples. Table 6.4 shows the information of samples. ECF ECK PPF PPH PYKHAK HPT EFP TRXPVOBLP PSE GV ECF 4.9 13.1 14.7 13.1 10 10 16.4 17.210.613.9 11.4 11.4 ECK 6 13.9 14.7 13.9 8.2 8.2 16.4 18 11.413.1 10 10 PPF 16 17 1.6 0.8 15.5 14.713.9 18 12.317.2 16.4 16.4 PPH 18 18 2 2.4 13.9 13.913.1 18.814.716.4 16.4 15.5 PYK 16 17 1 3 17.2 16.413.9 18 12.318 18 18 HAK 12 10 19 17 21 0.8 13.9 13.912.313.1 2.4 10 HPT 12 10 18 17 20 1 14.7 15.512.313.9 1.6 9 EFP 20 20 17 16 17 17 18 20.513.919.6 15.5 17.2 TRX 21 22 22 23 22 17 19 25 15.510 17.2 15.5 PVO 13 14 15 18 15 15 15 17 19 15.5 14.7 13.9 BLP 17 16 21 20 22 16 17 24 12 19 14.7 12.3 PSE 14 12 20 20 22 3 2 19 21 18 18 10 GV 14 12 20 19 22 12 11 21 19 17 15 12 The estimated divergence time between samples was calculated based on the percentage of the transversional substitutions at the third codon positions using the rate of divergence for the third codon positions of mammalian cytochrome b gene of ca. 0.5% *106 years and was presented in Table 6.9. With the inclusions of Glaucomys and Eoglaucomys all Chinese flying squirrels, including 7 genera and 10 species, were analyzed using morphometric methods as well. The results were showed in Table 6.10. In discriminant function analysis, function 1 that accounted for 89% of total variance separated most populations into genus-based groups. The mixed group included H. phayrei Pteromys volans, G. volans and B. pearsonii (Figure 6.10). Principal

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164 components analysis yielded the same results. The first two factors, accounting for 99% of the total variance, distinguished 12 populations as four major groups. Again, Hylopetes Pteromys volans, G. volans and B. pearsonii greatly overlapped with each other (Figure 6.11). Table 6.9 Estimated divergence time between samples based on the rate of the transversional substitutions at the third codon of cytochrome b sequences proposed by Irwine et al. (1991). See Table 6.4 for sample abbreviations. ECF ECK PPF PPH PYK HAK HPTEFPTRX PVO BLP PSE ECK 9.8 PPF 26.2 27.8 PPH29.4 29.4 3.2 PYK 26.2 27.8 1.6 4.8 HAK 20.0 16.4 31.0 27.8 34.4 HPT20.0 16.4 29.4 27.8 32.81.6 EFP 32.8 32.8 27.8 26.2 27.827.8 29.4 TRX 34.4 36.0 36.0 37.6 36.027.8 31.0 41.0 PVO 21.2 22.8 24.6 29.4 24.624.6 24.6 27.8 31.0 BLP27.8 26.2 34.4 32.8 36.026.2 27.8 39.2 20.0 31.0 PSE 22.8 20.0 32.8 32.8 36.04.8 3.2 31.0 34.4 29.4 29.4 GV 22.8 20.0 32.8 31.0 36.020.0 18.0 34.4 31.0 27.8 24.6 20.0

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165 Table 6.10 Multivariate analyses of Chinese flying squirrels Axis Eigenvalue Proportion Cumulative Discriminant function analysis CAN I 173.38 0.89 0.89 CAN II 14.33 0.07 0.96 CAN III 2.46 0.01 0.97 Principal components analysis PRIN I 873.17 0.97 0.97 PRIN II 11.81 0.01 0.98 PRIN III 3.75 0.01 0.99 6.4 Discussion 6.4.1 Phylogeny of the Trans-Himalayan Flying Squirrels The eastern trans-Himalayan flying squirrels include three genera, Eupetaurus, Petaurista and Hylopetes, which are distributed mainly in western Yunnan, China. Three genera, each containing one species, E. cinerus, P. albiventer and Eoglaucomys fimbriatus, occur in the western trans-Himalayas, mainly in N Pakistan. Usually, the populations of Eupetaurus and Petaurista in both regions are considered as the same species based on their morphological structures and external characters (Corbet and Hill, 1992; Nowak, 1999). Eogalucomys the small Kashmir flying squirrel, which used to be considered as a species of arrow-tailed flying squirrels ( Hylopetes ), is merited as an independent genus (Thorington et al., 1996; Nowak, 1999). My results from both the morphometric and molecular analyses strongly support the latest classifications.

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166 As discussed in Chapter 3, the populations of Eupetaurus in the eastern and the western trans-Himalayas are significantly different genetically. The genetic distance between these two clades is 11.0 – 13.3% (Table 3.4 and Table 6.7), implying two probable species. Unfortunately, since no skull was associated with the skins found in SW Yunnan of China, it is unreasonable to directly elevate these two populations as distinct species based only on the genetic information without evidence from morphological structures, especially from skull and teeth. The same situation exists between the populations of P. albiventer in Pakistan and W Yunnan, China, which are named as the same species or subspecies, P. albiventer or P. p. albiventer in recent references on the basis of their external and dental structures (Corbet and Hill, 1992; Wang, 2002). But the findings in both morphometric and molecular analyses in this study reveal that they are significantly different, at least at subspecies-level (See Chapter 4 for detail). When these two populations are compared with other trans-Himalayan flying squirrels, the morphometric results show that these two Petaurista populations are significantly different in cranial structures (Figure 6.4 and Figure 6.5). The principal components analysis indicates that their main differences are in skull size and the morphological structures of lower jaw. The population of P. albiventer in Pakistan shares more cranial characteristics with P. elegans and P. alborufus than with the population of P. albiventer in Yunnan, China. These two populations may occupy different habitats that result in different adaptations at different geographic locations during a long and complex geological, climatic, floristic and faunistic history. The arrow-tailed flying squirrel ( Hylopetes ) in China and SE Asia used to be considered as the same genus with the small Kashmir flying squirrel ( Eoglaucomys ) in

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167 the western trans-Himalayas (Corbet and Hill, 1992; Nowak, 1991; Roberts, 1997). They are elevated as two valid genera based on their different dental structures in recent references (Thorington et al., 1996; Nowak, 1999). The results in present study support their separation. The differences between these two groups are significant in both morphology (Figure 6.4 and Figure 6.5) and genetics (Table 6.7 and Table 6.9). They can be distinguished as two valid genera, namely Hylopetes in China and SE Asia, and Eoglaucomys in Pakistan and Kashmir. With inclusion of the populations of Eoglaucomys Hylopetes and Petaurista the differences among them are concordant with their geographical distributions. The present distributions of Eupetaurus, Petaurista, Hylopetes, and Eoglaucomys in these regions owe much to both major climatic changes in the late Pleistocene and the physical barriers to migration. The three flying squirrels in Pakistan occur sympatrically in some regions with the same living conditions, such as the Himalayan temperate forest with a mixture of deciduous and coniferous tree species, each occupying different microhabitat and having different food habits. Woolly flying squirrel ( Eupetaurus ) in Pakistan is confined to remote valleys in the extreme northern Himalayas. It lives in steppic mountainous conditions with isolated forest that consists of blue pine ( P. wallichiana ), edible seed or chilgoza pine ( P. gerardiana ), spruce ( Picea smithiana ), and Juniperus macropoda (Roberts, 1997) The unique tooth structures of Eupetaurus are closely associated with its special feeding habit. The developed molars suggest that Eupetaurus lives on a highly fibrous vegetable diet as its teeth are adapted to a high rate of abrasion and wear on their grinding surface (Woods and Howland, 1979). Giant flying squirrel in the western trans-Himalayas is distributed mainly in N Pakistan where the major habitat

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168 is the Himalayan moist temperate forest. This flying squirrel feeds on the fir and pine cones, the nuts, even the young twigs and tree buds, or the acorns of the hill oak ( Quercus dilatata ). The food compositions are partially different in Eupetaurus and Eoglaucomys (Roberts, 1997). Eoglaucomys lives in dry temperate coniferous forests and is confined to the more sheltered lower slopes, or the Himalayan moist temperate forest. Compared to Petaurista Eoglaucomys is apparently more adaptable to harsh conditions where the forest is predominantly coniferous, which has developed cell tissue with a high proportion of tough silicates. The morphological differences in skull size and the structures of lower jaw (Table 6.6) demonstrate that each of them has adapted to different living conditions. The similar sympatric distribution of E. cinereus, P. albiventer, and H. alboniger also exists in SW China, where the two Eupetaurus skins were collected. Unfortunately almost nothing is known about the habitat, feeding habits, and geographical distribution of Eupetaurus in China except for two skins. Hylopetes is distinguishable from Petaurista by smaller body size and relatively shorter but broader tail, which has the hairs spreading laterally in a feather shape. The geographical distribution of H. alboniger and P. petaurista is overlapped extensively at different elevations (500m to 3,500m) from nouthwestern Yunnan to southern Yunnan, China. The habitat is mountainous conditions with coniferous forest. According to local people, P. albiventer frequently feeds on walnuts, acorns, and even corns in farm; whereas H. alboniger is always staying on the top of trees, such as Yunnan pine and spruce, and feeds on pine seeds, and young twigs, which I have watched in the field in Gongshan of Yunnan, China. But nothing else is

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169 available about its feeding habits, geographic distribution, and food selection during different seasons. Geological changes have strongly affected the evolution and distribution of the mammals by creating dispersal barriers and corridors, as well as by forming optimal habitats in both extremes of the trans-Himalayas. In Pakistan, there is a rich and varied mammalian fauna, affinitive to two of the major faunal regions, the Palaearctic region west of the Indus and the Oriental region east of the Indus (Roberts, 1997). The great Indus River and its drainage basin form a dominant physiographic feature over a large part of the country. Southwest China is comprised the hills of the eastern Himalayas and the Tibet plateau, particularly the Hengduan and Min mountain systems. Elevation varies from below 1,000m in the valley floors to glacial peaks of over 6,000m. The subtropical to sub-alpine mountains are cut into a number of subunits by major river gorges. The vegetation on mountain slopes has a relatively narrow vertical zonation and the vegetation on the plains has a broad horizontal zonation. In eastern Tibet and western Sichuan, among mountains within reach of the southwest monsoon, forests grow up to an elevation of 4,100-4,500m. A mixed coniferous and broad-leaved forest composed predominantly of spruce, fir, and oak with an understory of Acer Lindera Litsea Rhododendron and other trees predominate. Hoffmann (2001) thought that the mammalian fauna in this region belongs to either the Palaearctic region or the Oriental region. Below about 1,500m, tropical and subtropical communities typical of the Indomalayan region predominate, and above 2,500m, cool temperate to boreal communities typical of the Palaearctic region dominate the landscape. The region around 2,000m is a transition zone in which animals from both regions occur in varying

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170 proportions depending on local environmental conditions. Accepting Hoffmann’s hypothesis, flying squirrels in both regions are the elements of the Oriental region. The genetic differences among these four genera are significant, too (Table 6.7). They diverged from their ancestor stock(s) about 20 to 32 million years ago (Table 6.9), the early Miocene. Migrations between the eastern and the western trans-Himalayan flying squirrels were after the secondary upheaval the great Himalayan mountain chain, which was about 13 million years ago. This dramatic tectonic movement led to an even more violent nature in Asia and Europe. Because of the changes of climate and environments that were influenced by geographical events, the populations in each genus were split into two branches. One branch migrated west into what is now Pakistan, Afghanistan, and NW India, another migrated eastward to what is now in SW China. During the smaller upheavals or movements of tectonic plates in the late Pliocene, flying squirrels in each region adapted to different habitats in the eastern and the western extremes of the Himalayan mountain chain. The same migrationspatterns also exist in some of the more primitive or earlier mammalian genera surviving today in Pakistan that came from the Oriental region, such as the grey goral, Himalayan langur, civet cats, and naked-tailed murid rodents (Roberts, 1997). 6.4.2 Systematics of Chinese Flying Squirrels In structure, the morphology of tree squirrels is primitive and the morphology of flying squirrels is derived (Thorington and Heaney, 1981). The ability to glide depends upon the existence of membranes at the sides of the body, which are most fully developed in Petaurista Based on the comparison of the cranial structures, all Chinese flying squirrels can be morphologically divided into four groups: Petaurista, Aeretes, Trogopterus and the mixed group containing Belomys, Petinomys, Hylopetes and

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171 Pteromys (Figure 6.10 and Figure 6.11). This result is close to McKenna’s (1962) and Mein’s (1970) classification, although the classification of this study may not be very accurate because less than 5 specimens of Belomys, Petinomys, Aeretes and Trogopterus were used in multivariate approaches. With the inclusion of Eupetaurus the molecular data partitions all Chinese flying squirrels as five groups: Petaurista, Pteromys, Eupetaurus Hylopetes and Petinomys and the mixed group including Trogopterus and Belomys The phylogentic reconstruction of Chinese flying squirrels (Figure 6.9) is in part identical to Oshida’s et al. (2000a) study. The major differentiation is the divergence time between species. According to the estimated time calculated with the transversional rate at the third codon positions of cytochrome b gene, the generic divergences of Chinese flying squirrels occurred about 27 to 38 million years ago, the late Eocene to middle Oligocene. Trogopterus originally contains two species, Pteromys xanthipes in China and Sciuropterus pearsonii in India for their similar external structures (Figure 6.13). Thomas (1888) erected Sciuropterus pearsonii as new genus Belomys The chief distinction between these two genera lies in the structure of the teeth. Trogopterus is distributed in the forests of northeastern Hupei, Ichang, the area upper Min River, and Yunnan, China. Externally the flying squirrels of this genus show no striking peculiarities but appear to be characterized by the presence of a small tuft of long black hair at the inner and another at the outer base of each ear (Allen, 1940). The notable enlargement of the posterior upper premolar and the enamel pattern in checkteeth are somewhat complex and irregular. Trogopterus feeds on oak leaves, and the specifications of teeth are obviously associated with its feeding habit. To date, three

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172 forms have been named, but it is probable that these are all races of a single wide-ranging species. Belomys pearsonii ranges from Nepal eastward into southern China and is represented in the island of Taiwan by a closely allied form. It is distinguished externally from Pteromys by the presence of a tuft of long delicate hair at the base of each ear (Figure 6.12). The results in this study support that Trogopeterus is better distinguished from Belomys by its actually and proportionately longer toothrow (Figure 6.10 and Figure 6.11). The molecular data suggest that Trogopterus was the first diverged flying squirrel. About 20 million years ago, the extant genera Trogopterus and Belomys were split from their common ancestor, inferring that the morphological differences in skull are the recent developments. But there are no available data from fossil records, ecological observations, and quantitative descriptions with which to compare the present morphological and molecular data. Based on the morphology of cheekteeth (the key characteristic in the classification of flying squirrels), Glaucomys Hylopetes Eoglaucomys and Petinomys are closely related (Allen, 1940). They have short and broad heads, complex molars, and a tail that is bushy, cylindrical, and as long or longer than the head and body (Figure 6.13). The teeth of Hylopetes are like those of Glaucomys in essential structure, with transverse ridges of the upper molars complete, and partly joined internally to the outer slope of the internal wall, and lacking any notch extending part way across the tooth from the inner side. Molecular results based on the cytochrome b sequences support this conclusion, however the morphometric study based on the cranial characters reveals that Eoglaucomys is morphologically different from the other flying squirrels with small body

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173 size (Figure 6.10 and Figure 6.11). This difference might be related to their different living conditions (see Chapter 5 for detail). The skull of Pteromys closely resembles in general the contour of the genus Hylopetes The species Pteromys volans in China is distributed mainly in SE China (Figure 6.3). Its developed wrist has a long rod that becomes more or less bony and serves to spread the anterior edge of the lateral parachute or membrane. Pteromys volans usually lives in a hollow tree and feeds on nuts and pine seeds; owls and the smaller cats frequently capture these squirrels at night (Nowak, 1999). Although Oshida’s et al. (2000a) research indicates that a close phylogenetic relationship exists between Pteromys and Petaurista the morphological and molecular data in this study present different results. Their cranial structures are significantly different in both the size and shape. Phylogenetic trees constructed with MP and NJ methods imply an early divergence of Pteromys volans from other flying squirrels (Figure 6.8). Aeretes is a medium-sized species with a bushy flattened tail about the same length as the head and body (Figure 6.14). A. melanopterus the species confined to northeastern China, has a very special skeleton, which shows a very long lumbar region, its eight vertebrae equaling in length the combined cervical and thoracic portions of the spine (Ellerman, 1940). The upper incisors are much broader than in any member of Petaurista or Trogopterus and have a well-marked groove running vertically down the outer three-quarters of the width. Because of these characters in skull and skeleton, it seems worthy of generic distinction from smaller flying squirrels. Nothing is recorded of its habits, but the broad incisors and shortened rostrum may indicate a different feeding

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174 habit from related species. My findings in morphometric analysis show that Aeretes apparently differs from the remaining flying squirrels, both large and small. The most severe climatic cooling in the trans-Himalayan region was at about 33 Myr, slightly after the Eocene-Oligocene boundary, and was characterized by a drop in the mean annual temperature and by changes in vegetation from dense forests in the Eocene to more open country in the Oligocene (Meng and McKenna, 1998). In Oligocene faunas large species were few, medium-sized were rare or absent, and small mammals, such as rodents, became dominant. The first uplift of the great Himalayan mountain chain occurred at about 50 million years ago. This upheaval led to formation of a land bridge in the Bay of Bengal, connecting Gondwandland with southeastern Asia, particularly the Sino-Malaysian region. The paleontological records indicate that by the late Miocene the geography of the trans-Himalayas was similar to that of today (Wang, 1984). In middle Pleistocene, the Tibetan plateau was 3,000-3,500 m in elevation, and upheaved to 4,500-5,500 m during Holocene (Xu. 1981). The presence of the Himalayas caused the diversification of climate, and it became an important regulator of the Asian environment. Climatic and floristic fluctuations have led to fluctuations of species limits in both elevation and latitude through the effect on vegetation. The characteristically oriental faunal species invaded into the subcontinent and spread westward across the interface between Asia and Gondwanaland. The three or four major glacial periods in Asia, separated by warmer, interglacial periods in Pliocene-Pleistocene, played an important role for the present distributions of flying squirrels. This is corroborated in part by the evidence of a much greater diversity of species in the eastern Himalayas, compared with the northwest in what is now Pakistan.

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175 The fossil records show that Sciurinae evolved from the Paramyinae, whereas the Petauristinae evolved from the Prosciurinae before the mid-Miocene (Mein, 1970). In Miocene, the European fauna evolved independently from the North American and IndoMalayan forms (Arbogast, 1999). During the Pliocene flying squirrels were dominant in Europe, supplanting other squirrels. Considering the early fossil remains of Petinomys found in Europe (Black, 1972), all Chinese extant stocks had diverged from their ancestor(s) in the middle Oligocene. During the Oligocene-Miocene radiation of flying squirrels in Europe, each stock has migrated towards south. Their present geographical distributions are apparently associated with the recent tectonic movements during Pleistocene. Several molecular studies have supported the general hypothesis of latest Pleistocene southward depression, followed by postglacial northward expansion of ranges in Palearctic and Nearctic taxa (Cooper et al., 1995). The levels of molecular divergence in several widespread neotropical bird and frog superspecies indicate that phylogenetic diversification preceded the latest Pleistocene by millions of years (Bush, 1994; Heyer and Maxson, 1982). If flying squirrels evolved from a tree squirrel during the Oligocene after the Chadeonian, 35 million years ago (Johnson-Murray, 1977), the molecular data in present study and in Arbogast’s (1999) study suggest that the great difference between Petaurista and Glaucomys indicate that either flying squirrels are diphyletic or the divergence of lineages occurred very early (Thorington et al., 1998). The evolutionary history of flying squirrels, at least the New World flying squirrels that occupy the boreal and deciduous forest habitat of North America has been shaped profoundly by the Pleistocene glacial interglacial cycles (Arbogast, 1999).

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176 To distinguish Chinese flying squirrels morphologically and externally, Table 6.11 is a genus-based key to identification of different genera. This key is based on the characters used for classification of flying squirrels by Ellerman (1940). Table 6.11 Key to the genera of Chinese flying squirrels Cheekteeth strongly hypsodont ……………………………………………… Eupetaurus Cheekteeth not strongly hypsodont Cheekteeth always in lower series and usually in the upper series characterized by signs of extreme complication due to wrinkling; the essential pattern of the cheekteeth usually more or less masked P4 conspicuously enlarged Checkteeth semi-hypsodont; P4 extremely enlarged. …………………. Trogopterus Cheekteeth brachyodont P4 more moderately enlarged .………………………………. Belomys P4 not specially enlarged; the basi-occipital narrowed……… Petaurista Cheekteeth with a more normal pattern, the wrinkling though sometimes traceable never excessive, and never masking the essential pattern Bullae low and flattened, scarcely rising above general level of the base of the skull ……………………….……. Petinomys Ballae without special peculiarities M3 with two clear ridges between the anterior and posterior margins of tooth; M3 lower with four ridges And three depressions, incisive foramina long…………. Pteromys M3 with only one ridge between anterior and posterior margins of tooth; M3 lower never with four ridges and three depressions; incisive foramina short. Cheekteeth relative simpler, with small extra ridges and depressions not or barely traceable ……… Eoglaucomys Cheekteeth relatively more complex, with small extra ridges and depressions normally present ………. Hylopetes 6.5 Summary This chapter deals with the phylogeny of the trans-Himalayan flying squirrels and the systematics of Chinese flying squirrels. The results of morphometric and molecular

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177 study provide some important insights into the complex evolutionary history of flying squirrels. 1. The genetic differences among Eupetaurus, Eoglaucomys Hylopetes and Petaurista are significant. They diverged from their ancestor stock(s) about 20 to 32 million years ago, the early Miocene. 2. The morphological and genetic differences among the populations of Eupetaurus, Eoglaucomys Hylopetes and Petaurista are concordant with their geographical variations along the trans-Himalayan mountain chain. The current distributions owe much to both major climatic changes in the late Pleistocene and the physical barriers to migration. 3. All Chinese flying squirrels can be genetically partitioned into five groups: Petaurista, Pteromys, Eupetaurus Hylopetes and Petinomys and the mixed group including Trogopterus and Belomys 4. The estimated times from cytochrome b gene show that the generic divergences of all Chinese extant stocks of flying squirrels diverged from their ancestor(s) in the middle Oligocene. The present geographical distributions are apparently associated with the recent tectonic movements during Pleistocene.

