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Population Genetics and Conservation of the Florida Manatee

Permanent Link: http://ufdc.ufl.edu/UFE0041115/00001

Material Information

Title: Population Genetics and Conservation of the Florida Manatee Past, Present, and Future
Physical Description: 1 online resource (144 p.)
Language: english
Creator: Bonde, Robert
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: behavior, biology, conservation, genetics, manatee, population, sirenia
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: POPULATION GENETICS AND CONSERVATION OF THE FLORIDA MANATEE: PAST, PRESENT, AND FUTURE Robert K. Bonde 352-264-3555; rbonde@usgs.gov Department of Physiological Sciences Dr. Peter M. McGuire Doctor of Philosophy December 2009 Examination of the biology and genetics of manatees helps to explain their pattern of dispersal through evolutionary time. The West Indian manatee reached peninsula Florida about 15,000 years ago between the last glacial ice age. Consequences related to founder effect and recent bottlenecks have reduced the genetic diversity in the population. Recent population growth may be short lived as questions remain about potential for gene flow and reproduction. Detailed genetic investigation of manatees in Florida reveals interesting traits related to behavior and distribution. Calves learn migratory and habitat use patterns from their mothers and pass this information on through generations. This information will help managers detect patterns in the Florida population of this endangered species. The future of the manatee in Florida teeters on their resilience and the ability to adapt despite rapid changes imposed by human population growth.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Robert Bonde.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: McGuire, Peter M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041115:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041115/00001

Material Information

Title: Population Genetics and Conservation of the Florida Manatee Past, Present, and Future
Physical Description: 1 online resource (144 p.)
Language: english
Creator: Bonde, Robert
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: behavior, biology, conservation, genetics, manatee, population, sirenia
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: POPULATION GENETICS AND CONSERVATION OF THE FLORIDA MANATEE: PAST, PRESENT, AND FUTURE Robert K. Bonde 352-264-3555; rbonde@usgs.gov Department of Physiological Sciences Dr. Peter M. McGuire Doctor of Philosophy December 2009 Examination of the biology and genetics of manatees helps to explain their pattern of dispersal through evolutionary time. The West Indian manatee reached peninsula Florida about 15,000 years ago between the last glacial ice age. Consequences related to founder effect and recent bottlenecks have reduced the genetic diversity in the population. Recent population growth may be short lived as questions remain about potential for gene flow and reproduction. Detailed genetic investigation of manatees in Florida reveals interesting traits related to behavior and distribution. Calves learn migratory and habitat use patterns from their mothers and pass this information on through generations. This information will help managers detect patterns in the Florida population of this endangered species. The future of the manatee in Florida teeters on their resilience and the ability to adapt despite rapid changes imposed by human population growth.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Robert Bonde.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: McGuire, Peter M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041115:00001


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1 POPULATION GENETICS AND CONSERVATION OF THE FLORIDA MANATEE: PAST, PRESENT, AND FUTURE By ROBERT KNUDSEN BONDE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Robert Knudsen Bonde

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3 To the perpetuation of mermaid myths

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4 ACKNOWLEDGMENTS I would like to thank my committee, especially Dr. Peter McGuire whose ideas, guidance, and direction were a major contri butor for all our present day unders tanding of sirenian genetics. It was through his unselfish determination and fortitude that all of his enthusiastic graduate students, me included, found their strengths. Dr. Roger Reep was the guiding light that led me into graduate school so many years after establis hing my career with the Si renia Project. Rogers encouragement, perseverance, and compassion helped mold my personality and way of approaching problems too. Dr. Lynn Lefebvre was my mentor and direct -line supervisor for much of my professional career with U. S. Geological Survey (USGS) Sirenia Project. Through her support, I was able to juggle the responsibil ities of my job with those required of my academic career. Dr. Tim King of the USGS Leetown Science Center whose expertise on population genetics is rivaled by few in the wo rld; Tim your guidance and advice are ingrained and treasured by all immersed in wildlife geneti cs today. Finally, Dr. Ruth Francis-Floyd, who joined my committee in the later stages and st ill provided upbeat enthusiasm, even when times appeared discouraging. Thanks to all. Of particular importance to me and my career is Dr. Maggie Ke llogg Hunter (fellow graduate student) who guided me through the final stages of my work and who will help lead future genetic efforts at the USGS Sirenia Project. Other stellar graduate students from UF who shared their knowledge are Dr. Angela Garcia -Rodriguez who designed the first studies and thinking relative to trichechid biogeography and la id the foundation for development of Florida manatee population genetics. Thanks to Dr. Kim Pause Tucker (University of South Florida) who carried the torch further and helped establish our present day understanding of Florida manatee genetics. None of these studies would have been possible without the knowledge and tutoring provided by AnnMarie Ginger Cl ark of the University of Florida (UF)