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178 Figure 6. 1 Phylogenetic reconstruction of flying squirrels, tree squirrels, and fossil squirrels based on 44 dental characteristics

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179 Figure 6.2 Chinese flying squirrels

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180 Figure 6.3 Trans-Himalayan flying squirrels

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181 Figure 6.4 Scatter-plot of discriminant function analysis of the trans-Himalayan flying squirrels onto the first two functions (CAN I and CAN II) -20 -15 -10 -5 0 5 10 15 20 -20-100102030CAN ICAN II P. albiventer in Yunnan P. yunanensis H. alboniger H. phayrei E. fimbriatus P. philippensis P. albiventer in Pakistan P. elegans P. alborufus

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182 -10 -5 0 5 10 15 -20-15-10-5051015202530 CAN ICAN III P. albiventer in Yunnan P. yunanensis H. alboniger H. phayrei E. fimbriatus P. philippensis P. albiventer in Pakistan P. elegans P. alborufus Figure 6.5 Scatter-plot of discriminant function analysis of the trans-Himalayan flying squirrels onto function 1 and function 3 (CAN I and CAN III)

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183 -30 -20 -10 0 10 20 30 -60-50-40-30-20-10010203040 PRIN IPRIN II P. albiventer in Yunnan P. yunanensis H. alboniger H. phayrei E. fimbriatus P. philippensis P. albiventer in Pakistan P. elegans P. alborufus Figure 6.6 Principal components analysis of the trans-Himalayan flying squirrels onto the first two factors (PRIN I and PRIN II)

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184 -8 -6 -4 -2 0 2 4 6 -60-50-40-30-20-10010203040PRIN IPRIN III P. albiventer in Yunnan P. yunanensis H. alboniger H. phayrei E. fimbriatus P. philippensis P. albiventer in Pakistan P. elegans P. alborufus Figure 6.7 Principal components analysis of the trans-Himalayan flying squirrels onto the first and the third factors (PRIN I and PRIN III)

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185 Figure 6.8 Phylogenetic relationships of all Chinese flying squirrels constructed using neighbor-joining (NJ) method. Scales in the tree represent branch length in terms of nucleotide substitutions per site. Sample abbreviations are defined in Table 6.4.

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186 Figure 6.9 Phylogenetic relationships of all Chinese flying squirrels constructed via the maximum parsimony method using heuristic search algorithm. Scales in the tree represent branch length in terms of nucleotide substitutions per site. Sample abbreviations are defined in Table 6.4.

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187 -8 -6 -4 -2 0 2 4 6 8 10 -20-15-10-50510152025CAN ICAN II A. melanopterus B. pearsoni T, xanthipes Pteromys volans Petinomys setosus G. volans P. albiventer P. yunanensis H. alboniger H. phayrei E. fimbriatus P. philippensis Figure 6.10 Plot of Chinese flying squirrels based on discriminant function analysis onto the function 1 and function 2 (CAN I and CAN II)

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188 -8 -6 -4 -2 0 2 4 6 8 -50-40-30-20-1001020304050 PRIN IPRIN II A. melanopterus B. pearsoni T. xanthipes Pteromys volans Petinomys setosus G. volans P. albiventer P. yunanensis H. alboniger H. phayrei E. fimbriatus P. philippensis Figure 6.11 Plot of Chinese flying squirrels based on principal components analysis onto the factor 1 and factor 2 (PRIN I and PRIN II)

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189 Figure 6.12 Trogopterus xanthipes and Pteromys volans

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190 Figure 6.13 Eoglaucomys Hylopetes and Glaucomys

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191 Figure 6.14 Aeretes mlanopterus and Belomys personii

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192 CHAPTER 7 SUMMARY AND FUTURE WORK 7.1 Summary The areas of the Himalayas where high mountain ranges meet the lowlands of Asia in a series of deep, narrow, and often xeric gorges are described as the “transHimalayas.” The systematics, geographical distributions, and conservation status of many species in these rugged and remote regions are poorly understood. Flying squirrels are especially poorly understood because they occur in deep forest habitats and are nocturnal in habits. Despite the abundant taxonomic, ecological, and morphological information available for some flying squirrels, the phylogenetic relationships of many taxa remain uncertain. This taxonomic uncertainty is especially true for Chinese flying squirrels. Analyses of the partial sequences of mitochondrial cytochrome b gene and of morphological data were used to study the systematics and biogeography of flying squirrels in the eastern and the western trans-Himalayas. Detailed phylogenetic analyses have been carried out on forms or populations of Eupetaurus Petaurista and Hylopetes ( Eoglaucomys ) that are distributed in SW China and Pakistan. Taken together, the major contributions of this dissertation are as follows: 1. The two specimens that were collected in northwestern Yunnan, China, are members of the same genus Eupetaurus There are significant differences in the population in the eastern and the western trans-Himalayas, indicating that two distinct species are present. 2. The possible divergence time of the two populations of Eupetaurus was at the end of Miocene, about ten million years ago. The glacial period and the uplift of the Himalayas and Qinghai-Tibet plateau during the Pliocene-Pleistocene period are

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193 the major factors that secondarily affected on the present distribution of Eupetaurus in the trans-Himalayas. 3. The population of P. petaurista ( albiventer ) in Pakistan is significantly different from the population in W Yunnan of China in morphology and genetics. It is probably a new subspecies, even a new species. 4. P. yunanensis is genetically distinctive from P philippensis and is a valid species. There is no basis for retaining P. hainana as a recognizable species; instead, it might be a subspecies or synonym of P philippensis P. xanthotis is a valid Chinese endemic species and shows a close phylogenetic relationship with P. leucogenys in Japan and China. 5. H. electilis is a possible valid species of Hylopetes based on its genetic characters, although it shares similar morphological characters with H. phayrei in skull. 6. Eoglaucomys in the western trans-Himalayas differs morphologically and genetically from Hylopetes in China and SE Asia. My data strongly support recognizing Eoglaucomys as a valid genus. Eoglaucomys diverged from other Hylopetes as early as 24 million years ago, the middle to late Oligocene. The migration of Hylopetes to the present geographical distribution is due to the tectonic movements of the Himalayas during the Pliocene-Pleistocene period. 7. All Chinese flying squirrels can be genetically partitioned into five groups: Petaurista, Pteromys, Eupetaurus Hylopetes and Petinomys and the mixed group including Trogopterus and Belomys The morphological and genetic characters of Eupetaurus, Eoglaucomys Hylopetes and Petaurista are concordant with their geographical variations along the great Himalayan mountain chain. The present distributions owe much to both major climatic changes in the late Pleistocene and the physical barriers to migration. 7.2 Future Work Despite the accomplishments of this dissertation, it is apparent that our understanding of the systematics of flying squirrels and the mechanisms responsible for their present geographical variations is far from complete and will continue to be an interesting area for future research. A few of further analyses I think would provide significant contributions to resolve the exact phylogenetic placement of Chinese flying squirrels.

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194 In Chapter 3, I performed the comparative study between the populations of Eupetaurus in the eastern and the western trans-Himalayas, but the populations between the Himalayan extremes such as those in India and Sikkim are not included. It would be useful if the molecular data of the populations between Pakistan and China could be analyzed. Finding or collecting a skull or a skeleton of Eupetaurus in the eastern transHimalayas is also crucial for identifying the taxonomic status of Eupetaurus populations, and in describing the probably new Eupetaurus species in SW China. South China is located at the crossroads of southeastern Asia and has been a bypass for animal dispersal from mainland Asia southward into the Indo-Malayan region. The further study of Petaurista and Hylopetes should focus on the populations between SW China and SE Asia, and between the eastern and the western trans-Himalayas, such as in north India, Myanmar, Thailand, Laos, and SE Asia. The inclusion of the population of H. phayrei in southeastern China will be significant for reconstructing the phylogenetic topology of Chinese Hylopetes by employing the similar analytical approaches. Although I was able to construct the phylogenetic relationships of the Chinese flying squirrels at the generic-level, further analyses are necessary. Because specimens of flying squirrels, especially skins and fluid preserved specimens, are not as common in museums or academic institutes as specimens of non-flying squirrels, many of the forms are very little known. The collection of additional specimens of flying squirrels is crucial. The taxonomic affinities and phylogenetic relationships of taxa such as Aeretes, Trogopterus, Belomys, H. electilis, H phayrei and P. petaurista in various geographical locations could be resolved if specimens and tissues were available.

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195 One of the most serious gaps in the knowledge of the Chinese flying squirrel is in the area of ecology, the study of other animals and plants in relation to each other and to its environment. The biology of the Chinese flying squirrels is still poorly known, with nearly every new collection or study revealing new distribution limits and providing very useful information. Further research will undoubtedly lead to changes in details. .

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196 APPENDIX GEOLOGICAL EPOCHS Geological epoch Time scale (million years) Holocene 0.1 present Pleistocene 1.8 – 0.1 Pliocene 5 – 1.8 Neogene Miocene 23 5 Oligocene 34 23 Eocene 55 34 Paleogene Paleocene 60 55 Cretaceous >65

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210 BIOGRAPHICAL SKETCH Fahong Yu was born in Lanzhou, Gansu province, China, and finished grade school in this city. Fahong graduated from Mingqing High School in 1979 and completed his teaching certification program from a professional school in 1981. By 1982 Fahong became a high school teacher in Mingqing County, Gansu province. In 1984, the Northwest Normal University accepted him as an undergraduate student and he received his B.A. in 1988. From 1989 to 1992, as a graduate student, Fahong studied the evolution of primates at the Kunming Institute of Zoology (KIZ), the Chinese Academy of Sciences, with Professor Yanzhang Peng. In May 1992, he graduated with a M.Sc. in biology from KIZ. Then he continued his research in biology as an assistant researcher in KIZ. In 1996, Fahong left KIZ to pursue his doctoral studies at the University of Florida, US. In the summer of 1999, when he became a Ph.D. candidate in zoology, he was also accepted as a graduate student at the Department of Computer and Information Science and Engineering (CISE). In 2000, he started his research with Dr. Stanley Su. In December 2001, he received his Master of Science from the Computer and Information Science and Engineering Department of the University of Florida. In 2002, under the guidance of Dr. Charles Woods, he was awarded the Ph.D. degree from the Department of Zoology, University of Florida.


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Copyright Date: 2002

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SYSTEMATICS AND BIOGEOGRAPHY OF FLYING SQUIRRELS IN THE
EASTERN AND THE WESTERN TRANS-HIMALAYAS












By

FAHONG YU


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2002




























Copyright 2002

by

FAHONG YU















ACKNOWLEDGMENTS

I am most grateful to my committee chair, Dr. Charles Woods, for his continuous

guidance, advice, and support throughout my graduate education. Certainly without his

support and guidance, I would not have been able to carry out this research. Charles has

had the most influence on the direction of my dissertation. He has spent all of his own

time to teach me new techniques and to honestly discuss my results. I especially thank

Dr. Brian McNab, cochairman of my supervisory committee, for his constant help

dealing with all kinds of paperwork and providing me good suggestions during my

graduate study. I am extremely thankful to Dr. William Kilpatrick for his creative and

unique perspective on issues concerning many aspects of my research. I also appreciate

the help and assistance of the other committee members, Dr. Ronald Wolff, Dr. Melvin

Sunquist, and Dr. Michael Miyamoto, who were willing to give me their time and offer

sound guidance.

My sincere appreciation goes to Professor Yingxiang Wang and Professor Yaping

Zhang in Kunming Institute of Zoology, China, for allowing me to access the collections

and to use equipment in their labs. Also, they provided valuable comments and

suggestions throughout the process of developing my dissertation and helped me to

significantly improve the quality of the work. I would like to thank Mr. Tian Ming and

Dr. Junfen Pang for their interest in my project and for their help when I worked at

Kunming. I am grateful for the generosity of Ms. Ginger Clark and Mr. Joel Ernst for

allowing me to use the equipment and supplies and to access the computer programs in









laboratory. They helped me solve the contamination problems in my molecular

experiments. I greatly appreciated the generosity of the collection managers of Mammal

Range of Florida Museum Natural History, Ms. Laurie Wilkins and Candace McCaffery,

for their unselfish help and for allowing me to continue accessing the equipment and

specimens.

My research was primarily supported by Charles Woods (National Fish and

Wildlife Foundation); the director foundation of Kunming Institute of Zoology and the

director foundation of the Laboratory of Cellular and Molecular Evolution of Kunming

Institute of Zoology, the Chinese Academy of Sciences; the visiting scholarship of the

American Museum of Natural History, New York. The Department of Zoology provided

partial funding of my project. My research project would not have been possible without

the support and permissions from the Nature Reserves in Lunchun, Gongshan,

Xishuangbaina of Yunnan. Field work by Dr. Shunqing Lu, Mr. Lin, Mr. He, and Mr.

Zhang was instrumental in my success. I also wish to thank the American Museum of

Natural History, New York; Florida Museum of Natural History, Gainesville; National

Museum of Natural History, Washington DC; Chinese Institute of Zoology, Beijing;

Kunming Institute of Zoology, Kunming; and Northwestern Institute of Biology,

Qinghai, for their permission to examine specimens and to collect tissues.

I also owe many thanks to Ms. Karen for her friendly help and service. I would

also like to express my appreciation of support and help provided by Professor Feng, Dr.

Hongshan Wang, Mr. Lin, and Ms. Linda Gordon, and other colleagues, faculty, graduate

students of the Department of Zoology, University of Florida.









My wholehearted gratitude goes to my parents, my wife, and my brother for their

unconditional love, encouragement, and financial support throughout my six years of

study.
















TABLE OF CONTENTS
page
A C K N O W L E D G M E N T S ...................... .. .. ............. ...................................................iii

LIST OF TABLES ...................... ....................... ........ .. ............ ix

LIST OF FIGURES ........................... ............................... .... xii

A B S T R A C T ................................................... .................. ................ x v ii

CHAPTERS

1 INTRODUCTION..................... ............. 1

1.1 Evolution of Flying Squirrels......................................... ............... .............. 2
1.2 Evolution of Chinese Flying Squirrels.. ....................... ...... ..............6
1.3 Objectives of This Study ..................................................................... 10


2 METHODS AND TECHNIQUES OF PHYLOGENETIC STUDY............................ 13

2 .1 M olecular Study .............. .. ........... .... .... ...................................... ..... ... 14
2.1.1 Mitochondrial Cytochrome b Gene................................................... 14
2 .1.2 P hylogenetic A naly sis ............................................................................ ... 16
2 .1.2 .1 P arsim ony m ethod ........................................................................ ... 17
2.1.2.2 L ikelihood m ethod ........................................ .......... .............. 17
2 .1.2 .3 D istan ce m eth od ......................................................................... ... 18
2.2 M orphom etric Study ............................................................. ............ 19


3 PHYLOGENY AND BIOGEOGRAPHY OF EUPETAURUS............................ 22

3.1 Introduction ............... ...... ......... ........... .. ........ ..... ........ 22
3.2 M materials and M methods ........... ....................................................... .............. 25
3.2.1 Samples ............. ..... ......... .................. 25
3.2.2 M methods ......... ............. ............................. ... .. .. ..... ......... 27
3.2.2.1 M itochondrial D N A isolation.................................... .................... 27
3.2.2.2 PCR am plification ................................................... ................. 27
3 .2 .2 .3 Sequ ence analy sis...................................................... .... .. .............. 2 8
3.3 R results .................................. .................................0
3.3.1 Phylogenetic Relationship of Eupetaurus between the Eastern and the
Western Trans-Himalayas ........... ...... ............................ 30









3.3.2 Phylogenetic Analysis between Eupetaurus and Petaurista...................... 31
3.4 D iscu ssion ..................... .... ........ ............ ..... ........ ...... .................. ... ... .. 33
3.4.1 Phylogenetic Status of the Population of Eupetaurus in the Eastern Trans-
H im alayas ................................... ...... ... ... ... .. ....... .............. 33
3.4.2 Phylogenetic Relationship between Eupetaurus and Petaurista.................. 35
3.5 Sum m ary .............. ..... ..... ............ ......... .. ......................................39


4 PHYLOGENY OF GIANT FLYING SQUIRREL PETAURISTAA) IN SW CHINA
AND PAKISTAN: IMPLICATIONS FOR DEVELOPMENT OF MOLECULAR
AND M ORPHOLOGICAL ANALYSIS ........................................... .................... 49

4.1 Introduction .............................. ..................... .... ........ 49
4 .2 M materials and M methods ......... ................. ....................................... .. ................... 54
4.2.1 Specimens in M orphometric Study ............ ....... .......................... 54
4.2.2 Species for M molecular A nalysis............................................... .... .. .............. 56
4.2.3 M orphom etric A analysis .......................................................... .......... .... 58
4.2.4 M olecular A nalysis...................................................... ........... .............. 59
4.3 Results ...................................... ..... ............................ 60
4.3.1 Phylogenetic Relationships of Chinese P. philippensis ............................ 60
4.3.1.1 M orphological data ........................................ ........................ .. 60
4 .3 .1.2 M olecular data ..................... ........ ...... ............... .................. 63
4.3.2 Phylogenetic Relationship between P. .\.xh,,tii and P. leucogenys........... 65
4.3.3 Phylogenetic Relationship of P. petaurista........ ..... .......................... 66
4.3.4 Phylogenetic Relationships of Chinese Petaurista................................. 70
4.4 D discussion .............. .... .. ... .... .... ... ....... ................................ .. 76
4.4.1 Phylogeny of the Trans-Himalayan P. petaurista (albiventer) ................. 76
4.4.2 Taxonomic Status of P. philippensis, P. yunanensis, and P. hainana .......... 80
4.4.3 Phylogenetic Relationship between P. .\xni,,ti\ and P. leucogenys........... 83
4.4.4 Systematics of Chinese Petaurista........ ................... ..... ............ 85
4.5 Summary ............................................ 89


5 PHYLOGENY OF EOGLAUCOMYS AND HYLOPETES IN THE EASTERN AND
THE WESTERN TRANS-HIMALAYAS AS INFERRED FROM MOLECULAR
AN D M ORPH OM ETRIC STUD Y ........................................................................... 114

5.1 Introduction .............. ...... .... ... .............................. ........ 114
5.2 M materials and M methods ........... ..................................................... .............. 117
5.2 .1 M materials ............. .......................................................................... ..... 117
5.2.2 M orphometric Analysis................................ .............. 119
5.2.3 B iochem ical Study ............................... ... ................................... 120
5.3 Results ....................................................................... ........ 121
5.3.1 Comparison between Eoglaucomys and the Chinese Hylopetes............... 121
5.3.1.1 Morphological data .............................. 121
5.3.1.2 M olecular data............... ................. ............ 123
5.3.2 Phylogenetic Relationships of Hylopetes ............ ............ ............. 124









5.4 Discussion ....................................................... ......... 128
5.4.1 Taxonomic Status of Eoglaucomys ............. ............. ............................ 128
5.4.2 Phylogenetic Status of H electilis........................................................... 130
5.4.3 Phylogenetics and Biogeography of Eoglaucomys and Hylopetes.............. 132
5 .5 C on clu sion............................................... ....... ......... ...... 13 5


6 PYLOGENY OF FLYING SQUIRRELS IN THE TRANS-HIMALAYAS AND
OTHER PARTS OF CHINA............................................ 152

6.1 Introduction ..................................... ............................... ......... 152
6.2 M materials and M methods ....................................................................... 155
6 .2 .1 Sp ecim en s ................... ........................................................ 15 5
6.2.2 M ethods of Phylogenetic Analyses ............. ......................... ................ 158
6.3 R results ....................... ............ ....... ................ ............. ..... .............. 158
6.3.1 Comparative Study of the Eastern and the Western Trans-Himalayan Flying
Squirrels ............. .. ........... ... .... ....................... .......... ............ 158
6.3.2 Phylogenetic Relationships among Flying Squirrels in China.................... 161
6.4 D discussion .................................... .. .. ................ .......... 165
6.4.1 Phylogeny of the Trans-Himalayan Flying Squirrels............................. 165
6.4.2 Systematics of Chinese Flying Squirrels.............. ..... ................. 170
6 .5 S u m m a ry ............................................................................................................... 1 7 6


7 SUMMARY AND FUTURE WORK............................... ................... 192

7 .1 S u m m ary ................................................................... 19 2
7.2 Future W ork ........................................ 193


APPENDIX

GEOLOGICAL EPOCHS................................ ............... .............. 196

LIST O F REFEREN CE S ................................................... ................................. 197

B IO G R A PH IC A L SK E T C H .......................................................................................... 2 10














viii
















LIST OF TABLES


Table page

1.1 McKenna's classification of flying squirrels in Petauristinae............... .................. 3

1.2 Mein's classification, determined from dental characters........................................... 4

1.3 The latest estimate of flying squirrels in Pteromystinae............................................. 4

1.4 Comparison of different classifications of flying squirrels................ .......... .... 5

1.5 Flying squirrels and their distributions ......................... ...................................... 6

1.6 C h in ese fly in g squ irrels....................................................... .................................... 8

3.1 Thirteen historical and 2 recent specimens of Eupetaurus ...................................... 24

3.2 Specimens of Eupetaurus and other flying squirrels examined in this study* ........... 26

3.3 Cycling program of PCR amplification ............................... .. .............. 28

3.4 Percentage differences of Eupetaurus and Petaurista based on the pairwise
comparisons of cytochrome b gene (390 bp) .......................................... .. 31

3.5 Transversional substitutions at the third codon positions of cytochrome b gene
between Eupetaurus and Petaurista (based on 390 bp).............. .................... 32

3.6 Estimated divergent times among Eupetaurus and Petaurista based on a rate of
divergence for the third codon positions of mammalian cytochrome b gene of ca.
0 .5% 106 y ears.................. ................................... .............. 33

4.1 Chinese P etaurista form s ........ ............ ................ ......................... .............. 50

4.2 F orm s of P etaurista ...................................................... .. .. .......... .. .. ........... 52

4.3 M ajor form s of P p hilipp ensis.......................................................... .................... 52

4.4 Species and localities of Petaurista populations examined in morphometric analysis55

4.5 V ariables in m orphom etric study ................................................................................ 56

4.6 Samples of Petaurista examined in molecular study ............................................... 57









4.7 Sequence data of Petaurista and Pteromys used in this study ............................... 58

4.8 Discriminant function analysis of five P. philippensis forms ................................ 62

4.9 Principal components analysis of five P. philippensis forms................................. 63

4.10 Pairwise comparison based on the partial sequences (409 bp) of cytochrome b gene
between five P. philippensis forms. Data below the diagonal are the numbers of
nucleotide substitutions, transitions vs. transversions. Data above the diagonal
represent the genetic differences between samples. The samples were defined in
Table 4.6 and 4.7. ......................................... ............ .............. 64

4.11 Percentage of genetic differences between P. .\xllhiii\ and other giant flying
squirrels based on pairwise comparison of the partial cytochrome b sequences
(409 bp). See Table 4.6 and Table 4.7 for sample information............................ 65

4.12 Discriminant function analysis of P. petaurista.................................... .............. 67

4.13 Principal components analysis of P. petaurista.................................... .............. 68

4.14 Percentage of differences and the numbers of transversional and transitional
substitutions between P. petaurista (albiventer) populations based on pairwise
comparison of the partial sequence (375 bp) of cytochrome b gene................... 69

4.15 Discriminant function analysis of Chinese Petaurista................... ............. 71

4.16 Principal components analysis of Chinese Petaurista ................................... 72

4.17 Pairwise comparison of Chinese Petaurista based on the partial sequences (380 bp)
of cytochrome b gene. Data above the diagonal were the percentage of genetic
differences between samples, and data below the diagonal were the numbers of
transitions vs. transversions between samples ............................................... ...... 74

4.18 Transversional substitutions at the third codon positions of the partial sequences
(375 bp) of cytochrome b gene in Petaurista...................... .............. 75

4.19 The estimated divergence time between species based on a divergence rate for the
third codon positions of mammalian cytochrome b gene of ca. 0.5% *106 years 75

5.1 Species of Hylopetes and Eoglaucomys .................. .... ... ........... ...... 116

5.2 Species and localities of Hylopetes and Eoglaucomys examined in morphometric
analysis ..................................... ................................. .......... 118

5.3 Samples of Eoglaucomys and Hylopetes examined in molecular study ................. 119

5.4 Discriminant function analysis between the Chinese Hylopetes and Eoglaucomys 122

5.5 Principal components analysis between Chinese Hylopetes and Eoglaucomys........ 123









5.6 Pairwise comparison of cytochrome b nucleotide sequences (400 bp) between
Chinese Hylopetes and Eoglaucomys.................... ......................... 124

5.7 Pairwise comparison of Hylopetes and Eoglaucomsy based on the partial cytochrome
b sequences (375 bp) .................. ............................ ................... 126

5.8 Transversional substitution rates at the third codon positions of the partial sequences
(375 bp) of cytochrome b gene between species........................................... 127

5.9 Estimated divergence time between species using the rate of the transversional
substitutions at the third codon positions of mammalian cytochrome b gene of ca.
0.5% *106 years...... ............................................................ 127

6.1 Classification of the extant flying squirrels determined from 44 dental characters.. 153

6.2 Chinese flying squirrels other than Petaurista................ ......... ............... 153

6.3 Species and localities of flying squirrels used in morphometric analysis ............... 156

6.4 Samples of flying squirrels used in molecular study ............................. .............. 157

6.5 Discriminant function analysis on the eastern and the western trans-Himalayan
flying squirrels............................................ ...... .......... ..... 159

6.6 Principal components analysis of trans-Himalayan flying squirrels on the first three
factors ................ ................................... ........................... 16 1

6.7 Pairwise comparison based on the partial cytochrome b sequences (375 bp) between
species. See Table 6.4 for the sample abbreviations........................................ 162

6.8 Pairwise comparison of the transversional substitutions at the third codon positions
of the partial cytochrome b sequences (366 bp) between samples. Data below
the diagonal are the numbers of transversions at the third codon positions. Data
above the diagonal represent the transversional percentage difference between
samples. Table 6.4 shows the information of samples ................................. 163

6.9 Estimated divergence time between samples based on the rate of the transversional
substitutions at the third codon of cytochrome b sequences proposed by Irwine et
al. (1991). See Table 6.4 for sample abbreviations.......................................... 164

6.10 M ultivariate analyses of Chinese flying squirrels................................................ 165

6.11 Key to the genera of Chinese flying squirrels ................................................... 176















LIST OF FIGURES


Figure page

3.1 Historical records of Eupetaurus specimens in the world. The numbers in the map
stand for the collecting localities of specimens, which are corresponding to the
num bers of Table 3.1 ................................................ ..... .. .. .......... .. 40

3.2 Phylogenetic tree of Eupetaurus reconstructed by the maximum parsimony (MP)
method. Numbers above branches indicate the bootstrap values (%). Sample
abbreviations are coded in Table 3.2........................................... ...... .............. 41

3.3 Phylogenetic tree of Eupetaurus reconstructed by the UPGMA method. Sample
abbreviations are coded in Table 3.2............................................... .. .............. 42

3.4 Phylogenetic tree of Eupetaurus and Petaurita constructed with UPGMA method.
Sample abbreviations are defined in Table 3.2. ............................................. ...... 43

3.5 Phylogenetic relationships ofEupetaurus, Petaurita, and G. volans constructed with
the parsimony maximum (MP) method. Numbers above branches indicate the
bootstrap values (%). Sample abbreviations are defined in Table 3.2 ............... 44

3.6 Phylogenetic relationships ofEupetaurus, Petaurita, and G. volans constructed with
the neighbor-joining (NJ) method. Scales in the tree represent branch length in
terms of nucleotide substitutions per site. Sample abbreviations are coded in
T ab le 3 .2 ............................................................................................ . 4 5

3.7 Eupetaurus cinereus in Pakistan and SW China ............................ 46

3.8 Ventral views of the skulls of E. cinereus, P. petaurista, and P. .x\,lli,,ti ................ 47

3.9 E. cinereus, P. petaurista, and P. .\ ,,i ............. ................................ ....... ....... 48

4.1 Chinese giant flying squirrels (Petaurista) .............. .................................... ........ 90

4.2 Plot of five P. philippensis forms onto discriminant function 1 (CAN I) and function
2 (C A N II) ............................................................... .. ..... ........ 91

4.3 Plot of five P. philippensis forms onto discriminant function 1 (CAN I) and function
3 (CAN III) ..................................... ................................ .......... 92









4.4 Principal components analysis of five P. philippensis forms onto factor 1 (PRIN I)
and factor 2 (PR IN II) ..................... ................ ...................................... 93

4.5 Principal components analysis of five P. philippensis forms onto factor 1 (PRIN I)
an d factor 3 (P R IN III) ................................................................................. 94

4.6 Phylogenetic relationships ofP. philippensis forms based on the cytochrome b gene
using maximum parsimony method (MP). Numbers above branches indicate the
bootstrap values (%). Sample abbreviations are defined in Table 4.6 and Table
4.7............ ......................................... ......... 95

4.7 Phylogenetic relationships ofP. philippensis forms based on the cytochrome b gene
using neighbor-joining method (NJ). Scales in the tree represent branch length in
terms of nucleotide substitutions per site. See sample abbreviations in Table 4.6
and T able 4.7. ....................... .................................................. 96

4.8 Phylogenetic tree of P. .\,hiiii1iii and other giant flying squirrels constructed using
maximum parsimony method (MP). Numbers above branches indicate the
bootstrap values (%). Sample abbreviations are defined in Table 4.6 and Table
4.7............ ......................................... ......... 97

4.9 Phylogenetic tree of P. .\,hiiii1iii and other giant flying squirrels constructed using
neighbor -joining method (NJ). Scales in the tree represent branch length in
terms of nucleotide substitutions per site. See sample codes in Table 4.6 and
Table 4.7 ................................... ................................. ......... 98

4.10 Plot of P. petaurista populations of discriminant function analysis onto the first and
the second discrimiant function (CAN I to CAN II)........................................... 99

4.11 Plot of P. petaurista populations of discriminant function analysis onto the first and
the third discrimiant function (CAN I to CAN III) ........................................... 100

4.12 Principal components analysis ofP. petaurista populations onto factor 1 and factor 2
(PR IN I to PR IN II) .................. ................................... .. .......... 101

4.13 Principal components analysis ofP. petaurista populations onto factor 1 and factor 3
(PRIN I to PRIN III)................ ...................................... .... ............ .. 102

4.14 Phylogenetic relationships within the populations of P. petaurista reconstructed by
maximum parsimony (MP) method with Pteromys volans as the outgroup.
Numbers above branches are the bootstrap values (%). The abbreviations of taxa
are defined in Table 4.6 and Table 4.7 ....... ........ ....................... .............. 103

4.15 Phylogenetic relationships within the populations of P. petaurista reconstructed by
neighbor-joining (NJ) method with Pteromys volans as the outgroup. Scales in
the tree represent branch length in terms of nucleotide substitutions per site. The
abbreviations oftaxa are defined in Table 4.6 and Table 4.7. .......................... 104









4.16 Discriminant function analysis of Chinese Petaurista on discriminant function 1
(CAN I) and function 2 (CAN II).............................. .............. 105

4.17 Discriminant function analysis of Chinese Petaurista on discriminant function 1
(CAN I) to function 3 (CA N III) ................................. ....................... .. ........ 106

4.18 Principal components analysis of Chinese Petaurista on factor 1 (PRIN I) and factor
2 (PRIN II) ..... ...................................... ............... 107

4.19 Principal components analysis of Chinese Petaurista on factor 1 (PRIN I) and factor
3 (PRIN III) ..................................... .............................. .......... 108

4.20 Phylogenetic topology of Petaurista based on the maximum parsimony method.
The number of bootstrap value (%) is given above each branch. The
abbreviations oftaxa are defined in Table 4.6 and Table 4.7. .......................... 109

4.21 Phylogenetic topology of Petaurista based on the neighbor-joining method. Scales
in the tree represent branch length in terms of nucleotide substitutions per site.
The abbreviations oftaxa are defined in Table 4.6 and Table 4.7. .................... 110

4.22 Phylogenetic topology of Petaurista based on the UPGMA method. The
abbreviations oftaxa are defined in Table 4.6 and Table 4.7. ...................................... 111

4.23 P. petaurista in Pakistan and W Yunnan, China.................................. 112

4.24 P. philippensis and P. yunanensis in Yunnan, China......................................... 113

5.1 Distribution of Chinese Hylopetes and Eoglaucomys........................... 136

5.2 Discriminant function analysis between Chinese Hylopetes and Eoglaucomys onto
function 1 and function 2 ............................ ..... ................................... 137

5.3 Discriminant function analysis between Chinese Hylopetes and Eoglaucomys onto
function 1 and function 3 ........................... ..... .................................. 138

5.4 Principal components analysis of Chinese Hylopetes and Eoglaucomys onto factor 1
and factor 2 ........................................... .................. ................ 139

5.5 Principal components analysis of Chinese Hylopetes and Eoglaucomys onto factor 1
and factor 3 ........................................... ................. ................ 140

5.6 Phylogenetic topology of Eoglaucomys and Chinese Hylopetes constructed using the
neighbor-joining method based on the partial sequences (400 bp) of cytochrome b
gene. Scales in the tree represent branch length in terms of nucleotide
substitutions per site. Sample abbreviations are coded in Table 5.3................. 141

5.7 Scatter-plot of Hylopetes and Eoglaucomys along the first two discriminant
functions ..................................... ................................. ......... 142









5.8 Scatter-plot ofHylopetes and Eoglaucomys onto the first and the third discriminant
function............................................ .......... 143

5.9 Plot of principal components analysis of Hylopetes and Eoglaucomys onto factor 1
and factor 2 .......................................... .................. ................ 144

5.10 Plot of principal components analysis of Hylopetes and Eoglaucomys onto factor 1
and factor 3 .............................................. ................. .... .. ..... 14 5

5.11 Phylogenetic tree of Hylopetes and Eoglaucomys generated by MP method on
partial sequences (375 bp) of cytochrome b gene. Numbers above branches are
the bootstrap values (%). Sample abbreviations are coded in Table 5.3............. 146

5.12 Phylogenetic tree of Hylopetes and Eoglaucomys generated by NJ method on partial
sequences (375 bp) of cytochrome b gene. Scales in the tree represent branch
length in terms of nucleotide substitutions per site. See sample abbreviations in
T ab le 5 .3 ................. ................................. ....... .......... ..... 14 7

5.13 Eoglaucomysfimbriatus and Hylopetes alboniger............................................ 148

5.14 Hylopetes electilis and Hylopetes phayrei .................. ........... ............ .. 149

5.15 Hylopetes electilis and Hylopetes nigripes........................................................ 150

5.16 Hylopetes lepidus and Hylopetes spadiceus ........ ............................................ 151

6. 1 Phylogenetic reconstruction of flying squirrels, tree squirrels, and fossil squirrels
based on 44 dental characteristics ............................................. .................. 178

6.2 Chinese flying squirrels.. ....................................... 179

6.3 Trans-Himalayan flying squirrels........................... ......... ............. ...... 180

6.4 Scatter-plot of discriminant function analysis of the trans-Himalayan flying squirrels
onto the first two functions (CAN I and CAN II) .............................................. 181

6.5 Scatter-plot of discriminant function analysis of the trans-Himalayan flying squirrels
onto function 1 and function 3 (CAN I and CAN III)............... ................... 182

6.6 Principal components analysis of the trans-Himalayan flying squirrels onto the first
two factors (PRIN I and PRIN II) .............. ...... ....................................... 183

6.7 Principal components analysis of the trans-Himalayan flying squirrels onto the first
and the third factors (PRIN I and PRIN III).................................................... 184

6.8 Phylogenetic relationships of all Chinese flying squirrels constructed using
neighbor-joining (NJ) method. Scales in the tree represent branch length in
terms of nucleotide substitutions per site. Sample abbreviations are defined in
T ab le 6 .4 .................. ................................. ...... .......... ..... 18 5









6.9 Phylogenetic relationships of all Chinese flying squirrels constructed via the
maximum parsimony method using heuristic search algorithm. Scales in the tree
represent branch length in terms of nucleotide substitutions per site. Sample
abbreviations are defined in Table 6.4. ........................................ .............. 186

6.10 Plot of Chinese flying squirrels based on discriminant function analysis onto the
function 1 and function 2 (CAN I and CAN II)............................................... 187

6.11 Plot of Chinese flying squirrels based on principal components analysis onto the
factor 1 and factor 2 (PRIN I and PRIN II)..................................................... 188

6.12 Trogopterus .\x ul/il,/e\ and Pteromys volans ............................. .............. 189

6.13 Eoglaucomys, Hylopetes, and Glaucomys........ ................................. 190

6.14 Aeretes mlanopterus and Belomys personii ..................................................... 191















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

SYSTEMATICS AND BIOGEOGRAPHY OF FLYING SQUIRRELS IN THE
EASTERN AND THE WESTERN TRANS-HIMALAYAS

By

Fahong Yu

December 2002


Chair: Charles A. Woods
Cochair: Brian K. McNab
Major Department: Zoology

The areas of the Himalayas where high mountain ranges meet the lowlands of

Asia in a series of deep, narrow, and often xeric gorges are described as the "trans-

Himalayas," including the eastern extreme (SW China, Burma) and the western extreme

(Pakistan and Afghanistan). The systematics and biogeography of many flying squirrels

in this region, however, are poorly understood and remain uncertain. This is especially

true for Chinese flying squirrels. Analyses of the partial sequences of mitochondrial

cytochrome b gene and morphological data were performed for investigating the

phylogenetic relationships of forms or populations of Eupetaurus, Petaurista, and

Hylopetes (Eoglaucomys) along the trans-Himalayas.

First, the molecular data revealed that the two specimens in SW China are

Eupetaurus, which differs significantly from the population in Pakistan, suggesting two

distinct species. They diverged at the end of the Miocene. The glaciations and the uplift









of the Himalayas during the Pliocene-Pleistocene period are the major factors that

affected the present distribution of Eupetaurus.

Second, morphological and molecular data suggested that the population of P.

petaurista in Pakistan apparently differentiated from the population in W Yunnan of

China. P. yunanensis is distinctive from P. philippensis and is a valid species. There is

no basis for retaining P. hainana as a recognizable species, however. P. .\xm/il,i\, is a

valid Chinese endemic species and has a close phylogenetic relationship with P.

leucogenys in Japan and China.

Third, the comparative study of Eoglaucomys and Hylopetes in the eastern and the

western trans-Himalayas showed that Eoglaucomys is morphologically and genetically

distinct from Hylopetes and is a valid genus. H. electilis on the island of Hainan, China,

is a valid species of Hylopetes, although it shares similar morphological characters with

H. phayrei in skull.

Last, all Chinese flying squirrels can be divided into five groups: 1) Petaurista; 2)

Pteromys; 3) Eupetaurus; 4) Hylopetes and Petinomys; and 5) Trogopterus and Belomys.

The morphological and molecular data of this study support that the current distributions

of Chinese flying squirrels owe much to both major climatic changes in the late

Pleistocene and the physical barriers to migration.


xviii














CHAPTER 1
INTRODUCTION

The areas of the Himalayas where high mountain ranges meet the lowlands of

Asia in a series of deep, narrow, and often xeric gorges are described as the "trans-

Himalayas." Its eastern and western extremes are considered as the eastern and the

western trans-Himalayas. The former includes southwestern Yunnan, eastern Tibet,

southern Yunnan, northern Burma and India, while the latter consists of northwestern

India, Pakistan, and Afghanistan. They form, in effect, the left and right sides of an open

book, with the Tibet Plateau as the center.

The eastern trans-Himalayas consists of several distinct topographic regions

defined by drainage patterns associated with the parallel mountain chains. These deep

gorges have only eastern and southern outlets. The collections of plants and animals

made by Pere Armand David, a French missionary-naturalist, were made in the eastern

trans-Himalayas, an area also known as the western Chinese highland (Allen, 1940). In

this great area the forests are largely of fir and spruce with hardwoods at middle

elevations. The high ridges (2,800 to 3,000m) are characterized by thickets of small

bamboo and rhododendron. These old north-south orientated ranges are isolated by deep

ravines and characterized by diversified habitats which afford asylum for many peculiar,

often primitive, types of mammals unknown elsewhere in the world today. However,

many of these species are now threatened by logging, over-hunting, trapping, fuel

collecting, mushroom rearing, and overgrazing. Deforestation is now widespread in this

region where critical habitats for many species are becoming fragmental. The plant and









animal species associated with the once broadly interconnected inter-mountain

ecosystems are now fragmented into patterns of disjunctive distributions. More work in

western and southern China, as well as adjacent Myanmar, and in Assam, Bhutan, Nepal,

northern India and Pakistan, to shed more light on species distributions in this area of

great geographic complexity and high relief is clearly desirable (Hoffmann, 2001). The

systematics, geographical distributions, and conservation status of many species in these

rugged and remote regions are poorly understood. Flying squirrels are especially poorly

understood because they occur in deep forest habitats and are nocturnal in habits.

1.1 Evolution of Flying Squirrels

Since Cuvier (1798) separated flying squirrels (=Volant) from the non-volant

squirrels, placing them in the single genus Pteromys, several different revisions of the

classification and the distribution of flying squirrels have been proposed based on

geographical distributions and external structures (Anderson, 1878; Allen, 1940;

Ellerman, 1940; Zahler and Woods, 1997), morphological features (Shaub, 1958;

McKenna, 1962; Mein, 1970; Johnson-Murray, 1977; Thorington, 1984; Thorington and

Heaney, 1981; Thorington and Darrow, 1996; Thorington et al., 1996, 1997, 1998;

Thorington and Stafford, 2001), and biochemical and molecular analyses (Arbogast,

1999; Oshida and Masuda, 2000; Oshida et al., 2000a, 2000b, 2001). By studying the

skull morphology and color variation of the flying squirrels in western Yunnan, Anderson

(1878) put all 29 species of flying squirrels into the same genus, Pteromys, of the family

Sciuridae. Allen (1940) classified flying squirrels as an independent family,

Petauristidae, based on their unique parachute membrane extending from the ankles to

the wrists and characteristics of the skull, such as the short, triangular and slightly raised

postorbital processes, and the distinct depression between the orbits, a result of the large









crepuscular eyes. Ellerman (1940) comprehensively summarized the early evolutionary

history of the flying squirrels in the book The Families and Genera of Living Rodents.

McKenna (1962) categorized the living petauristine sciurids into five major tribes,

Glaucomys, Iomys, Petinomys, Trogopterus, and Petaurisa, on the basis of the

differences of the dentitions, auditory regions, and bacula, paralleling the classification of

the subfamily Sciurinae (Table 1.1). Later on, McKenna et al. (1977) put all flying

squirrels in Pteromyinae instead of Petauristinae because of the membrane.



Table 1.1 McKenna's classification of flying squirrels in Petauristinae
Group Genus
Glaucomys Eoglaucomys, Glaucomys, Pteromys, Petaurillus

Iomys Iomys

Petinomys Aeromys, Petinomys, Hylopetes

Trogopterus Pteromyscus, Belomys, Trogopterus

Petaurista Aeretes, Petaurista, Eupetaurus



Mein (1970) made comparisons between the dentition of fossil forms and of

modern forms of flying squirrels. Based on his study and description of dental

characters, he categorized flying squirrels into three different groups (Table 1.2). The

distinct anatomical structures of the wrist support the hypothesis that flying squirrels and

non-flying squirrels have different phylogenetic histories (Oshida et al., 2000c, 2000d;

Thorington and Darrow, 2001). Corbet and Hill (1992) promoted flying squirrels as a

separate family, Pteromyidae. More recently, at the mammal meeting in Seattle,

Washington, based on the muscular and skeletal features, Thorington (personal comm.,









1998) suggested the possibility that all flying squirrels belong to two groups: Glaucomys-

like forms and Petaurista-like forms, each consisting of different subgroups (Table 1.3).


Table 1.2 Mein's classification, determined from dental characters
Group Genus
I Glaucomys, Eoglaucomys, and Iomys

II Pteromys, Trogopterus, Pteromyscus, Belomys, Aeretes,
Petaurista, and Eupetaurus
III Petinomys, Hylopetes, Aeromys


Table 1.3 The latest estimate of flying squirrels in Pteromystinae
Group name Subgroup and genus
Gs Subgroup I: Glaucomys, Eoglaucomys
Subgroup II: Hylopetes, Petinomys, Petaurillus, Iomys
Subgroup I: Petaurista, Aeretes
Subgroup II: Trogopterus, Belomys, Pteromyscus
Petaurista Subgroup III: Eupetaurus
Subgroup IV: Aeromys
Subgroup V: Pteromys


All extant flying squirrels whether in Petauristinae, a subfamily of Sciuridae

(Nowak, 1991, 1999; Wilson and Reeder, 1992), or in Pteromyidae (Corbet and Hill,

1992) belong to 14 or 15 genera and 37-52 species (Table 1.4) found in both the Old and

New World. Of them, one genus, Glaucomys, is confined to North American evergreen

and deciduous forests, and the others are centered chiefly in the subtropical forests of the

oriental region (Table 1.5). With few exceptions, such as Chakraborty (1981) and

Thorington et al. (1996), who considered Eoglaucomys as a valid genus distinct from

Hylopetes, the current classification of flying squirrels at the generic level is widely

accepted (Bruijin and Uney, 1989; Corbet and Hill, 1992; Wilson and Reeder, 1992).









The taxonomic controversies of flying squirrels at the species level are mainly within the

genera of Hylopetes and Petaurista.


Table 1.4 Comparison of different classifications of flying squirrels
Number of Species
Genus Ellerman Nowak Wilson and Corbet and Hill
1940 1991 Reeder 1992 1991
Petaurista 11 6 8 8
Aeromys 2 2 2 2
Pteromys 4 2 2 2
Glaucomys 2 2 2 2
Hyloptetes 13 8 10 11
S- synonymy of synonymy of
Eoglaucomys 1 -
Hylopetes Hylopetes
Petinomys 11 7 8 8
Petaurillus 3 3 3 2
Aeretes 1 1 1
Trogopterus 1 1 1 1
Belomys 1 1 1 1
Pteromyscus 1 1 1 1
Iomys 1 1 2 3
Biswanmoy- 1 1 1
opterus
Eupetaurus 1 1 1 1
Total 52 37 43 44


However, these classifications are mainly based on the shared primitive features

(= plesiomorphic characters) and as a result there is great confusion. Many species or

forms are frequently referenced as different species, subspecies or synonyms (Allen,

1940; Corbet and Hill, 1991, 1992; Nowak, 1991; Wilson and Reeder, 1992). Despite the

high level of taxonomic, ecological, and morphological information available for some

species of flying squirrels, the phylogenetic relationships of many taxa remain uncertain.

This taxonomic uncertainty is especially true for Chinese flying squirrels.














Table 1.5 Flying squirrels and their distributions
Genus Common name Distribution

Kashmir, northern Indochina, Malay
Petaurista Giant flying squirrel peninsula, Sumatra, Java, Borneo,
Japan, Korea, Manchuria, Taiwan
Aeromys Large black flying squirrel Malaya, Sumatra, and Borneo
SConiferous forest zone of Eurasia
Pteromys Old World flying squirrel (Japan, Finland to Korea)
(Japan, Finland to Korea)
Glaucomys New World flying squirrel Canada, W and E USA, Honduras
Northern India, Thailand, south
Hyloptetes Arrow-tailed flying squirrel China, Indochina, Sumatra, Java,
Borneo, and Naruna Islands
Eoglaucomys Small Kashmir flying squirrel Afghanistan, Pakistan, Kashmir
n s D f fng s l Java, Malaya, Sumatra, Borneo,
Petinomys Dwarf flying squirrel P... S India, Sri Lanka.
Philippines, S India, Sn Lanka
Petaurillus Pygmy flying squirrel Borneo and Malaya

Aeretes Groove-toothed flying squirrel NE China and Sichuan, China

Trogopterus Complex-toothed flying squirrel China, Himalayas and Indochina

Belomys Hairy-footed flying squirrel E Nepal Indochina, Taiwan

Pteromyscus Smoky flying squirrel S Thailand Sumatra, Borneo

lomys Horsfield's flying squirrel Malaya Java, Borneo, Sumatra
Bswanmoyo Namdapha flying squirrel NE India, Southeast Asia
pterus
Pakistan, SW China, North India,
Eupetaurus Woolly flying squirrel Sikkim



1.2 Evolution of Chinese Flying Squirrels

The flying squirrels distributed in China belong to seven genera and 14 or 15

recognized species (Allen, 1940; Corbet and Hill, 1991, 1992; Wilson and Reeder, 1992;

Nowak, 1999). The most comprehensive discussions of Chinese flying squirrels are

found in Allen's (1940) The Mammals of China and Mongolia, Ellerman and Morrison-

Scott's (1966) Checklist of Palaearctic and Indian Mammals, and Corbet and Hill's









(1992) The Mammals of the Indomalayan Region. Chinese flying squirrels belong to four

geographical regions: 1) southwestern China (Tibet, Yunnan, and Sichuan); 2) southern

China including the island of Hainan; 3) northern China; and 4) central China (Table 1.6).

The region favored by most flying squirrels is southwestern China where there are more

than 10 species.

Southwestern China is the main part of the eastern trans-Himalayas. It comprises

a total area of 767,000 km2, stretching from the southeast corner of Tibet through central

and northern Yunnan, western Sichuan, and the hills of the eastern Tibet plateau,

particularly the Hengduan and Min mountain systems. Elevation in this region varies

from below 1,000 m on the valley floors to over 6,000m on the highest snow covered

ridges. The topographical features of this region are very complicated. The limestone

bedrock forms diverse landforms including karst landscapes, sharp peaks, intermountane

basins, rocky gorges, grottos, and underground rivers. This area can be divided into three

great steps with increasing altitudes from low hills at 1,200m in the southeast to high

mountain peaks at 3,000-4,000m in the northwest. The vegetation is highly diversified

and changes progressively from southeast to northwest. The transition of plant

communities in this zone is more altitudinal than latitudinal. The three parallel rivers are

the Yangtze, Mekong, and Salaween and are separated by snow-capped mountains.

Mountains are vertically stratified with distinct vegetation and typically show a complete

spectrum from subtropical evergreen broadleaf forests at lower altitudes to deciduous

temperate broadleaf forests, mixed broadleaf coniferous forests at middle levels, and

coniferous subalpine forests with dense bamboo and rhododendron associated with alpine

meadows at higher altitudes. As a result of the influence of the continental monsoon









from the north and the maritime monsoons from the southwest and southeast, winters are

generally cool and dry while summers are warm and wet.


Table 1.6 Chinese flying squirrels
Genus Species Distribution

Aeretes A. melanopterus NE Hebei and Sichuan

Belomys B. pearsonii Hunan, SW China, Hainan, and Guizhou

Trogopterus T. .\ihin,, Heber, Huber, Yunnan, Sichuan
N. cereus Pakistan, Kashmir to Sikkim (India),
Eupetaurus E. cinereus
Yunnan and Tibet
H. alboniger Sichuan, Yunnan, and Hainan
Hylopetes
H. phayrei Fukien and Hainan

P. alborufus Sichuan, and S and C China, Taiwan

P. elegans Sichuan and Yunnan

P. leucogenys Gansu, Sichuan, and Yunnan
Petaurista
PP. .\xn1thti, Sichuan, Tibet, Gansu, and Yunnan

P. petaurista Sichuan, Yunnan and Fukien

P. philippensis Taiwan, South China and Hainan

P. magnificus Tibet
Pteromys P. volans North China and West China


Southwestern China is the most interesting and remarkable of the Chinese faunal

and floral divisions and has the highest level of biodiversity among Chinese provinces.

More than half of the country's protected or endangered mammals, including 25 species

under the first class list and 29 of the second, and 50% of the country's total flora,

including four first class protected species and 60 of the second class, are found here

(Mackinnon et al., 1996). This region provides an ideal habitat for flying squirrels and it

is possible that the region is the center of the radiation of flying squirrels. However,









because the region is remote and economic conditions are very poor, the local people and

government officially (or unofficially) harvest Yunnan pine, firs, and spruce trees at a

very high rate. As a consequence of the rapidly disappearing pine and spruce trees upon

which flying squirrels feed and which provide important habitats for flying squirrels,

most species are threatened. Eupetaurus, Petaurista and Hylopetes are the taxa most

threatened, and are the genera where most of the species-level taxonomic controversies

exist.

Eupetaurus was previously thought to occur only in northern Pakistan and to be

very rare or even extinct. However, a review of museum specimens suggests a historical

distribution that also includes India, Tibet, Sikkim, and SW China. Zahler (1996) and

Zahler and Woods (1997) documented the continued existence of E. cinereus in northern

Pakistan. Two "skins" (only skins, no skulls) represented in the collection at the

Kunming Institute of Zoology (KIZ) of the Chinese Academy of Sciences have been

identified as Eupetaurus based on the pelage color and external features (Wang and

Yang, 1986; Corbet and Hill, 1991, 1992). I have found no further evidence of its

presence or distribution in southwestern China. Comparisons between Eupetaurus

cinereus from Pakistan and the "skins" collected in SW China are important in order to

understand the radiation and taxonomy of the genus Eupetaurus and to clarify the

taxonomic and phylogenetic status of Eupetaurus within flying squirrels.

The separation of Eoglaucomys from Hylopetes in the western trans-Himalayas is

based on dental differences (Ellerman, 1947, 1963; Chakraborty, 1981; Thorington et al.,

1996). Some authors do not accept Eoglaucomys as a valid genus (Wilson and Reeder,

1992; Corbet and Hill, 1992) and consider that H. alboniger and H. phayrei are the









species of Hylopetes distributed in China. I believe that an examination of the validity of

Eoglaucomys based on molecular analysis rather than morphometric is critical for the

clarification of the taxonomy of Hylopetes group. As part of this analysis I will examine

Hylopetes populations distributed in the eastern trans-Himalayas. The validity of H.

electilis in Hainan, China, and the phylogenetic relationships among H. electilis, H.

alboniger and H. phayrei are as well interesting topics to elucidate the phylogenetic

relationship of Chinese flying squirrels.

Petaurista is a polymorphic genus with considerable variation in pelage coloring.

More than 10 species of Petaurista have been recognized (Corbet and Hill, 1991, 1992;

Nowak, 1991, 1999; Wilson and Reeder, 1992; Zhang et al. 1997; Wang, 2002). Of

various species included within this genus, it is difficult to resolve the numerous

intraspecific and interspecific taxonomic and phylogenetic problems. Various authors

still express serious doubts concerning the validity of P. xanthotis, P. philipensis, P.

hainana, and P. yunanensis (Nowak, 1999; Wilson and Reeder, 1992; Corbet and Hill,

1992, Wang, 2002). To resolve the affinities of these complex taxa, a comprehensive

revision to clarify the relationships among these forms is essential because the available

morphometric and molecular data are too scanty to throw any light on the problems.

1.3 Objectives of This Study

Most recent phylogenetic studies have concentrated on the analysis of molecular

data, particularly DNA sequences. But the combination of both molecular and

morphological analyses in systematics has been attracting more and more attention from

both systematists and evolutionary biologists (Minelli, 1998; Schierwater and Kuhn,

1998; Benton, 1998; Hugot, 1998; Flynn and Nedbal, 1998; Baker et al., 1998; Smith,

1998; Smith and Patton, 1991; Staongnhope et al., 1998; Barome et al., 1998; Ruedi et









al., 1998; Shoshani and McKenna, 1998; Goodman et al., 1998; Huelsnebeck et al.,

1996). Despite the increasing use of DNA sequence data, morphometric analysis still

remains one of the most useful techniques available to investigate phylogenetic

relationships between taxa (Sanderson et al., 1993).

Flying squirrels exhibit a high level of geographic variation in morphological

characters such as pelage color, cranium morphology and dental structures.

Opportunities for studying flying squirrels traditionally have been limited to studying

specimens represented in museums and institutes. In part, this is reflected in the

continuing problems of flying squirrel classification. Despite the agreements or

disagreements on the taxonomic status of different nominate genera and species in the

literature, flying squirrels have not been the main subject of any comprehensive

systematic revision. The phylogenetic analysis of mtDNA in rodents has centered largely

on murids (Ferris et al., 1983; Smith and Patton, 1991). The overall phylogenetic

relationship of flying squirrels remains poorly understood because they are nocturnal,

elusive, and difficult to capture. No attempt has been made to investigate the systematics

of Chinese flying squirrels.

There appear to be similar patterns of speciation and distribution of flying

squirrels in the eastern and the western trans-Himalayas. A comprehensive and

comparative analysis of the phylogeography and systematic relationships of flying

squirrels in the eastern and the western trans-Himalayas will be extremely valuable. In

this study, molecular analyses of partial sequences of mitochondrial cytochrome b gene

and morphometric study of skull data were performed for the flying squirrels distributed

in various areas of China and Pakistan. I also discuss the biogeographic history and









phylogenetic relationships among them as well as the taxonomic status of the groups or

forms in Eupetaurus, Petaurista, and Hylopetes (Eoglaucomys) from SW China and

Pakistan. The objectives of this study are to seek the answers for the following questions:

1. Search for confirmation of the continued presence of the "lost species" --
Eupetaurus cinereus, in SW China and investigate the taxonomic status of genus
Eupetaurus.

2. Examine the taxonomic and phylogenetic relationship between Hylopetes and
Eoglaucomys, and the taxonomic validity ofH. electilis.

3. Determine if the Petauristapetaurista (sensu lato) groups in different localities
along the eastern and western trans-Himalayas form a single species P. petaurista
(albiventer), or a complex of species. Confirm the validity ofP. xanthotis, P.
philipensis, P. hainana, and P. yunanensis and reconstruct the phylogenetic
relationships among Petaurista groups distributed in SW China and Pakistan.

4. Investigate what the systematic relationships are among Chinese flying squirrels,
including Petaurista, Eupetaurus, Trogopterus, Hylopetes, Eoglaucomys,
Belomys, and Pteromys.

In the remaining chapters, I will attempt to answer the above questions. In

Chapter 2, I will briefly discuss the techniques and methods of phylogenetic analysis used

in the present study. Chapter 3 is about the phylogeny and zoogeography of Eupetaurus

inferred from an analysis of the cytochrome b gene. Chapter 4 will focus on the

phylogenetic relationships of Peaturista distributed in SW China and Pakistan based on a

molecular analysis and morphometric study. The phylogenetic relationship

between/within Hylopetes and Eoglaucomys is discussed in Chapter 5. Chapter 6 is a

discussion of the systematics and biogeography of the trans-Himalayan flying squirrels.

The results and the tentative future work are summarized in Chapter 7.














CHAPTER 2
METHODS AND TECHNIQUES OF PHYLOGENETIC STUDY

There is little doubt that the introduction of molecular techniques has already

significantly enhanced the capacity to address fundamental questions in phylogenetic

relationships and will make an even greater contribution in future. Theoretically the

phylogenetic analysis of molecular level can provide different and well-corroborated

estimates of phylogenetic relationships. It is possible to compare and contrast

phylogenies based on morphological data with biochemical data such as genome data,

globins, or cytochrome b (Benton, 1998). Messenger and McGuire's (1998) study of

cetaceans showed that combined analyses of the morphological and molecular data

provide a well-supported phylogenetic estimate consistent with that based on the

morphological data alone. But, intraspecific variation is ubiquitous in systematic

characters, including morphology, allozymes, and DNA sequences. Sometimes the

analysis of one data set (i.e., molecular) provides one highly corroborated phylogeny;

whereas analysis of another data set (i.e., morphological) provides a different highly

corroborated phylogeny. The phylogenetic inferences based on characters derived from

morphology are corroborated by molecular evidence in some mammal groups (Flynn and

Ndebal, 1998; Shoshani and McKenna, 1998). However the integration of distinct data

sets, such as the molecular data and morphological data in phylogenetic analysis, has

caused considerable debate among evolutionary biologists in recent years (William and

Ballard, 1996; Wiens, 1998a, 1998b; Wiens and Servedio, 1998).









2.1 Molecular Study

The molecular biological revolution of the last several decades has reached into

every conceivable corner of biological investigation (McKenzie and Batterham, 1994;

Poinar, 1999). Molecular sequences are information-rich and there are many different

ways of extracting useful information from them. Molecular phylogenetic analysis has

been developed to infer the branching pattern of different taxa from their common

ancestor and the sequential dates when such branching events or cladogenic speciation

events occurred. The results of DNA sequence data have been successfully used to

reconstruct the phylogenetic relationship in rodents, which provides strong support for

the monophyly of rodents, a conclusion that has considerable support from morphology

(Honeycutt and Adkins, 1993; Luckeet and Hartenberger, 1993; Frye and Hedges, 1995).

2.1.1 Mitochondrial Cytochrome b Gene

The molecular methods used to detect genetic variation within and between

species have led to exciting advances in studies of historical biogeography. Molecular

survey of DNA sequence data is one of the popular techniques used to quantitatively

measure genetic variations among taxa (Storfer, 1996). In principle, any part of the

genome can be used for DNA studies. Over the past few years, a variety of different

observations have challenged some notions of mitochondrial biology, such as the variable

rates of mitochondrial DNA (mtDNA) sequence evolution among taxa (Rand, 1994).

However mtDNA is by far the most commonly used methodology in phylogenetic

analysis and evolutionary biology since it features several advantages that make it the

usual choice for population-level questions (Meyer, 1994). For example, mtDNA

sequence data can provide the perspective of a maternally inherited marker on patterns

and levels of geographic structuring and the analyses of polymorphic restriction sites. On









the basis of the gene sequences of mtDNA, researchers obtained valuable information on

evolutionary relationships and divergence times for mammalian subspecies, species, and

higher level taxa (Ferris et al., 1983; Brown et al., 1979; Zhang and Ryder, 1993;

Avise,1987, 1994; Janke et al., 1994; Miyamoto and Fitch, 1995; Wettstein et al., 1995;

Miyamoto, 1996).

To extract historical information from molecular data, it is important to

understand the dynamic nature of the sequences and know how molecular sequences

change over geological time. Variation of evolutionary rates occurs at several levels in

DNA sequences: among the sites (e.g., the second position vs. the third position), among

the kinds of substitutions (e. g., transition vs. transversion, or silent vs. replacement), and

among regions of the molecule (Ferris et al., 1983; Avise, 1994). At the nucleotide level,

which is the most fundamental level for any mutation, there are 12 possible changes, with

four being transitional changes and eight being transversional changes. In general, recent

divergences are related to rapidly evolving changes and older divergences are related to

slowly evolving changes (Graybeal, 1993). These patterns hold for cytochrome b gene,

with studies at the population and species level using all informative characters (Smith

and Patton, 1991; Moritz, 1994).

The mitochondrial cytochrome b gene is one valuable molecule for evolutionary

relationship reconstruction among populations, species, and higher taxa in animals and

has been used extensively in molecular phylogenetic studies. Because it is slow in terms

of amino acid substitutions and the rate of evolution for silent substitutions at the third

codon positions is similar to that of other mitochondrial genes, the cytochrome b gene

that facilitates the alignment of sequences permits comparisons among widely divergent









taxa (Kocher et al., 1989; Irwin et al., 1991). Using the partial or complete sequences of

the mitochondrial cytochrome b gene, researchers and scientists successfully determined

the interspecific phylogenies in a wide range of mammalian taxa, such as the

phylogenetic analysis of relationships in bats and some murids (Wright et al., 1999;

ludica, 2000, Barome et al., 1998), and genetic analysis of intrageneric relationships of

some squirrels and flying squirrels (i.e., Petaurista, Glaucomys) (Hafner, et al., 1994;

Oshida and Obara, 1992; Oshida and Yoshida, 1999; Oshida et al., 1996, 2000a, 2000b,

2001; Arbogast, 1999).

2.1.2 Phylogenetic Analysis

As far as phylogeny is concerned, finding the best trees and using the best tree to

reconstruct the phylogenetic relationship are main objectives. With the development of

the polymerase chain reaction (PCR), it is possible to recover genetic information from

even severely degraded tissues, such as hair, old skin, and excrement. The widespread

successful application of molecular analysis in animal phylogenetic reconstruction and

evolutionary biology can be attributed partly to the discovery of this versatile PCR

technique. The use of DNA from museum skins can reconstruct phylogenetic trees

among organisms and develop conservation policies for endangered species.

A typical phylogenetic analysis involves a bewildering array of decisions,

including what type of data to sample (molecular or morphological), what phylogenetic

methods to apply (distance, likelihood, and parsimony), whether or not to order or weight

characters, and which taxa and characters to include or exclude. The common methods

used for molecular phylogenetic reconstruction include distance method (e.g., the

unweighted pair-group method with arithmetic mean (UPGMA), the neighbor-joining, or









the more complicated Fitch-Margoliash method), parsimony method, and likelihood

method.

2.1.2.1 Parsimony method

Parsimony is a character-based analysis. Each character is considered

independent of its neighbor, but only informative sites are considered during the

calculation. The parsimony method calculates only the order of the branches of the tree

and does not give branch-length estimates. Maximum parsimony is the easiest and most

practical to implement, and when evolutionary times are short, maximum parsimony,

maximum likelihood, and compatibility tend to yield the same estimated phylogeny

(Crandall et al., 1994). The accuracy of parsimony depends largely on how polymorphic

characters are coded and the sample size (individuals per species), which is usually an

important component of phylogenetic accuracy (Wiens and Servedio, 1998). The

advantage of this method is that it uses a logical model and the calculations are rapid.

However, a major shortcoming of the method is that a large amount of data that are not

informative are discarded.

2.1.2.2 Likelihood method

In likelihood method every site of sequences is considered and the likelihood of

the replacement of a particular nucleotide from pools of nucleotides is calculated. It is

based on random similarity rather than on common descent, and increases with increasing

divergence between the outgroup and the ingroup taxa (Milinkovitch and Lyons-Weiler,

1998). Likelihood method considers every site including unchanged sites and gives an

accurate estimate of branch lengths. The maximum parsimony method can be viewed as

an approximation to the maximum likelihood method, which has been used extensively









for parameter estimation in molecular data analysis. The disadvantage is that this method

is very time-consuming.

2.1.2.3 Distance method

Distance methods calculate the total number of changes, scored according to the

type of change, between every pair of sequences in the alignment. The method is based

on distance to calculate branch length that visually represents the number of changes

between sequences with consideration of unchanged characters and ambiguous

alignments.

In practice, the choice among all methods is a serious concern because the

intraspecific and interspecific variation is so widespread that the application of different

methods can give radically different trees for the same data set. Even subtle differences

in how polymorphism is treated can have a significant impact on tree topology (Wiens,

1995; Wiens and Servedio, 1998). UPGMA unweightedd pair group method using

arithmetic averages) is the most commonly used clustering method, in which the

averaging of the distances is based on the total number of taxa in the cluster analysis

(Swofford et al., 1996; Swofford, 2000). Like the likelihood method, it generally gives

the most accurate results compared to other techniques (Wiens and Servedio, 1998; Rohlf

and Wooten, 1988).

In this study, the partial mitochondrial cytochrome b genes (315 420 bp) were

amplified with PCR technique. Some sequence data are retrieved from the GenBank of

NCBI (National Center for Biotechnology Information). Maximum parsimony (MP),

neighbor-joining (NJ), and UPGMA methods were used to reconstruct the molecular

phylogeny of Chinese flying squirrels to determine whether the specific genetic structures









and geographic patterns are correlative within each group and to reveal their systematic

relationships.

2.2 Morphometric Study

Most recent phylogenetic studies focus mainly on the analysis of molecular data,

but tracing changes in morphological characters is also an important way to evaluate the

distribution of the characters on which those taxonomic units are based. The quantitative

description, analysis, and interpretation of shape and shape variation in biology are a

fundamental area of research. Taxonomic modifications and reinterpretation of

morphological characters in the context of the molecular tree requires further scrutiny.

Studies of morphology contribute in different ways to the understanding of

evolutionary patterns and processes. Ideally a functional morphological study provides

information about the interdependency of characters, and also a transformation scheme of

characters that is biomechanically feasible (Galis, 1996). Morphometric studies have

applied univariate analyses to differentiate morphotypes (Lee and Cheng., 1996), and

multivariate analyses were used to produce an overview of the associations between

variables and species patterns (Gauch, 1982).

The common multivariate analyses include principal components analysis (PCA),

discriminant function analysis, and cluster analysis (Manly, 1994). In multivariate

analysis, the selection and the number of the characters used are critical since the

interpretation of the results is based on them. Sometimes, raw data probably are

transformed by logarithm function to reduce the skewness of original data, make their

variances homogeneous, and correct the heterogeneity in magnitude of variables. If there

are adequate sample sizes, multivariate analyses allow one to make overall tests as well

as a proper posterior test of sets of variables.









Discriminant function analysis is to portray the relationships based on the

canonical variables, and to develop linear models of variables to maximize the separation

of groups. The discriminant functions are extracted from the between-group covariance

matrix standardized by the within-group covariance matrix. Mahalanobis distances are

used to measure phenetic distances, which are the indices to evaluate the overlap between

pairs of populations, and to transform original distances to maximize power of

differentiations between individual specimens to describe the relationship among species.

Principal components analysis (PCA) is based upon the variance-covariance

matrix of the log-transformed variables. It is performed to identify variables that account

for maximum variation in data and to produce a smaller number of uncorrelated factors

that are linear combinations of original variables. The first axis lies in the direction of the

greatest variability between the sample means, and each succeeding axis lies in the

direction of the next greatest variability. Factor loadings, describing the relative

contribution of each variable to the principal components, are used to compare the

morphological structures between samples.

The discrepancy between cluster analysis and other analysis becomes

understandable and less important when characteristics of various statistical techniques

are considered (Sneath and Sokal, 1973). Cluster accurately represents distance between

adjacent groups. Euclidean distance between centroids and an unweighted pair-group

method using arithmetic average (UPGMA) clustering algorithm is usually applied to

generate a phenogram, depicting morphological relationships among taxa.

In this study, all related specimens were pooled together for univariate analysis,

which is restricted to one-way analysis of variance (ANOVA) and F-test to calculate the






21


mean, standard deviation (SD), and the significant test among variables for species. The

multivariate analyses including discriminant function analysis, principal components

analysis and cluster analysis are used to determine how the groups are related when all

the characters are considered simultaneously. Because these multivariate methods are

unable to deal with missing data, or else they deal with missing information in a rather

arbitrary manner (Rohlf and Marcus, 1990), in this study, only those variables that are

available in all sampled groups are selected for further analysis.














CHAPTER 3
PHYLOGENY AND BIOGEOGRAPHY OF EUPETAURUS

3.1 Introduction

Eupetaurus cinereus, the woolly flying squirrel, is one of the most unusual and

least known species in the world (Chakraborty and Agrawal, 1977; Zahler, 1996; Zahler

and Woods, 1997). Because observations in the wild have been precluded by the rarity of

specimens, virtually, nothing is known of its food habits, reproduction, distribution,

habitat preference, behavior, anatomy, or systematics. It is considered among the most

endangered mammals (IUCN No.: EN 8 A2ce, B 1+2cd1) (Baillie and Groombridge,

1996), probably the most threatened of all flying squirrels.

Eupetaurus is a crucial genus in the phylogenetic study of flying squirrels. The

cheekteeth of E. cinereus are hypsodont and share many characteristics with rodents that

have high-crowned teeth with flat surfaces, such as capromyids (hutias) from the West

Indies, thryonomyids (cane rats) from Africa, and New World echimyids (spiny rats),

rather than other members of Sciuridae (McKenna, 1962). The highly specialized

grinding teeth feature many advantages that allow it to live in relatively treeless rocky

areas and possibly supplement its diet during the winter months by eating some abrasive

material. Since the structure of teeth is so divergent from other flying squirrels, Schaub

(1958), and Grasse and Dekeyser (1955) placed Eupetaurus in its own rodent family,


1 EN: the category of threat is endangered; A2ce: Population decline projected in the
future; Bl+2cd: small distribution and decline because of the severely fragmented
population and habitat; C2a: Small population size and continuing decline by fragmented
habitat.









Eupetauridae. By comparing the dentition with the giant flying squirrel (Petaurista

.\X uthri,), McKenna (1962) proposed that Eupetaurus is a very high-crown flying

squirrel and demonstrably a petauristine sciurid on the basis of a large number of

characters other than the dentition, and returned it to the sciurid subfamily Petauristinae.

Its present taxonomical status is as follows:

Order Rodentia Bowdich, 1821

Suborder Sciuromorph Brandt, 1855

Superfamily Sciuroidea Gill, 1872

Family Sciuridae Gray, 1821

Subfamily Petauristinae Simpson, 1945

Genus Eupetaurus Thomas, 1888

Species Eupetaurus cinereus Thomas, 1888

Eupetaurus had been considered to be very rare or even extinct until a live

specimen was captured in northern Pakistan in 1994, which confirmed the existence of

woolly flying squirrel. Zahler and Woods (1997) summarized are of the available

information on the ecology and conservation of Eupetaurus in Pakistan. Eupetaurus in

Pakistan is limited to the region of the Sai Valley in the central Indus River Valley near

Nanga Parbat, the most westerly main massif in the Himalayan Range. It lives in caves

of high alpine zones that are characterized as high, cold desert dominated by Artemisia

and Juniperus above 2,000 m, and apparently shows quite unique ecological adaptations

for surviving in regions that are inhospitable to any other flying squirrels. The present

estimate of the number living in Pakistan is between 1,000 and 3,000 (Zahler and Woods,

1997).










Eupetaurus was historically found from Pakistan, to India, Tibet, Sikkim and SW

China, based on 13 available museum specimens in London (British Museum of Natural

History), Netherlands (Laiden Museum), Bombay (=Mumbai, Bombay Natural History

Society), Calcutta (Indian Museum), and China (Kunming Institute of Zoology, China,

KIZ), which confirms presence of Eupetaurus from Pakistan to China (Figure 3.1 and

Table 3.1).


Table 3.1 Thirteen historical and 2 recent specimens of Eupetaurus
Locality Museum ID Collector and date Additional information
1 Tibet, China LM: 19524 Anderson, J. 1878 Skin and skull
2 Yunnan, China KIZ: 73372 Wang, Y. X. 1984 Skin only
3 Yunnan, China KIZ: 73921 Wang, Y. X. 1984 Skin only
4 Sikkim IM: 19103 Gill, J. S. Skin only
5 Gilgit, Pakistan IM: 9492 M. Miles, 1887 Skin and skull
MacPherson, M. A. .
6 Gilgit, Pakistan BNHS: 7107 MacPherson, M. A. Skin only
1916
The collection site is
SSaiNalah, : 71 Lorimer, Lt. C. D. not associated with the
Pakistan 1924 specimen. Skin is in
very poor shape.
8 Chitral, Pakistan BNHS: 7109 Fulton, H. T. Skin only
Sai Valley, MajL. .
9 Valley, BNHS: 7110 a. Skin only
Pakistan MacKenzie, 1924
Co-type with a skin and
10 Astor, Pakistan MH 1 Purchased by fragmentary snout. The
10 Astor, Pakistan BMNH: 88.9.29.1
Lydekker, R. 1879 location is said to be
from the Astor District.
Gyantse Bazar,
11 se Bazar BMNH: 23.11.10.2 No record Purchased from Tibet
Tibet________
12 Gilgit, Pakistan No record No record Partial skin
13 Chitral, Pakistan No record No record Partial skin
14 Gilgit, Pakistan UF: 26583 Woods, C. 1996 Partial skin
15 Gilgit, Pakistan UF: 28620 Woods, C. 1996 Skin and skull
Note: BNHS: Bombay Natural History Society, India; BMNH: British Museum
of Natural History, England; IM: Indian Museum in Calcutta, India; LM:
Leiden Museum, the Netherlands; KIZ: Kunming Institute of Zoology,
China.









However, it is not possible to establish with certainty that all specimens are the

same species, since the majority of the specimens available were collected at the

beginning of the last century and some specimens are incomplete and lack skulls. The

collecting sites of some specimens are not conclusively associated with individual

specimens since the data were recorded solely according to the description of dealers.

Therefore taxonomic and phylogenetic studies have been hampered by questionable

records and the paucity of Eupetaurus specimens in collections. For example, questions

still remain concerning the exact collecting site of the two skins from China represented

in the KIZ and the specimen from Sikkim. The phylogentic status of Eupetaurus and the

phylogenetic relationships between Eupetaurus and other flying squirrels, such as

Petaurista, are not yet well understood.

In this study, Eupetaurus specimens from different localities were compared with

new specimens obtained from Pakistan by analyzing the sequence data from

mitochondrial cytochrome b gene using parsimony and distance methods. Here I

compare the taxonomic status of Eupetaurus populations in Pakistan and SW China,

determine how much variety exists within the genus Eupetaurus along its extensive

distribution, and reconstruct the phylogenetic relationship between Eupetaurus and

Petaurista.

3.2 Materials and Methods

3.2.1 Samples

Among those 15 known Eupetaurus specimens represented in museums and

institutes, some skins were in very poor shape and without the corresponding skulls;

some records were not precisely associated with the specimens; and some specimens

were only purchased or shipped by the dealers or a third party. Of 10 available skin









samples obtained from museums and institutes, eight specimens have reliable data

associated with them. The rest were described mainly based on comments from either

the medicinal dealers or the intermediate persons who shipped or purchased the

specimens from different locations.


Table 3.2 Specimens ofEupetaurus and other flying squirrels
examined in this study*
Species Code Collecting locality Museum ID
ECL Tibet, China LM: 19524
ECK1 YUNNAN, CHINA KIZ: 73372
ECK2 Yunnan, China KIZ: 73921
ECI Sikkim IM: 19103
ECB1 Gilgit, Pakistan BNHS: 7107
E. cinereus
ECB2 Chitral, Pakistan BNHS: 7109
ECB3 Sai Valley, Pakistan BNHS: 7108
ECF1 Gilgit, Pakistan UF: 26583
ECF2 Gilgit, Pakistan UF: 28620

ECD Pakistan Sequence provided by Dr.
Roth

Other flying squirrels used in this study
PPF Gilgit, Pakistan UF ID: 28236
P.petaursta PPY Yunnan, China KIZ: 353209

P. .\x n,'ii' PTK Gansu, China NBI ID: 85063

G. volans GV Tennessee, US Sequence #: AF063066

Note: The abbreviations of the museums and Institutes see Table 3.1.
NIB: Northwestern Biological Institute, China


In this study, two samples (ECB3 and ECI) from Pakistan and Sikkim (Table 3.1)

were not included because of their highly degraded sequences. The samples that are in

good condition and recorded by collectors with certainty were used for molecular









analysis. The detailed information ofEupetaurus and other flying squirrels examined is

given in Table 3.2. Dr. Louise Roth, who is an associate professor of biology at Duke

University, provided the sequence data for the sample ECD from Pakistan.

3.2.2 Methods

3.2.2.1 Mitochondrial DNA isolation

Total DNAs of all samples used were extracted from dry skins using the DNeasy

tissue kit (QIAGEN Inc., Valencia, CA91355-1106) and the protocol for animal tissue

recommended by the manufacturer. Initially, 180 |pl of buffer ATL and 20 |pl of

proteinase K (20 mg/ml) were added to the 2 ml tube containing the decalcified material.

The sample was mixed and placed into a 550C H20 bath for 48 hours. After vortexed for

15 seconds, 200 apl of buffer AL was added. Then it was heated at 700C for 10 minutes.

After added 200 apl of 100% ethanol, the sample was applied to a Dneasy tissue kit-mini

column. During the elution step, 100 apl of buffer AE was added. After incubated at

room temperature for 1 minute, it was centrifuged and stored at -40C for PCR.

3.2.2.2 PCR amplification

The following primers were used to amplify the partial nucleotide sequence (315-

402 bp) of the mitochondrial DNA (mtDNA) with polymerase chain reaction (PCR) at

the interdisciplinary center for biotechnology research (ICBR), University of Florida

(UF), and Kunming Institute of Zoology, Kunming, China:

L14725 5'-CGA AGC TTG ATA TGA AAA ACC ATC GTT G-3'

L14841 5'-AAA AAG CTT CCA TCC AAC ATC TCA GCA TGA TGA AA-3'

H15149 5'- AAA CTG CAG CCC CTC AGA ATG ATA TTT GTC CTC A-3'.









Primer names correspond to the light (L) and heavy (H) strand and the 3' end-

position of the primers in the human mtDNA sequence (Anderson et al., 1981). All

primers were synthesized at either ICBR of UF or Shanghai of China.

The 25 |pl of PCR reaction mixture contained 2 pLl 10ng of genomic DNA, 2.5 pl

of each primer (H15149 and L14725 or L14841), 3.0 pl 10x PCR buffer (100 mM Tris-

HC1, pH 8.3 at 250C, 500 mM KC1, 15 mM MgC12, 0.01% gelatin), 4.0 pl of dNTPs (10

mmol), 3.0 pl MgCl2 (25 mmol), and 0.2 pl Tag (Sigma tag DNA polymerase (Sigma

Chemical CO., St Louis, MO 63178)). The cycling program in Table 3.3 was used for

PCR amplification.



Table 3.3 Cycling program of PCR amplification
Step Temperature (C) Time Cycles
Initial denaturing 94 5 minute 1
Denaturing 94 1 minute
Annealing 55 1 minute 38
Extension 72 1 minute 5 second
Final extension 72 5 minute 1


PCR products were purified with the Qia-quick PCR purification kit protocol

(QIAGEN Inc., Valencia, CA91355-1106). Automatic sequencing was performed with

an automated DNA sequencer at University of Florida. The variant sites of sequences

were rechecked by comparing the four-color electromorph of sequencing data against the

computer results.

3.2.2.3 Sequence analysis

Cladistic analysis was performed using the phylogenetic analysis using parsimony

(PAUP version 4.0) (Swofford, 2000). Both distance and parsimony methods were

applied for phylogenetic analysis to infer gene genealogies of mtDNA. The maximum









parsimony (MP) method using the branch and bound search algorithm (Penny and

Hendy, 1986) with the 50% majority-rule consensus, the neighbor-joining (NJ) method

(Nei, 1987; Saitou and Nei, 1987), and the UPGMA method (Swofford et al., 1996) were

used to reconstruct the phylogenetic trees. In NJ method, numbers of nucleotide

substitutions per site were estimated for multiple substitutions by the Kimura's two-

parameter method (Kimura, 1980). Branch-and-bound search was performed to ensure

that all minimum-length trees were identified (Zhang and Ryder, 1994). If all characters

change sufficiently slowly, they may be equally weighted during phylogenetic inference,

even though they do not change actually with equal probability (Felsenstein, 1981,1983,

Graham et al., 1998). The MP trees were generated by equal weighted parsimony and the

bootstrap values (Felsenstein, 1985) were derived from 1000 heuristic replicates.

Quantitative pairwise comparisons between all taxa under the two-parameter

model of Kimura (1980) were made for the partial cytochrome b gene sequences. The

proportions of nucleotide substitutions between all pairs of sequences were calculated,

including the percentage of sequence divergence within and between taxa, the ratio

between the transitions and transversions, and the transverstional substitutions at the third

codon positions between taxa. Relying on dates of divergence estimated from fossil

material for a number of mammalian taxa, Irwin et al. (1991) thought that the average

rate of sequence divergence at third positions of the cytochromoe-b gene of mammals is

about 10% per million years. But the relative rate of molecular evolution in rodents has

been estimated to be ca. 1.5-2 times faster than that of other mammalian lineages

(Britten, 1986; Dewalt et al., 1993; Li et al., 1990). In this study, the divergence time

between taxa was estimated according to the transverstional substitution rate at the third









codon positions of mammalian cytochrome b gene proposed by Irwin et al. (1991), which

was 0.5% per million years.

Petauristapetaurista (albiventer) from Pakistan and NW China, and P. .\xmiii\

from NW China, which had been considered as the close relatives of Eupetaurus, were

used for phylogenetic analyses. Glaucomys volans from North America was used as the

out-group for phylogenetic reconstruction. The DNA sequence data of G. volans were

quoted from GeneBank that is submitted by Arbogast (1999).

3.3 Results

The specimens of woolly flying squirrels collected around the turn of the century

were mostly from the general region of Gilgit in the area of the confluence of the

Himalayan, Karakoram, and Hindu Kush mountain ranges in northern Pakistan. Because

some samples were collected more than 100 years ago and some are in very poor

condition, the recovered sequences were short and fragmental, usually between 300 to

400 base-pair longs.

3.3.1 Phylogenetic Relationship of Eupetaurus between the Eastern and the Western
Trans-Himalayas

The partial sequences (389 bp) of cytochrome b gene were successfully

determined for seven Eupetaurus samples. The genetic differences obtained from the

pairwise comparison (Table 3.4) separated the woolly flying squirrels as two distinct

groups. The first group was the eastern tans-Himalayan group (Eupetaurus I) including

samples from Tibet (ECL) and Yunnan (ECK1 and ECK2) in China. The second group

was the western trans-Himalayan group (Eupetaurus II) including samples from Chitral

(ECB2) and Gilgit (ECB 1, ECD, ECF1, and ECF2) in Pakistan. The genetic difference

between these two groups was about 12%. The maximum parsimony (MP) and UPGMA










analysis based on all Eupetaurus individuals generated the similar branching in trees, all

of which contained the same two major clades (Figure 3.2 and Figure 3.3). In MP

analysis, only one most parsimonious phylogenetic tree was produced with high bootstrap

value (99 100%).



Table 3.4 Percentage differences of Eupetaurus and Petaurista based on the
pairwise comparisons of cytochrome b gene (390 bp)
ECL ECK ECK ECB ECB ECF ECF ECD PPY PPF PT
ECL ECD PPY PPF PTK
1 2 1 2 1 2
ECL 3.8 3.8 8.4 11.36 11.0 11.0 11.0 16.7 15.9 17.0
ECK1 9/6 0 13.4 14.0 13.3 13.3 13.3 17.9 17.2 19.4
ECK2 9/6 0 13.4 14.0 13.3 13.3 13.3 17.9 17.2 19.4
ECB1 25/6 37/13 37/13 3.6 3.8 3.8 3.8 17.1 16.2 17.5
ECB2 32/11 42/16 42/16 7/4 0 0 0 18.1 17.0 18.7
ECF1 32/11 41/11 41/11 8/6 0/1 0 0 16.9 16.7 19.1
ECF2 32/11 41/11 41/11 8/6 0/1 0 0 16.9 16.7 19.1
ECD 32/11 41/11 41/11 8/6 0/1 0 0 16.9 16.7 19.2
PPY 39/26 44/26 44/26 36/30 42/27 41/25 41/25 41/25 5.2 14.0
PPF 38/24 43/24 43/24 33/30 38/27 40/25 40/25 40/25 18/2 12.1
PTK 41/26 48/28 48/28 35/32 40/32 46/29 46/29 46/29 43/12 37/10
Note: Data below the diagonal are the numbers of nucleotide substitutions,
transitions vs. transversions. Data above the diagonal represent the
genetic differences between samples.



3.3.2 Phylogenetic Analysis between Eupetaurus and Petaurista

Although P. petaurista had been thought as the closest relative of Eupetaurus for

long time, Mckenna (1962) regarded P. .\inlhitii as the closest living relative of E.

cinereus for their similar dental structures. The genetic discrepancies between

Eupetaurus and Petaurista based on the pairwise comparisons of cytochrome b gene

were not consistent with Mckenna's hypothesis (Table 3.4). The genetic difference

between P. .\inll,,ti\ and Eupetaurus was 17.0 19.4%, which was higher than that









between P. petaurista and Eupetaurus (16.7 18.1%). The phylogenetic tree did not

support his conclusion either, in which all samples were clustered into two genetic clades

(Figure 3.4).

The estimated dates of divergence among mtDNA clades of Eupetaurus and

Petaurista were calculated based on their rates of divergence for the third codon positions

of cytochrome b gene (Table 3.5 and Table 3.6). The approximate divergent time

between the eastern and the western trans-Himalayan Eupetaurus was about 10 million

years ago, and approximately 29.2 32.2 million years ago between Eupetaurus and

Petaurista.



Table 3.5 Transversional substitutions at the third codon positions of cytochrome
b gene between Eupetaurus and Petaurista (based on 390 bp)
Eupetaurus I Eupetaurus II PPF PPY PTK

Eupetaurus I 5.4 14.6 14.6 14.6
Eupetaurus II 7 15.4 15.4 16.1
PPF 19 20 1.0 6.1
PPY 19 20 1 5.4
PTK 19 21 8 7

Note: Data below the diagonal are the numbers of transversions at the third
codon positions. Data above the diagonal represent the
transversional percentage difference between samples.


With Glaucomys volans as the outgroup taxon, the phylogenetic relationships

among Eupetaurus, P. petaurista and P. .\xiiiti\ were reconstructed using maximum

parsimony and neighbor-joining methods. Three major clades were formed in both MP

and NJ trees (Figure 3.5 and Figure 3.6). The first dichotomy isolated Petaurista

including PPY, PPF, and PTK from Eupetaurus group. Then all individuals of









Eupetaurus were clustered as two groups: one was the Pakistan Eupetaurus (ECF1,

ECF2, ECB1, ECB2, and ECD); another was the Chinese Eupetaurus (ECK1, ECK2, and

ECL).



Table 3.6 Estimated divergent times among Eupetaurus and Petaurista
based on a rate of divergence for the third codon positions of
mammalian cytochrome b gene of ca. 0.5% *106 years
Estimated date of
mtDNA clades
divergence (1.0*10 years)
P. petaurista (albiventer) 29.2
Eupetaurus I vs. P. \.iinlhtii 29.2
Eupetaurus II 10.8
Eupetaurus II vs. P. petaurista (albiventer) 30.8
P. .x\,1,aiii 32.2
P. petaurista vs. P..iixnhtii 10.8- 12.2


3.4 Discussion

3.4.1 Phylogenetic Status of the Population of Eupetaurus in the Eastern Trans-
Himalayas

The available morphological comparisons and taxonomic studies of Eupetaurus

were solely based on the pelage characteristics and the dental forms of the specimens

represented in museums and institutes. The two specimens collected in the deep gorge

country of SW Yunnan near the Thailand, Burma, and Tibet border by KIZ were

recognized as E. cinereus on the basis of the pelage color and the feet (Wang and Yang,

1986; Corbert and Hill, 1992; Zahler and Woods, 1997), which are externally similar to

those in Pakistan (Figure 3.7). The molecular data of this study strongly support their

identification. These two Chinese Eupetaurus show the similar molecular features with

the population of Eupetaurus distributed in Tibet. The genetic difference is 3.8% (Table

3.4), less than the intraspecific cytochrome b differences of squirrels (Oshida and









Yoshida, 1999). This confirms the presence of Eupetaurus in southwestern China,

although lack the morphological data from these specimens.

Eupetaurus was commonly considered as a monotypic genus, consisting of a

single species, E. cinereus. However, the genetic distances in NJ tree and the

polycotomy in MP tree of the present study suggest that the populations in the eastern

and the western trans-Himalayas can be divided into two major mtDNA clades (Figure

3.2 and Figure 3.3). The genetic distance between these two clades was 11.0 13.3%

(Table 3.4). Since the reported intraspecific cytochrome b differences of squirrels was <

3.0%, and was applied to other flying squirrels, such as Glaucomys (Arbogast, 1999) and

Petaurista (Oshida and Yoshida, 1999), the genetic difference between Eupetaurus I and

Eupetaurus II can be referred as the interspecific variation. This implies that the

Eupetaurus populations in the western (Pakistan) and the eastern (China) trans-

Himalayas might be two distinct species.

Using the substitution rate at the third codon positions estimated from the

mammalian cytochrome b genes (Irwin et al., 1991), the divergence between the two

groups could have occurred approximately 10.8 Myr (million years) ago (Table 3.8),

suggesting that the two populations diverged early before the glacial period and the uplift

of the Himalayas and Qinghai-Tibet plateau during Pliocene Pleistocene (see

APPENDIX for time scales). It is inferred that the ancestor stock ofEupetaurus

originated somewhere along the Himalayas mountain chain. Before the glacial eustacy,

the eastern and the western trans-Himalayan Eupetaurus diverged from the ancestral

Eupetaurus and separately migrated to their present locations after the closure of the

Tethys Sea at the end of Miocene. During the glacial period of the Pliocene, they became









adapted to the cold environments of mountain rallies. During inter-glacial times, they

migrated to higher elevations to avoid warmer conditions. The subsequent glaciations

and the uplift of the Himalayas and Qinghai-Tibet plateau in Pleistocene (Xu, 1981;

Zheng et al., 2000) caused a great change of climate and ecological system along the

Himalayas, which consisted of several distinct topographic regions determined by

drainage patterns and the parallel mountain chains in both the western and the eastern

trans-Himalayan regions, such as the Yangtze river system, Salaween river system, and

Mekong river system. These tectonic events led to southwestern China, also possibly

northern Pakistan, becoming refuges for some special mammals, such as Eupetaurus. It

is possible that the present distribution of Eupetaurus in the trans-Himalayas is

secondarily related to the tectonic activities of the Cenozoic that caused dramatic changes

of environment in the Eurasian continent (Wang, 1984).

In Pakistan, the distributions of E. cinereus, P. petaurista, and Eoglaucomys

fimbriatus are sympatic, which correspond to different ecological habitats, indicating

their different feeding habits. A similar pattern of sympatric distribution of E. cinereus,

P. peturista, and Hylopetes alboniger was also found in SW China where the two

Eupetaurus skins were collected. But, without sufficient morphological evidence, it is

premature to raise these two groups as distinct species, although it is noteworthy that the

genetic characteristics between these two groups are corresponding to their geographic

distances of sampling localities.

3.4.2 Phylogenetic Relationship between Eupetaurus and Petaurista

Petaurista and Eupetaurus have been considered as the closely related species for

a long time because of their similar external structures and sympatric distribution (Figure

4.8). Their common ancestor was likely to have originated in the eastern Himalayas and









Indo-China (Zahler and Woods, 1997). According to McKenna (1962), the

differentiation of the recent genus Eupetaurus from a Petaurista-like sciurid provided a

significant parallel to the derivation of various dentally high-crowned rodents from

sciurid and paramyid stock in the early and middle Tertiary (Wood, 1962). The

differences between Petaurista and Eupetaurus are primarily the result of the high-

crowned teeth (Figure 3.8).

Eupetaurus has high-crowned teeth and survives in more restricted areas that

appear to meet their unique habitat requirements. Thomas (1888) believed that woolly

flying squirrels fed mainly on lichens, mosses, and other plants associated with rocky

areas. Local people in Pakistan believe that Eupetaurus feeds on seeds, needles, bark of

conifers, spruce buds, and some abrasive materials (Zahler and Woods, 1997). In eastern

Tibet and NW Yunnan, forests grow up to an elevation of 3,500-4,500 m, where there is a

mixed coniferous and broad-leaved forest that composes predominantly of spruce, fir,

and oak. It is the optimum habitat for Eupetaurus. The adaptive shift of the feeding

mechanism is analogous to the shifts that led to the distinctive morphology of the

dentition of beavers, mylagaulids, eutypomyids, and numerous other high-crown rodents

(McKenna, 1962). But, the pattern of the dentition in Eupetaurus is unlike that of any

other known rodent, either fossil or recent. A good ecomorph of E. cinereus is

Plagiodontia aedium from Hispaniola, which also lives in rock crevices and caves,

located in rocky areas at high elevation (3,000m). Both forms probably feed upon the

similar abrasive materials for their similar modifications to the masticator apparatus

(Woods and Howland, 1979).









Since Eupetaurus is so distinct from other flying squirrels, it appears that E.

cinereus is morphologically convergent with capromyids as well as cane rats and New

World spiny rats, rather than with other petauristines. The unique dental form of

Eupetaurus might be due to isolation in marginal habitats or the strong competitive

pressure from P. petaurista and Eoglaucomysfimbriatus or H. alboniger. The present

molecular findings indicate that Eupetaurus had diverged from Petaurista before the

Oligocene-Miocene radiation of giant flying squirrels in Europe (Oshida, et al., 2000a),

and that Eupetaurus is a specialized species that genetically differs from Petaurista

(Figure 3.5 and Figure 3.6).

Petaurista .\ixtriti and P. petaurista were considered as the closest living

relative of Eupetaurus (Mckenna, 1962; Zahler and Woods, 1996) (Figure 3.9), but the

genetic data of the present study reveals that the isolation between the ancestors of

Eupetaurus and Petaurista occurred approximately at the end of Oligocene, about 30.8 -

32.2 Myr ago (Table 3.4 and Table 3.5). The phylogenetic reconstruction also

demonstrates that Eupetaurus and Petaurista are different clades with significant genetic

distances, 15.9 19.4% (Table 3.3). Considering their divergence time, Eupetaurus

diverged from Petaurista-stock much earlier than the formation of P. xanthotis, which

diverged from Petaurista about 11 Myr ago. The present distribution of P. petaurista

(albiventer) in western trans-Himalayas is a recent dispersal.

Petaurista .\ixti,1ni is a Chinese endemic species and is found living from Tsing

Hai and Kansu, southeastward to Hubei and southward to Sichuna and Yunnan, China.

The elevation ranges from 2,000m in Gansu to 3,300m in Yunnan where the eastern

extremes of the Himalayas and the Tibetan Plateau separate the range of P. .\,iiiiihtii









from Eupetaurus. P. .\xnhi,,ti\ has semi-hypsodont molariform teeth, which is

significantly different from P. petaurista (Figure 3.8). The principal differences between

P. .x\ mih,,ti and Eupetaurus are the result either of the increased height of crown of its

molar teeth of Eupetaurus or of a few minor changes in molar crown pattern acquired by

P. .\xnithiti, Mckenna (1962) proposed that the lineage leading to P. .vtxth'1,ti had

changed little and E. cinereus had modified the dentition and the masticatory musculature

to a considerable degree. The similar dental structure between Eupetaurus and P.

.\(I1thitii might be the convergent adaptation to the similar feeding resources.

According to the paleontological records, by the late Miocene the geography of

the trans-Himalayas was similar to that of today (Wang, 1984). The presence of the

Himalayas helped cause the diversification of climate, and it became an important

regulator of the Asian environment. There were either three or four major glacial periods

in Europe and Asia, separated by warmer, interglacial periods in Pliocene-Pleistocene.

Eupetaurus migrated to its current geographical distribution in the early Miocene when

the radiation of Petaurista occurred in the Eurasian continent (Oshida et al., 2000a).

After diverging from the ancestral Petaurista in the middle Miocene, P. .\vnth,,ti\ was

restricted to the southern parts of the Eurasian continent. During glacial spisodes, it

adapted to cold environments and high elevations, and its feeding habits became

specialized. With the retreating of glaciers and the uprising of the Himalayas, it

subsequently expanded into northward, where it inhabited in the temperate forest. The

present distribution of P..\xinthitii is mainly due to the geographic events of the

Pliocene-Pleistocene. The molecular data here indicate that the phylogenetic relationship









between Eupetaurus and P. .\inl,,iti\ or P. petaurista is not as close as indicated from

morphology.

3.5 Summary

In this study, the partial cytochrome b gene sequences (390 bp) of Eupetaurus

were analyzed to elucidate the phylogenetic status of Eupetaurus in the eastern and the

western trans-Himalayas. I also discussed the phylogenetic relationship between

Eupetaurus and Petaurista. The phylogenetic trees were reconstructed using

neighboring-joining and maximum parsimony methods. The following results were

concluded in the present study:

1. The two specimens that were collected in northwestern Yunnan, China, are
Eupetaurus.

2. The Eupetaurus populations in the eastern and the western trans-Himalayas are
significantly different (>13%). The genetic characters between these two
populations are corresponding to their geographic distribution. They are two
distinct species.

3. The divergence time of the two Eupetaurus populations was at the end of
Miocene. The glacial period and the uplift of the Himalayas and Qinghai-Tibet
plateau during the Pliocene-Pleistocene period are the major factors that
secondarily affected on the present distribution of Eupetaurus in trans-Himalayas.

4. There is not a very close phylogenetic relationship between Eupetaurus and P.
\X.i\h,,iti%. The similar dental characters might be of the convergent adaptation to
the similar food resource.



















I;. .


bw DhNJi


.i .c


- wu


&stakzu


Figure 3.1 Historical records of Eupetaurus specimens in the world. The

numbers in the map stand for the collecting localities of specimens,

which are corresponding to the numbers of Table 3.1.


"r I
r

;: r
:n


















ECB2


ECF2


99


ECD





ECF1





ECK2


100


ECK1
100




ECL





ECB1








Figure 3.2 Phylogenetic tree of Eupetaurus reconstructed by the maximum
parsimony (MP) method. Numbers above branches indicate the
bootstrap values (%). Sample abbreviations are coded in Table 3.2.





















ECB2





ECF2





ECD





ECF1





ECB1





ECK2





ECK1





ECL









Figure 3.3 Phylogenetic tree of Eupetaurus reconstructed by the UPGMA
method. Sample abbreviations are coded in Table 3.2.


















ECF2




ECD




ECF1




ECB2




ECB1




ECK1



ECK2




ECL




PPF




PPY




PTK









Figure 3.4 Phylogenetic tree of Eupetaurus and Petaurita constructed with
UPGMA method. Sample abbreviations are defined in Table 3.2.


















ECF1



78 ECF2



100 ECD



99 ECB2



ECB1

59

ECK2
100

ECK1
99


ECL



PPY

97

PPF
88


PTK



GV









Figure 3.5 Phylogenetic relationships ofEupetaurus, Petaurita, and G.
volans constructed with the parsimony maximum (MP) method.
Numbers above branches indicate the bootstrap values (%). Sample
abbreviations are defined in Table 3.2.

















ECB2



ECF1



ECF2



ECD



ECB1



ECK2



ECK1



ECL



PPY



PPF



PTK



GV
0.01 substitutions/site







Figure 3.6 Phylogenetic relationships of Eupetaurus, Petaurita, and G.
volans constructed with the neighbor-joining (NJ) method. Scales
in the tree represent branch length in terms of nucleotide
substitutions per site. Sample abbreviations are coded in Table 3.2.




















































L ilemfl n Pktolatn


RL cBwirw i Yrm


Figure 3.7 Eupetaurus cinereus in Pakistan and SW China















































Figure 3.8 Ventral views of the skulls of E. cinereus, P. petaurista, and P. .\xinlvi


P!











































Si nd s iii.



P pmfatmyivri Idtjl ood L cine irms Irlghll


Figure 3.9 E. cinereus, P. petaurista, and P. .\x,miti\


R.mntdRAil














CHAPTER 4
PHYLOGENY OF GIANT FLYING SQUIRREL PETAURISTAA) IN SW CHINA AND
PAKISTAN: IMPLICATIONS FOR DEVELOPMENT OF MOLECULAR AND
MORPHOLOGICAL ANALYSIS

4.1 Introduction

Giant flying squirrel, Petaurista, contains many recognized distinct species

occupying different habitats. They inhabit various kinds of forests either in lowlands or

in mountains up to 4,000 meters in elevation from Pakistan, Kashmir to China, northern

Indochina, the Malayan Peninsula, Sumatra, Java, Borneo, Japan, Korea, and

Manchuria. Several taxonomic revisions have been proposed on the basis of dental and

cranial characteristics and external structures (Allen, 1940; Ellerman, 1940; Corbet and

Hill, 1991, 1992; Nowak, 1991; Wilson and Reeder, 1992). More than 18 forms in

Petaurista have been described as valid species, but some of them are only referenced

with very few specimens; some are based solely on skins with no corresponding skulls

(Allen, 1940; Ellerman, 1940); and some actually are the synonyms or subspecies of

other valid species (Corbet and Hill, 1992). The populations of Petuarista that are

distributed in China and occupy different habitats are recognized as ten distinct species

by recent authorities (Corbet and Hill, 1992; Zhang et al., 1997) (Table 4.1 and Table

4.2). Figure 4.1 depicted the geographic distributions of Chinese Petaurista. In

Petaurista, the species P. elegans, P. petaurista, P. alborufus, P. magnificus, P.

philippensis, and P. leucogeny are commonly accepted as valid species, with each

intricately divided into various forms or subspecies (Corbet and Hill, 1991, 1992;

Nowak, 1991, 1999; Wilson and Reeder, 1992; Zhang et al., 1977; Wang, 2002).









However, various species with significantly geographical variations are included within

this genus, the taxonomy and the intra- and the inter-specific phylogenetic relationships

are confusing and inconclusive, especially the giant flying squirrels that are distributed

in the eastern and western trans-Himalayas.


Table 4.1 Chinese Petaurista forms
Species Subspecies Distribution Habitat

P. p. grandis Taiwan

P. p. miloni Guangxi

P. petaurista P. p. rufipes Fujian, Guangdong Tropical and
subtropical forest
P. p. rubicundus Sichuan, Gansu

P. p. nigra W Yunnan

P. a. alborufus Shanxi, W Sichuan

P. a. lena Taiwan T l
Tropical and
P. a. castaneus Hubei, Sichuan, Guizhou subtropical forest

P. a. ochraspis Yunnan, Guangxi

P. elegans P. e. clarkei Yunnan, Guizhou
Forest
P. e. gorkhal S Xizang Forest

P. yunanensis Yunnan, Guangxi, Tibet Tropical forest

P. hainana Hainan Tropical forest

P. pectoralis Taiwan Tropical forest

,/i, Gansu, Qinghai, Yunnan, Mountain
Xizang coniferous
P. philippensis Yunnan, Guizhou Forest

P. magnificus Xizang Mountain forest

P. marica Yunnan, Guangxi Tropical forest

NOTE: THE ASSIGNMENTS ARE DESCRIBED BY ZHANG ET AL. (1997)









Ellerman (1940), Corbet and Hill (1992), and Wang (2002) had comprehensively

studied Chinese Petaurista based on the morphological and external characteristics, but

there is not sufficient evidence from either morphometric study or molecular analysis to

ascertain conclusively these specific conclusions. The major problems of Chinese

Petaurista are about the conspecific relations within P. petaurista (sensu stricto, the

taxonomic statuses of P. philippensis, P. xanthotis, P. hainana, and P. yunanensis, and

the phylogenetic relationships of Petaurista at the specific level.

Petauristapetaurista is a polymorphic species with considerable variation in

pelage coloring (Allen, 1940). This species mainly occurs in plantations as well as in

forest in southern China. Its broad distribution is beyond China including northern

India, Bhutan, Nepal, Pakistan, northern Afghanistan, and the Greater Sunda Islands. A

dozen of nominal subspecies have been described (Allen, 1940; Ellerman, 1940;

Ellerman and Morrison-Scott, 1966). Corbet and Hill (1992) put all Petaurista

populations into seven major forms (Table 4.2). However, from our comparative study

ofEupetaurus (Chapter 3) and observations of some other rodents distributed in the

western and eastern trans-Himalayas, it seems extreme to allocate the populations of

Petaurista in Pakistan and W Yunnan to the same form, P. p. albiventer. Further study

needs to be done to clarify the inter-group relations for their various geographical

distributions.

With few exceptions (Nowak, 1991), P. philippensis and P. .\x,1h,,ii\ have been

merited as valid species based on their distinct external and dental structures (Corbet and

Hill, 1991, 1992; Wilson and Reeder, 1992). P. philippensis is a polymorphic species

with extensive geographical variations and used to be included in P. petaurista









(Ellerman, 1940; Ellerman and Morrison-Scott, 1966). Because this species is clearly

separable from P. petaurista by the external structures, Corbet and Hill (1991, 1992)

ranked P. philippensis as a valid species and classified its populations as seven major

forms (Table 4.3). All populations distributed in China including the island of Hainan

were recognized as P. philippensis yunanensis. Without further evidence, much remains

to be done to clarify their taxonomic relationships. Some forms included by Corbet and

Hill (1992) may warrant separate specific rank.


Table 4.2 Forms of Petaurista
Form (subspecies) Distribution
P. p. albiventer W Pakistan, W Yunnan, China
P. p. petaurista Java
P. p. marchio Borneo and Malayan Peninsula
P. p. batuana W Sumatra
P. p. terutaus S Thailand
P. p. taylori S Burma and W Thailand
P. p. candidula Burma and Thailand


Table 4.3 Major forms of P. philippensis
Subspecies Distribution
P. p. philippensis Peninsular India and Sri Lanka
P. p. cineraceus W Burma
P. p. mergulus Mergui Is, Burma
P. p. lylei E Burma, Thailand
P. p. annamensis Vietnam, S China
P. p. yunnanensis SW CHINA, HAINAN, N
P. p. grandis ASSAM
Taiwan


Petaurista .x\nthitii is a Chinese endemic species with an extensive range, from

the spruce forests of northwestern Kansu southward in the highlands of western China to

the Likiang region. P. .x\,lih,,ti was considered as a form of Japanese giant flying









squirrel, P. leucogenys, by Ellerman and Morrison-Scott (1966), but it was elevated as a

distinct species for its much complex cheek-teeth (Corbet and Hill, 1991, 1992, Nowak,

1999). Except for a morphological illustration of teeth by McKenna (1962), almost

nothing is known about this species.

Petaurista yunanensis and P. hainana are generally treated either as the

subspecies (Corbet and Hill, 1992) or as the synonyms (Wilson and Reeder, 1992) of P.

philippensis based on the dental structures and pelage coloration, although both were

merited as two valid species of Petaurista early by Anderson (1878) and Allen (1940).

The former occurs from extreme southwestern Yunnan probably into Burma and

Indochina, and the latter is only distributed in Hamfong, Hainan, China (Figure 4.1).

Very little seems to be known as to their taxonomic statuses and the inter- or

intraspecific relationships with P. philippensis. A comprehensive study on the basis of

morphometric and molecular analysis is much needed.

Mitochondrial DNA (mtDNA) is one of those valuable molecules for

evolutionary relationship reconstruction among populations, subspecies, and species.

Using the polymerase chain reaction (PCR), it is possible to recover genetic information

from severely degraded tissues. With the mtDNA information it is able to infer the

inter- and intra-specific relationships, to investigate the genetic differentiation, and to

reconstruct the phylogenetic topology of the controversial taxa. Although some

molecular data and geographical and morphological variations involving morphology,

myology, and karyology have been intensively used for studying the species, subspecies

or forms of Petaurista (Cuvier, 1856; Bryant, 1945; Harrison, 1960; Johnson-Murray,

1977; Throington and Heaney, 1981; Oshida et al., 1992; Oshida et al., 1996; Oshida









and Masuda, 2000; Oshida et al., 2000a, 2000b), it is too scanty to throw any light on the

problems. There are still arguments on the validity of a number of species or forms in

Petaurista, and very little is known on the phylogenetic relationships within the genus

Petaurista.

In attempting to resolve the affinities of the complex taxa, a comprehensive study

based on both morphological data including 14 measurements of skull and molecular

data using partial sequences (375 400 bp) of mitochondrial cytochrome b gene were

conducted in this study. The objectives were to answer the following questions:

1. Do the populations of P. petaurista (albiventer) along the eastern and the western
trans-Himalayas form a single species -- Petaurista albiventer, or a complex of
species?

2. Are P. philippensis, P. xanthotis, P. hainana, and P. yunanensis valid species,
subspecies, or synonyms?

3. What are the phylogenetic relationships among the populations of Chinese
Petaurista and the populations of Petaurista in Pakistan and SE Asia?

4.2 Materials and Methods

4.2.1 Specimens in Morphometric Study

A total of 193 recent specimens of giant flying squirrels (Petaurista) were used

in morphometric analysis (Table 4.4). Specimens were conventional museum specimens

preserved as skulls, skeletons, and fluid. These specimens are deposited in the following

collections: American Museum of Natural History, New York (AMNH), Florida

Museum of Natural History, University of Florida, Gainesville (FLMNH), National

Museum of Natural History, Smithsonian Institution, Washington, DC (USNM),

Chinese Institute of Zoology, Beijing (China, BIZ), Northwestern Institute of Biology,

Qinhai (China, NIB), and Kunming Institute of Zoology, Kunming (China, KIZ).










Fourteen cranial measurements were taken with digital caliper to the nearest 0.01 mm.

The definitions and abbreviations of measurements were given in Table 4.5.






Table 4.4 Species and localities of Petaurista populations examined in
morphometric analysis
Species Subspecies Specimens Locality Museum

P. p. albiventer 10 (5F, 5M) NW Yunnan, China KIZ, AMNH
NWFP, Pakistan, KIZ,
P. p. albiventer 31 (14F, 7M) Kashmir NHs
Kashmir USMNH
Lakhuni, India,
P. p. candidula 4 (2F, 2M) Lakhuni, India, AMNH
P. petaurista Chiengmail, Thailand
S24 12E Sumatra, Borneo, USMNH,
P. p. melanotus 24 (12F, 12M)insula MNH
Malavan Peninsula AMNH
P. p. petaurista 10 (6F, 4 M) Java AMNH

P. p. batuana 11 (6F, 5M) W Sumatra AMNH
P p nsis 9 (F, 6 M Yunnan, Guangxi, China KIZ, BIZ,
Sphilippenss 9 (3F, 6 M)AMNH

P. philippensis P. p. lyei 10 (6F, 4M) Indochina, Thailand USMNH
MNH,
P. p. grandis 14 (3F, 11U) Taipei, Taiwan AMNH
FLMNH
P. yunanensis 15 (5F, 3M) Burma, Yunnan, China BIZ, AMNH
P. hainana 9 (5F, 4M) Namfong, Hainan AMNH

P. elegans 12 (6F, 6 M) Yunnan, Guangxi, China KIZ

P. alborufus 29 (5M, 24U) Taiwan, Yunnan, Burma KIZ, AMNH
P. xanthotis 5 (3F, 2 M) Yunnan, Qinhai, China KIZ, NIB

Total 193 F = Female, M = Male, U=Unknown sex

Note: Assignments of species and subspecies were based upon the classification of
Corbet and Hill (1992) and Zhang et al. (1997).













Table 4.5 Variables in morphometric study
Variable Definition

Cranial length from the tip of the occipital protuberance to the
CRANL
alveolar
BCASEL Braincase length from the tip of the occipital protuberance to orbit
Cranial width, distance between the points on the left and right
CRANW
superametal crests above the external acousticmeatus

BPORW Width between the left and right zygomatic arch
Postorbital constriction, the least distance across the top to skull
POCL
posterior to the postorbital process
PA Distance from the base of the anterior surface of the postglenoid
PGA
process to the M3
NAL Nasal length
TBL Tympanic bullae length
DSL Diastema length between the second incisor and the first premolar
MTRL Length of the maxillary tooth row
MTRW Width of the maxillary tooth row at M2
LMDL Maximum mandible length
LMDH Maximum height of low jaw
LMTL Length of the maxillary tooth row of lower jaw


4.2.2 Species for Molecular Analysis

Species of giant flying squirrels (Petaurista) used in molecular analysis were

listed in Table 4.6. All skin tissues were collected from AMNH, USNM, BIZ, NIB,

FLMNH, and KIZ. Fresh tissues were either obtained from the Cellbank of Kunming

Institute of Zoology, the Chinese Academy of Sciences, or collected from the locality in

where the species is distributed. To reconstruct the phylogenetic relationships, the









sequence data of P. petaurista and Pteromys volans were quoted from the GenBank of

NCBI. The detailed information was given in Table 4.7.


Table 4.6 Samples of Petaurista examined in molecular study
Species Code Museum ID Collecting locality
PPF1 FLMNH: 28236 Pakistan

P. p. albiventer PPF2 USMNH: 353209 Pakistan
(PPF and PPY) PPY1 KIZ: 640229 Yunnan, China
PPY2 KIZ: 73382 Yunnan, China
P. .\xR//,//i/ PTK1 QIZ: 85063 Gansu, China
(PTK) PTK2 QIZ: 984 Gansu, China

PPH1 KIZ: Fresh tissue Pianma,Yunnan, China

P. philippensis PPH2 KIZ: 620028 Yunnan, China
(PPH) PPH3 KIZ: 74540 Yunnan, China
PPH4 KIZ Yunnan, China
PYK1 KIZ: Fresh tissue W Yunnan, China
PYK2 KIZ: Fresh tissue Gongshan, Yunnan, China
P. yunanensis PYK3 KIZ: Fresh tissue Gongshan, Yunnan, China
(PYK) P 4 K: 7 G Y
PYK4 KIZ: 73270 Gongshan, Yunnan, China
PYK5 KIZ: Gongshan, Yunnan, China
PHK1 KIZ: 22686 Hainan, China
P. hainana (P ) PHK2 KIZ: 259442 Namfong, Hainan, China

PAK1 KIZ: 006679 Sichuan, China
PAK2 KIZ: 43178 Likiang, China
P. alborufus (PAK) .
P. alborufus (PAK) PAK3 KIZ: 73369 Yunnan, China

PAK4 KIZ: 64042 Luodian, Guizhou, China
PEK1 KIZ: 84354 Mile, Yunnan, China
P. elegans (PEK) PEK2 KIZ: 73375 Lijiang, Yunnan, China
PEK3 KIZ: 73369 Bijiang, Yunnan, China













Table 4.7 Sequence data of Petaurista and Pteromys used in this study
Species Code Locality Accession No

P. p. petaurista** PPB Borneo, E Malaysia AF063067

PPM1 Laos AB023908
P. p. melanotus
P. p. melanotus PPM2 S China AB023909

P. philippensis grandis PPG Natou, Taiwan AB023907

PLL1 Japan AB023903
P. leucogenys leucogenys PLL2 Japan AB023904

PLN1 Japan AB023905
P. leucogenys nikkonisJapan AB023
PLN12 Japan AB023906

PAC1 S China AB023898

P. alborufus castaneus PAC2 S China AB023899
PAC3 S China AB023900

PALI Nantou, Taiwan AB023901
P. alborufus lena
SPAL2 Hualien, Taiwan AB023902

Pteromys volan PVO Japan AB023910


Note: Most sequence data were quoted from GenBank of NCBI, which
were published by Oshida et al. (2000a).
** The sequence was quoted from GenBank of NCBI, which was pub
by Arbogast (1999).


4.2.3 Morphometric Analysis

Statistical analyses in morphometric study were performed using the SAS (SAS

Institute, 1982) or Statmost program (StatMost, 1995). The associations of cranial

characters and species were assessed by discriminant function analysis and principal


component analysis (PCA).









Discriminant function analysis carried out a multiple discriminant analysis in a

stepwise manner, selecting the variable entered by finding the variable with the greatest

F-value. This method was used to determine which of two or more groups a given

individual should be assigned to and placed them with the group to which they were

nearest on the discriminant functions. The relationships between groups onto the first

three discriminant functions were plotted. Principal components analysis was performed

to identify variables that account for maximum variation in data set and to accurately

represent distances between major groups, in assessing the specific relationships among

and between pooled individuals. The loadings or the eigenvector scores describing the

relative significance of each variable to principal components were used to compare the

morphological similarity and difference of skull. The projections of the row-points on

the first three factors were depicted to describe the morphological relationships among

samples examined.

4.2.4 Molecular Analysis

Because some samples were collected more than 50 years ago, the recovered

sequences were usually between 300 to 400 base-pair longs. The following primers

were used to amplify the partial nucleotide sequence (300 425 bp) of mitochondrial

DNA (mtDNA) with polymerase chain reaction (PCR):

L14725 5'- CGA AGC TTG ATA TGA AAA ACC ATC GTT G -3'

L14841 5'- AAA AAG CTT CCA TCC AAC ATC TCA GCA TGA TGA AA -3'

H15149 5'- AAA CTG CAG CCC CTC AGA ATG ATA TTT GTC CTCA -3'.

ICBR and KIZ synthesized all primers. The techniques and protocols used for DNA

isolation, PCR amplification and purification, sequencing analysis, and phylogenetic

reconstruction were the same as those in Chapter 3. Most molecular work was done at









the interdisciplinary center for biotechnology research (ICBR), University of Florida,

and Kunming Institute of Zoology (KIZ), the Chinese Academy of Sciences, China.

Some PCR products were sequenced at the University of Vermont, US.

One sample ofPteromys volans from NE China was used as the outgroup taxon

for constructing phylogenetic trees.

4.3 Results

All measurements of skull were taken by author at museums and institutes in

China and US, thereby avoiding the statistical complications and other associated

problems of inter-observer error in a multivariate data analysis. A percent difference

analysis was performed on 15 randomly chosen individuals to obtain an estimate of the

amount of intra-observer error involved in measuring the specimens. In molecular

study, because the recovered sequences of some old skins were highly degraded, the

sequence sizes of individuals used for building phylogenetic trees were different.

4.3.1 Phylogenetic Relationships of Chinese P. philippensis

Following the taxonomic assignments of Corbet and Hill (1992), five forms of P.

philippensis, including 57 specimens of P. p. philippensis (SW China), P. p. lyei

(Thailand), P. p. grandis (Taiwan), P. yunanensis, and P. hainana, were selected in

morphormetric analysis. The molecular analyses were based on the partial sequences

(409 bp) of cytochrome b gene. One individual of P. petaurista from Borneo was used

as an out-group.

4.3.1.1 Morphological data

Multivariate analyses revealed highly significant differences in five forms of P.

philippensis. The results of discriminant analysis were given in Table 4.8. The first

three axes accounted for 97% of the total variance. The forms in Thailand (P. p. lyei),









Taiwan (P. p. grandis), and Hainan (China, P. hainana) were distinguished as distinct

groups without overlapping with other forms on discriminant function 1 (CAN I) (Figure

4.2). The populations from Yunnan, China, including P. philippensis and P. yunanensis

shared similar morphological structures in skull and were clustered together. All

characters except for the variable BCASEL (braincase length), PORCL (postorbital

constriction), and LMTL (length of the maxillary tooth row of lower jaw) gave high

contributions to CAN I. Inspection of the plot based on the discriminant function 1 and

3 revealed that P. p. grandis and P. hainana were still separated as distinct groups

(Figure 4.3). BCASEL and MTRWL (Width of the maxillary tooth row) were the

variables having the highest canonical scores on CAN II and CAN III, respectively.

The PCA results in the first three factors and the eigenvector scores of variables

were presented in Table 4.9. Along the first principal component (PRIN I) that

accounted for 80% of the original total sample variance, all eigenvector coefficients of

variables were positive. The primary separation of taxa along this axis was among P. p.

lylei, P. p. grandis, and P. hainana. The population of P. philippensis distributed in

Yunnan was overlapped with P. yunanensis (Figure 4.4). The morphological variables

mainly responsible for this segregation were CRANL and LMDL. In the scatter-plot of

PRIN I against PRIN III, P. p. grandis, and P. hainana were separated as distinct groups

once again, but P. p. lylei, P. yunanensis and P. philippensis in Yunnan shared similar

cranial structures and were greatly overlapped (Figure 4.5). The variable BCASEL and

LMDL were strongly correlated with PRIN II and PRIN III, respectively. The results of

PCA were consistent with those of disrciminant function analysis.














Table 4.8 Discriminant function analysis of five P. philippensis forms
CAN Eigenvalue Proportion Cumulative

I 15.94 0.72 0.72

II 4.37 0.20 0.92

III 1.14 0.05 0.97

Canonical score
Variable CAN I CAN II CAN III


CRANL

BCASEL

CRANW

BPORW

PORCL

PGA

NAL

TBL

DSL

MTRL

MTRW

LMDL

LMDH

LMTL


0.95

0.54

0.94

0.94

0.66

0.94

0.78

0.70

0.85

0.84

0.76

0.94

0.84

0.65


0.02

0.59

0.05

-0.05

-0.08

0.12

0.03

0.40

-0.09

-0.18

-0.22

0.08

-0.03

-0.03


-0.11

0.03

-0.17

0.03

-0.19

0.02

-0.07

0.07

-0.01

0.18

0.31

0.20

0.30

-0.02














Table 4.9 Principal components analysis of five P. philippensis forms
PRIN Eigenvalue Proportion Cumulative

I 87.96 0.80 0.80

II 9.14 0.08 0.89

III 3.24 0.03 0.92

Eigenvector score
Variables PRIN I PRIN II PRIN II

CRANL 0.57 -0.06 -0.51

BCASEL 0.23 0.24 0.13

CRANW 0.26 -0.01 -0.13

BPORW 0.38 -0.10 -0.17

PORCL 0.08 -0.14 -0.05

PGA 0.24 -0.02 -0.05

NAL 0.17 -0.02 -0.22

TBL 0.10 0.00 0.03

DSL 0.13 -0.07 -0.06

MTRL 0.10 -0.04 0.09

MTRW 0.10 -0.02 0.01

LMDL 0.48 -0.26 0.75

LMDH 0.17 0.07 0.15

LMTL 0.11 0.00 0.14



4.3.1.2 Molecular data

Totally 12 samples of five forms ofP. philippensis were examined to reconstruct

the phylogenetic relationships. P. petaurista from southeastern Asia was used as an

outgroup. Table 4.10 showed the sequences differences that were corrected by Kimura's










two-parameter model (1980), and the numbers of transversions and transitions obtained

from pairwise comparison between samples.


Table 4.10 Pairwise comparison based on the partial sequences (409 bp) of
cytochrome b gene between five P. philippensis forms. Data below the
diagonal are the numbers of nucleotide substitutions, transitions vs.
transversions. Data above the diagonal represent the genetic differences
between samples. The samples were defined in Table 4.6 and 4.7.
PHK
PPH1 PPH2 PPH3 PPH4 PHK1 PYK1 PYK2 PYK3 PYK4 PYK5 PPG PPB
2

PPH1 1.3 1.7 1.5 5.6 4.7 8.3 8.6 8.3 8.3 8.3 12.5 5.7

PPH2 4/1 1.0 1.2 5.9 3.9 9.3 9.4 9.0 9.0 9.0 11.4 6.4

PPH3 5/1 4/0 0 7.1 5.1 9.7 9.7 9.3 9.3 9.3 11.7 6.8

PPH4 4/0 4/0 0 6.6 4.8 9.4 9.4 9.1 9.1 9.1 11.3 6.8

PHK1 21/1 20/2 25/2 25/2 2.2 10.0 9.6 9.9 10.0 10.0 12.9 8.0

PHK2 19/0 15/0 20/0 19/0 5/1 9.4 8.6 9.0 9.0 9.0 12.9 6.3

PYK1 31/3 33/4 35/4 35/3 35/4 35/3 0.5 0.5 0.5 0.5 13.8 8.6

PYK2 31/4 34/4 35/4 35/3 33/6 35/3 1/1 0.2 0.3 0.2 14.1 8.9

PYK3 30/4 32/4 33/4 33/3 34/6 33/3 1/1 1/0 0 0 13.8 8.3

PYK4 30/4 32/4 33/4 33/3 34/6 33/3 1/1 1/0 0 0 13.8 8.6

PYK5 30/4 32/4 33/4 33/3 34/6 33/3 1/1 1/0 0 0 13.8 8.3

PPG 42/8 37/7 37/8 37/8 43/8 43/8 46/9 46/10 45/10 46/10 45/10 12.1

PPB 23/0 21/2 25/2 27/0 30/2 24/0 32/2 32/2 31/3 30/3 31/3 41/8


The considerable sequence variations existed among all forms. The form from

Taiwan (P. p. grandis) was significantly different from the forms in mainland with high

genetic differences (11.3 14.1%). The form P. yunanensis apparently differed from P.

philippensis and P. hainana with 8.3 10% differences in sequence. The genetic

difference between P. philippensis and P. hainana was about 5%. The phylogenetic

reconstructions using maximum parsimony (MP) and neighbor-joining distance (NJ)









methods resulted in essentially the same branching patterns. Each of five forms was

formed as distinct clade (Figure 4.6 and Figure 4.7). The bootstrap values to support

these branching orders were high, ranging from 87% to 100%.

4.3.2 Phylogenetic Relationship between P. xanthotis and P. leucogenys

A partial sequence (409 bp) of cytochrome b gene of P..\,xanitii was successfully

sequenced from two museum specimens. The sequence data of Japanese P. leucogenys

were retrieved from GenBank of NCBI.



Table 4.11 Percentage of genetic differences between P. .\xanthtii and other giant
flying squirrels based on pairwise comparison of the partial cytochrome b
sequences (409 bp). See Table 4.6 and Table 4.7 for sample information.
PTK1 PTK2 PLL1 PLL2 PLN1 PLN2 PYK PPH PHK PPB

PTK1 3.8 14.1 12.9 13.2 13.2 14.7 15.5 13.4 14.3
PTK2 11/3 11.7 10.2 9.2 9.2 11.94 11.57 12.7 12.5
PLL1 41/16 34/12 1.3 2.8 2.1 15.4 14.4 15.1 16.0
PLL2 37/15 27/12 4/1 2.6 1.8 14.9 13.6 14.4 15.0
PLN1 37/16 24/12 11/0 9/1 1.0 14.6 13.1 13.5 14.3
PLN2 37/16 24/12 8/0 6/1 3/0 14.3 13.3 14.1 14.3
PYK 46/13 40/8 51/11 47/13 47/11 46/11 8.6 9.6 8.9
PPH 52/10 38/8 49/9 46/8 43/9 44/9 31/4 5.6 5.7
PHK 43/9 41/8 50/11 48/10 43/11 46/11 33/6 21/1 8.1
PPB 47/9 43/7 52/8 50/7 46/8 46/8 32/3 23/0 26/1
Note: Data below the diagonal are the numbers of nucleotide substitutions,
transitions vs. transversions. Data above the diagonal represent the
genetic differences between samples.


For the sake of comparison, P. philippensis, P. yunanensis, and P. hainana were

used to build their phylogenetic relationships with P. petaurista distributed in

southeastern Asia as an outgroup. Table 4.11 presented the percentage differences that









were corrected by Kimura's two-parameter model (1980), and the transversional and

transitional numbers between samples using pairwise comparison. P..x\,nthiii

significantly differentiated from other Petaurista forms for their highly genetic

differences, varying from 9.2% to 15.5%. The phylogenetic trees generated with MP

and NJ methods were concordant with the results of pairwise comparison. Compared to

P. philippensis, P. yunanensis, and P. hainana, there was a closed relationship between

P. .\xthi,,i\ and P. leucogenys, although each of them formed as a distinct clade.

(Figure 4.8 and Figure 4.9). The bootstrap value in MP tree for branching was 99%.

4.3.3 Phylogenetic Relationship of P. petaurista

The morphological study of P. p. albiventer between the eastern and the western

trans-Himalayan populations was based on 41 specimens from N Pakistan and W

Yunnan, China (Table 4.4). The populations from India, E and W Sumatra, Borneo,

Java, and Malayan Peninsula (49 specimens) were included to investigate the

morphological relationships among populations of P. petaurista. Table 4.12 showed the

results of discriminant function analysis on the first three functions, which were outlined

graphically in Figure 4.10 and Figure 4.11. The first discriminant function that

accounted for 68% of the total variance separated the population in W Yunnan from the

rest groups (Figure 4.10). CRANW, BPORW, and LMDL were the major variables

contributing to CAN I. The morphological variables contributing to CAN II included

LMTL, LMDH, and BCASEL. In the plot of CAN I to CAN III (Figure 4.11), the

populations distributed in Yunnan (SW China) and Burma were distinguished as

different assemblages. The rest populations were overlapped as a complicated

assemblage, although the overlap was little between the populations in Java and in

Pakistan. BCASEL was the dominant variable on CAN III.














Table 4.12 Discriminant function analysis of P. petaurista
CAN Eigenvalue Proportion Cumulative

I 20.43 0.68 0.68
II
1 5.42 0.18 0.86

4.16 0.14 1.00

CANONICAL SCORE

Variable CAN I CAN II CAN III

CRANL 0.76 -0.44 0.38

BCASEL 0.40 -0.57 0.64

CRANW 0.81 -0.03 0.31

BPORW 0.85 -0.21 0.37

PORCL 0.32 0.45 0.25

PGA 0.68 -0.29 0.48

NAL 0.71 -0.54 0.09

TBL 0.02 -0.28 0.14

DSL 0.73 -0.04 0.51

MTRL 0.72 -0.38 0.17

MTRW 0.70 -0.59 0.06

LMDL 0.81 -0.13 -0.26

LMDH 0.68 -0.57 0.06

LMTL 0.64 -0.60 0.07


The results of principal components analysis of P. p. albiventer were presented

in Table 4.13. The first principal component factor that accounted for 76% of the total

variance isolated the W Yunnan and Burma populations from others, forming two

distinct clusters (Figure 4.12). The main determinants were CRANL on PRIN I, and









BCASEL and LMDL on PRIN II. All specimens in the plot of the fist principal factor

and the third principal factor were clustered as two groups (Figure 4.13). The first group

consisted of the populations in Yunnan and Burma, and the second group included the

remaining populations. The main variable contributing to the observed association was

LMDL.


Table 4.13 Principal components analysis of P. petaurista

PRIN Eigenvalue Proportion Cumulative

I 50.07 0.76 0.76

II 6.52 0.10 0.85

III 3.46 0.05 0.91
Eigenvector score

Variables PRIN I PRIN II PRIN III

CRANL 0.63 0.17 0.04

BCASEL 0.20 0.75 -0.17

CRANW 0.24 -0.16 0.37

BPORW 0.37 -0.16 0.38

PORCL 0.04 -0.06 0.38

PGA 0.24 0.15 0.15

NAL 0.24 -0.03 -0.22

TBL 0.03 0.09 -0.06

DSL 0.18 -0.02 0.33

MTRL 0.14 -0.03 -0.08

MTRW 0.14 -0.03 -0.12

LMDL 0.27 -0.56 -0.35

LMDH 0.28 -0.09 -0.43

LMTL 0.16 -0.01 -0.17









The molecular study of the populations of P. p. albiventer was conducted based

on the partial sequences (375 bp) that were obtained from 4 museum skin samples. The

sequence data ofP. p. petaurista from Java and P. p. melantous from Malaya Peninsular

were used to build the phylogenetic topology.



Table 4.14 Percentage of differences and the numbers of transversional and
transitional substitutions between P. petaurista (albiventer) populations
based on pairwise comparison of the partial sequence (375 bp) of
cytochrome b gene.
PPF1 FPPF2 PPY1 PPY2 PPB PPM1 PPM2 PVO
PPF1 0.5 8.4 8.8 8.9 13.4 13.8 17.1
PPF2 2/0 8.3 8.6 8.9 13.3 13.8 17.7
PPY1 29/2 29/2 0.5 6.3 11.6 12.2 16.8
PPY2 32/2 32/2 3/0 6.3 11.6 12.2 16.8
PPB 32/1 32/2 24/0 24/1 11.5 11.5 15.9
PPM1 41/7 41/7 37/7 37/7 34/7 0.5 15.9
PPM2 44/7 44/7 39/8 39/8 34/7 2/0 15.9
PVO 43/19 46/19 37/21 39/20 37/20 37/20 37/20

Note: Data above the diagonal were the percentage of genetic differences
between samples and data below the diagonal were the numbers of
transitions vs. transversions between samples. See Table 4.4 for
sample abbreviations.


Table 4.14 was the percentage differences and the numbers of transversional and

transitional substitutions on the pairwise comparison between samples. The population

ofP. p. albiventer in Pakistan was apparently different from that in Yunnan with the

genetic distance, 8.6 8.9%. The population in Java showed similar genetic characters

with the population in Yunnan, China. Both the MP and NJ trees generated the similar

branching patterns (Figure 4.14 and Figure 4.15). The populations in Yunnan and in









Pakistan were separated as distinct branches with a high bootstrap value (98%). The

population in Malaysia, P. p. melantosus, had an early divergence from other groups.

4.3.4 Phylogenetic Relationships of Chinese Petaurista

The morphometric analyses were performed on 65 specimens including 5

species, P. alborufus, P. elegans, P..i\ilintii, P. philippensis, and P. petaurista

(albiventer). The results of discriminant function analysis and principal components

analysis were displayed in Table 4.15 and Table 4.16, respectively. The first

discriminant function (CAN I) accounted for 60% of the original sample variance.

Along CAN I, five Petaurista species were assembled as three clusters: P. alborufus was

one group, P. elegans and P..x\iainitii formed one group, and P. philippensis and P. p.

albiventer formed the third group (Figure 4.16). This separation was attributed to the

morphological variable PORCL, DSL, and LMDL on CAN I, and BCASEL on CAN II,

which accounted for 32% of the total variance. On the graph plotted on CAN I to

CANIII, P. alborufus, P. elegans, and P..\.ilnh,,ti\ were successfully distinguished as

distinct groups. The individuals of P. philippensis and P. p. albiventer were still widely

overlapped, showing similar morphological structures (Figure 4.17). The major

variables that contributed to CAN III were TBL and MTRW.














Table 4.15 Discriminant function analysis of Chinese Petaurista
CAN Eigenvalue Proportion Cumulative

I 18.39 0.60 0.60

II 9.67 0.32 0.92

III 1.85 0.06 0.98

Canonical score
Variable CAN I CAN II CAN III

CRANL 0.54 0.78 0.01

BCASEL -0.16 0.95 0.04

CRANW 0.58 0.74 -0.24

BPORW 0.47 0.83 0.06

PORCL 0.68 0.42 -0.07

PGA 0.55 0.70 -0.06

NAL 0.39 0.71 0.19

TBL 0.24 0.13 0.57

DSL 0.68 0.51 0.01

MTRL 0.42 0.77 0.24

MTRW 0.57 0.58 0.35

LMDL 0.68 0.60 0.14

LMDH 0.39 0.76 0.29

LMTL 0.29 0.55 0.26


In PCA, the first principal factor was the most important, which accounted for

80% of the total variable variance, with the cranial length (CRANL) having the highest

eigenvector value (Table 4.16). Along PRIN I, all specimens of Petaurista were

identified as three different groups (Figure 4.18), which were consistent with the result









of discriminant function analysis (see Figure 4.16). P. alborufus was firstly separated as

a group, and the rest four made up other two groups: one group comprising P. elegans

and P. .\x mil,,i\, and another group including P. philippensis and P. petaurista.

BCASEL with negative eigenvector score was the dominant variable that contributed to

PRIN II.


Table 4.16 Principal components analysis of Chinese Petaurista
PRIN Eigenvalue Proportion Cumulative

I 85.29 0.80 0.80

II 9.63 0.09 0.89

III 2.73 0.03 0.91

Eigenvector score
Variables PRIN I PRIN II PRIN II

CRANL 0.52 0.13 -0.05

BCASEL 0.37 -0.87 -0.07

CRANW 0.27 0.14 -0.56

BPORW 0.41 0.05 -0.07

PORCL 0.10 0.18 -0.32

PGA 0.22 0.06 -0.19

NAL 0.17 -0.01 0.19

TBL 0.03 0.03 0.17

DSL 0.11 0.15 -0.07

MTRL 0.13 0.00 0.19

MTRW 0.15 0.12 0.21

LMDL 0.38 0.37 0.23

LMDH 0.22 -0.03 0.37

LMTL 0.12 0.00 0.44









When all specimens were plotted onto PRIN I and PRIN III, except for P.

petaurista and P. philippensis that extensively overlapped, P. alborufus, P. xanthotis,

and P. elegans could be identified as distinct groups, although there existed a few

overlaps between each other (Figure 4.19). The most contributions to PRIN III were

from the morphological variables CRANW and LMTL.

In molecular analyses, the partial sequences (380 bp) were isolated from 8

species (senso lato), including P. alborufus, P. elegans, P. yunanensis, P. hainana, P.

philippensis, P. xanthtis, and P. petaurista (Zhang et al., 1997; Wang, 2002). The

sequence data of P. a. castaneus, P. a. lena, and P. 1. leucogenys (Oshida et al., 2000)

were quoted from GenBank ofNCBI. Pteromys volans was assigned as an outgroup to

reconstruct the phylogeny. Table 4.17 showed the genetic results from pairwise

comparisons between samples. P. elegans and P. .\txihlii\ were the two most diverse

species in Petaurista. The genetic differences between P. elegans and other Petaurista

groups were 9.2% 15.0%. P. xanthtis showed 12.8% 15.4% differences from other

groups. The population of P. alborufus in China (PAC) was apparently different from

that in Japan (PAL) with sequence difference at 12.5% level. The phylogenetic

reconstructions using UPGMA, MP and NJ methods yielded the similar topological

patterns (Figure 4.20, Figure 4.21, and Figure 4.22). P. .\xlin,,i\, P. elegans, and the

population of P. alborufus in Japan formed one group. The population of P. petaurista

in Pakistan and P. yunanensis formed another group. And the rest were clustered

together as the third group, including the population of P. alborufus in Yunna, P.

philippensis, the population of P. petaurista in Yunnan, and P. hainana.







74


Table 4.17 Pairwise comparison of Chinese Petaurista based on the partial sequences
(380 bp) of cytochrome b gene. Data above the diagonal were the percentage of
genetic differences between samples, and data below the diagonal were the
numbers of transitions vs. transversions between samples.
PAK1 PAK2 PAK3 PAC PHK PPH PPF PYK PPY PEK1 PEK2 PEK3 PAL PTK PVO
PAK1 0.5 0.6 0.6 8.5 6.3 9.7 8.4 6.4 13.7 13.1 13.7 12.5 14.4 14.8

PAK2 2/0 0 0 8.2 5.7 9.7 8.4 6.1 13.2 12.8 13.1 12.5 13.8 14.7

PAK3 2/0 0 0 8.2 5.4 9.3 8.4 5.9 13.3 12.8 13.1 12.7 13.7 14.5

PAC 2/0 0 0 8.4 5.9 10.0 8.7 6.0 12.9 12.8 13.1 12.5 14.2 14.7

PHK 30/1 29/1 29/1 30/1 5.6 9.7 9.6 5.3 12.8 12.8 12.9 15.1 13.4 17.0

PPH 24/0 22/0 20/0 22/0 19/1 9.1 8.9 1.8 10.8 9.2 9.9 14.7 15.4 16.4

PPF 35/2 35/2 33/2 36/2 33/3 33/2 6.1 8.3 13.4 13.1 14.0 14.5 12.8 17.0

PYK 28/4 28/4 27/4 28/4 31/5 30/4 21/2 8.9 14.2 14.5 15.0 15.6 14.6 17.6

PPY 24/0 23/0 23/0 22/0 19/1 5/0 30/2 30/4 9.8 9.9 9.9 12.9 14.4 15.8

PEK1 44/8 42/8 42/8 40/8 40/8 33/8 41/10 42/12 30/8 4.5 4.4 16.1 14.1 18.1

PEK2 41/8 40/8 40/8 39/8 38/10 26/8 41/8 47/8 29/8 16/1 1.1 13.9 13.5 17.0

PEK3 44/7 41/8 41/8 39/8 37/9 30/7 45/7 49/7 29/8 14/2 4/0 14.5 15.1 17.2

PAL 38/8 38/8 39/9 37/8 46/10 47/8 43/11 48/10 44/5 48/13 38/12 42/12 14.1 15.8

PTK 44/10 42/10 41/9 43/10 40/9 48/10 38/10 43/12 47/8 41/12 41/9 46/10 38/15 19.3

PVO 33/22 33/22 32/22 33/22 40/24 39/22 43/20 46/20 38/23 43/25 42/21 42/21 38/20 50/23


To estimate the divergence time between species and populations, the

transversional substitutions at the third codon positions were obtained from pairwise

comparison (Table 4.18). The divergence times were calculated using the rate of

divergence for the third codon positions of mammalian cytochrome b gene of ca. 0.5%

*106 (Table 4.19). P. .xltlh,,ili and P. elegans were the earliest species that diverged

from Petaurista, approximately 11.2 to 13 million years ago. In P. petaurista, the

divergence time between the population in Pakistan and the population in W Yunnan

was about 1.2 3.2 million years ago.









Table 4.18 Transversional substitutions at the third codon positions of the partial
sequences (375 bp) of cytochrome b gene in Petaurista.
PAK PYK PPH PHK PEK PTK PPF PPB PPY PVO
PAK 2.4 0 0 4.8 5.6 1.6 0 0 15.4

PYK 3 2.4 2.4 6.5 5.6 0.8 1.6 2.4 13.8

PPH 0 3 0 4.8 5.6 0.8 0 0 15.4

PHK 0 3 0 5.6 5.6 1.6 0 0 15.4

PEK 6 8 6 7 6.5 7.3 5.6 4.8 16.3

PTK 7 7 7 7 8 5.6 6.5 5.6 13.8

PPF 2 1 1 2 9 7 0.8 0.8 13

PPB 0 2 0 0 7 8 1 0 15.4

PPY 0 3 0 0 6 7 1 0 15.4

PVO 19 17 19 19 20 17 16 17 19

Note: Data below the diagonal are the transversional numbers at the third codon
positions. Data above the diagonal represent the transversional
percentage difference between samples. The sample abbreviations were
defined in Table 4.6.


Table 4.19 The estimated divergence time between species based on a
divergence rate for the third codon positions of mammalian
cytochrome b gene of ca. 0.5% *106 years
PAK PYK PPH PHK PEK PTK PPF PPB PPY
PYK 4.8

PPH 0 4.8

PHK 0 4.8 0

PEK 9.6 13 9.6 11.2

PTK 11.2 11.2 11.2 11.2 13

PPF 3.2 1.6 1.6 3.2 14.6 11.2

PPB 0 3.2 0 0 11.2 13 1.6

PPY 0 4.8 0 0 9.6 11.2 1.6 0

PVO 30.8 27.6 30.8 30.8 32.6 27.6 26 30.8 30.8









4.4 Discussion

4.4.1 Phylogeny of the Trans-Himalayan P. petaurista (albiventer)

Petauristapetaurista is extensively distributed in southeastern China, Sichuan,

Yunnan and Fukien. This species has a broad distribution beyond China including

northern India, Bhutan, Nepal, Pakistan, northern Afghanistan, and southeastern Asia.

The populations of P. petaurista in Pakistan and W Yunnan, China, are named as the

same subspecies, P. p. albiventer (Corbet and Hill, 1992; Zhang et al., 1997) since their

similar external and dental structures (Figure 4.23). Wang (2002) elevated the

populations of P. petaurista in Pakistan and W Yunnan (China) as a valid species, P.

albiventer. The present study based on the morphological and molecular analyses reveal

that these two populations are significantly different. The principal components analysis

indicates that their main differences are in skull size (The eigenvector scores are all

positive on the first principal component factor) and the morphological structure of

lower jaw, which apparently separate it from the rest populations (Figure 4.10 and

Figure 4.12). When compared with other populations, the population in Pakistan shares

more cranial characteristics with the populations from SE Asian rather than with P.

albiventer in Yunnan, China. This can be inferred that these two populations may

occupy different habitats that result in different adaptations. This inference agrees with

Ellerman's (1940) study, which shows that the forms candidula, barroni, and taylori of

P. petaurista in SE Asia are much similar to the Himalayan forms (P. p. albiventer).

The only species or subspecies of Petaurista distributed in Pakistan is the

Himalayan giant flying squirrel, P. (p.) albiventer. The area of suitable forest where this

giant flying squirrel can be found is comparatively limited. In Pakistan, it mainly occurs

in Himalayan moist temperate forest, extending in the northwest of Pakistan into Deodar









(Cedrus deodara) forest or subtropical pine (Pinus roxburghii) zone, elevation from

about 1,350 m to upper limit of the tree line at about 3,000 m (Roberts, 1997). P.

petaurista is sympatrically distributed in N Pakistan with E. cinereus and Eoglaucomys

fimbriatus. The competition for food resources among them is unavoidable. Since they

have different morphological dental structures, the feeding strategies and food selection

are different. The giant flying squirrel selectively feeds upon the young green leaves,

the fir and pine cones, the nuts, even the young twigs and tree buds (Quercus dilatata),

which are partially different from Eupetaurus and Eoglaucomys (Roberts, 1997).

Himalayan moist temperate forest commonly consisting of the hill oak (Quercus

dilatata), horse chestnuts (Aesculus indica), and walnuts (Juglans regia) supplies this

species with sufficient food resource. Since partitioning of microhabitats among

competing species is thought to contribute to coexistence among rodent species (Price,

1978), the different habitat selections of these three flying squirrels are a major

contributor to this coexistence.

The similar sympatric distribution of E. cinereus, P. petaurista, and Hylopetes

alboniger are also found in W Yunnan, China. P. p. albiventer in W Yunnan is widely

overlapped with H. alboniger from NW Yunnan toward southern Yunnan at different

elevations (500m to 3500m) (Zhang et al., 1997). These areas have typically high

diversity of plant species that correspond with the mixed dietary habits of these flying

squirrels. According to local people, both flying squirrels mainly inhabit in coniferous

forest. P. petaurista frequently feeds on walnuts, acorns, and corns, and occasionally

descends into farm to feed on corn at dawn. This is a little different from the population

of Pakistan, indicating that the Pakistan giant flying squirrel is more adaptable to harsh









or less mesic conditions than the giant flying squirrel in W Yunnan. Their

morphological differentiations, such as skin (Figure 4.23), are apparently associated with

their different living conditions.

The molecular data are partially consistent with the morphological data. The

difference in cytochrome b gene between the eastern and the western trans-Himalayan

giant flying squirrel (P. petaurista) is significant (Figure 4.13 and Figure 4.14). The

genetic distance is about 8.9%, above the subspecies-level (Table 4.14). Geologic

history has been a major factor in understanding evolution of flying squirrels. The

geotectonic and paleoclimatic records reveal a series of episodic landscape

transformations throughout the past millions of years coincident with changes in taxa

and ecological diversity. Based on the rate at the third codon positions of cytochrome b

gene (Table 4.18), the Pakistan population of P. petaurista diverged from the Yunnan

population about 1.6 million years ago (Table 4.19). The distinct phylogeographic

discontinuity between the eastern and the western lineages of P. petaurista suggests a

major environmental impediment to gene flow. The possibility that the coincident

pattern between these two populations were caused by a shared historical dispersal event

is supported by a diverse array of vertebrate taxa that exhibit a similar genetic

discontinuity in the trans-Himalayas (Woods, personal communication). The

populations of P. petaurista delineated by large phylogenetic gaps are obviously

associated with the biogeographic barrier of the great Himalayan chain to gene flow.

The climatic changes could lead to an expansion of the Palearctic fauna at the

expense of the Oriental fauna, though some generalized species or population of Oriental

forms would undoubtedly be able to adapt to the new conditions. The results of the









present study show that the genetic differences among the populations in SW China, SE

Asia, and Pakistan are not closely associated with the geographic distances of sampling

localities. This implies that P. petaurista rapidly extended into SW China, Pakistan, and

SE Asia in a short time during the southward expansion of temperate forests during the

glacial stage of Pleistocene. The estimations of the third codon positions of cytochrome

b sequences indicate that all populations of P. petaurista diverged from each other

during Pleistocene and Holocene. When all populations split from their common

ancestor in Pleistocene, one branch migrated to the western side what is now in Pakistan

where the climate in northern part and indeed the whole Indus plain, was evidently

warmer and more humid in early Pleistocene times (Roberts, 1997). One moved to the

eastern side what is now in SW China, and the third branch migrated to SE Asia. During

the subsequent glaciation in the late Pleistocene and Holocene that was intervened with

shorter periods of warmer moister climatic change, all populations gradually adapted to

their present habitats. The similar cranial structures among the populations in SE Asia

are apparently the adaptations of similar living conditions.

P. petaurista is a polymorphic species and is extensively distributed in various

geographical locations. Like many wide-ranging taxa, it is divided into separate species

or subspecies by zones of hybridization (Bull, 1991). Because hybrid zones involve

closely-related taxa at various stages of speciation, they represent natural settings for

study of speciation, gene flow, adaptation, and reinforcement of isolating mechanisms

(Baker et al., 1989; Harrison, 1990, 1993; Bendict, 1999). Therefore, the further study

of P. petaurista should focus on the populations between SW China and SE Asia, and









between the eastern and the western trans-Himalayas, such as the populations in Burma,

India, Thailand, and Laos.

4.4.2 Taxonomic Status of P. philippensis, P. yunanensis, and P. hainana

P. hainana (Hainan giant flying squirrel) in Hainan, P. yunanensis (Yunnan

flying squirrel) in Yunnan, and P. grandis (Taiwan giant flying squirrel) in Taiwan were

referenced as valid species by Allen (1940) and Ellerman (1940), but Hoffmann et al.

(1992) and Nowak (1999) regarded them as the subspecies or synonyms of P.

philippensis (gray-backed giant flying squirrel). By checking the collections in Beijing

and Yunnan, China, Zhang et al. (1997) and Wang (2002) elevate them as distinct

species recently. Taiwan is an island situated on the continental slope of mainland China

and separated by the 150 km strait. It is known to have been connected by a landbridge

to mainland China several times during the Pleistocene. In the last warm glacial period

(10, 000 to 56, 000 years ago), sea levels decreased by 80 to 150m (Lin, 1966). This

indicates that P. grandis migrated to Taiwan from south China during Pleistocene. The

findings from both molecular and morphological data in this study support P. p. grandis

to be a distinct species (Figure 4.2 and Figure 4.4), which are consistent with Oshida's et

al. (2000a) result and suggest P. grandis to be a valid species.

The molecular and morphometric analyses on the remaining populations show

some controversial results. The multivariate analyses indicate that P. hainana

apparently differs from the rest populations in cranial morphology and P. philippensis in

Yunnan is morphologically similar to P. yunanensis (Figure 4.2 to Figure 4.5). Whereas

the molecular data suggest that P. hainana is genetically close to P. philippensis with

short genetic distance (- 4 5%). P. yunanensis is evidently distinguishable from P.









hainana and P. philippensis with genetic differences about 10.9% and 8.6%,

respectively (Table 4.10).

Yunnan flying squirrel P. yunanensis is a very handsome maroon flying squirrel

with white speckling over the back (Figure 4.24). The type specimen is a skin without a

corresponding skull. It was collected from Momein (=Tengyueh), southwestern

Yunnan, China, and is represented in the Indian Museum, Calcutta with a catalog

number 9486. P. yunanensis resembles P. alborufus in size, but is morphologically

similar to the population of P. philippensis in Yunnan in the general deep bay coloring

(Figure 4.24). Both skulls are large but apparently show no special peculiarities. The

distribution of P. yunanensis is from the extreme southwestern Yunnan probably into

Burma and Indochina, extensively sympatric with P. philippensis in SW China (Zhang

et al., 1997). Although at present it is not clear to their feeding habits, their

morphological similarity in skull is clearly due to the adaptations to similar habitats.

The similar morphological characters and the distinct genetic characters between them

suggest that conservative systematic traditions or morphological stasis may be involved.

According to the estimated divergence time, P. yunanensis diverged from the stock of P.

philippensis about 4.8 million years ago, the early Pliocene. There are known to have

been three major periods of successive prolonged glaciation when sea levels sank and

huge ice caps developed over all the great mountain massifs during Pliocene, which

dramatically affected the climate, biogeographic structures, and flauristic environments

along the great Himalayan mountain chain. As a result, the eastern and the western

extremes of Himalayas became the optimal shelter of refugees. A quite unique

mammalian assemblage including flying squirrels migrates and survives today in the









western side of Yunnan of China. Although the range of P. yunanensis is overlapped

with P. philippensis in Yunnan, there is no indication that the two interbreed. The

significant differentiation in genetic characters suggest that P. yunanensis is a distinct

species, notwithstanding the general similarity in size and body coloration with P.

philippensis.

The type specimen ofP. hainana is an adult female skin with the corresponding

skull, which was collected from Namfong, the island of Hainan, China, on February 19,

1923, by Clifford H. Pope (Allen, 1940). It is now represented in the collection of

American Museum of Natural history. P. hainana is a tropical species that reaches the

northern limit of its range in extreme southern China. Allen (1940) predicted that no

doubt Hainan giant flying squirrel (P. hainana) would be found to show relationship to

some forms of the Indo-Chinese mainland. The present study demonstrates that P.

hainana is phylogenetically related to P. philippensis in Yunnan. P. philippensis is

distributed in mountainous coniferous forests at different elevations in W Yunnan;

whereas P. hainana is confined to tropical forest on Hainan. Either the feeding habits or

the living habitats are significantly different. The morphological difference between

them is probably associated with their geographical variations and living conditions.

But the close genetic distance between P. hainana and P. philippensis 5 7%, is not

consistent with their geographic distances of sampling localities. The conflicting

placement on the molecular and morphological trees is difficult to reconcile because the

phylogenetic hypothesis suggested by the molecular tree requires convergent evolution

in morphology and osteology to reflect similarities in these characters among the clades

(Austin, 1996). The estimation based on the third codon positions of cytochrome b gene