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5 Interdisciplinary Center for Bi otechnology Research genetics lab. Ginger is a great friend and resource that we have all become accustomed to use during times of uncertainty. Fellow student, Coralie Nourisson, who is conducting similar stud ies on manatee genetics in Mexico, also is thanked. Good luck with your research Coralie. I also thank Marta Rodriguez-Lopez from Puerto Rico who developed a pioneering genetic study on Antillean manatees a number of years ago. Dr. Juliana Vianna was also very helpful in sharing her study of manatee populations from throughout their range using mtDNA and microsatellite analysis in Brazil. The critical thinking about evolution by Dr. Daryl Domning of Howard University, who was responsible for much of the formulation leading to the marriage (dispa rities) between paleontology and genetics, is greatly appreciated. International researchers that have aided the sirenian gene tics programs with valuable sample collection include Nicole Auil, Belize; Dr. Miriam Marmontel, Brazil; Dr. Antonio Mignucci-Giannoni, Puerto Rico; Drs. Janet Lanyon and Helene Marsh, Australia; and Dr. Benjamin Morales-Vela, Mexico. A sincere than ks to my colleague and dear friend, Dr. James Buddy Powell with Sea to Shore Alliance. A ll the manatee rescue teams and rehabilitation facilities also are owed a great deal of gratitude for assistance with genetic sample collection, as well as Kit Curtin for her efforts in South Florid a. Federal research permits and encouragement was extended through many of our colleagues in management, especially the U.S. Fish and Wildlife Service. Particular thanks are exte nded to Monica Farris, Nicole Adimey, Dawn Jennings, and Jim Valade (thanks Jim, who purchased my first PCR machine which is still in use today), as well as all the members of the Coor dinated Florida Manatee Genetics Working Group. This project would not have been possible wi thout the foresight and stalwart efforts of many dedicated people, especially the staff of friends and colleagues at the USGS, Sirenia

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6 Project. Cathy Beck, Jim Reid, Susan Butle r, Amy Teague, Gaia Meigs-Friend, Howard Kochman, Dr. Cathy KB Langti mm, Dr. Tom OShea, Dr. Gale n Rathbun, Dr. Lynn Lefebvre, and Dr. Jeff Keay. Thanks to Dr. Kay Briggs (USGS), Dr. Daryl Domning (Howard University), Dr. Bill Farmerie (UF), Sean McCann (UF), and Dr. John Reynolds III (Mote Marine Lab). A special thanks to all our colleagues at the Fl orida Fish and Wildlife Conservation Commission, especially Michelle Davis, Dr. Charles Chip Deutsch, Dr. Martine de Wit, Andy Garrett, Tom Pitchford, Dr. Mike Tringali, and Leslie Ward-Geiger. A special thanks to my wife best friend, and companion during our journey with the manatees, Cathy Beck, and to our children, Michae l and Julie, that through their eyes we hope to be able to continue to carry on the pu rsuit of science and understanding.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF FIGURES .......................................................................................................................11LIST OF ABBREVIATIONS ........................................................................................................ 13Organizations ..........................................................................................................................13General Abbreviations ............................................................................................................13Laboratory .................................................................................................................... ...........14Genetics ...................................................................................................................... ............14Genetic Analyses ....................................................................................................................15ABSTRACT ...................................................................................................................... .............17CHAPTER 1 INTRODUCTION .................................................................................................................. 19Project Summary ............................................................................................................... .....19The Manatee ...........................................................................................................................20Sirenian Evolution ...........................................................................................................20Florida Manatee Biology .................................................................................................21Overview of Manatee Status, Research, and Management ....................................................24Why Do We Need to Study Manatee Genetics? .....................................................................292 TRICHECHID EVOLUTION ................................................................................................ 35Systematic Characters ......................................................................................................... ....35Manatee Evolution ..................................................................................................................36Population Expansion .......................................................................................................... ...41Molecular Status .............................................................................................................. .......44Extinction Processes ...............................................................................................................453 CRYSTAL RIVER MANATEE FINE-SCAL E Population ANALYSIS .............................. 53Introduction .................................................................................................................. ...........53Materials and Methods ...........................................................................................................58Sample Collection and DNA Extraction ......................................................................... 58DNA Isolation .................................................................................................................58Microsatellite Analysis ....................................................................................................58Statistical Analysis .......................................................................................................... 59Neighbor-joining trees ..............................................................................................60Analysis using STRUCTURE ......................................................................................61Multivariate analysis ................................................................................................ 61

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8 Results .....................................................................................................................................62Microsatellite Marker Analysis .......................................................................................62Cluster Analysis Using Multi-locus Genotypes .............................................................. 62Neighbor-joining trees ..............................................................................................63Analysis using STRUCTURE ......................................................................................64Multivariate analysis ................................................................................................ 64Individual identity ....................................................................................................64Cow/Calf Relationships ...................................................................................................65Grandmother Pedigrees ...................................................................................................66Discussion .................................................................................................................... ...........66Population Biology .......................................................................................................... 66Individual Identity ...........................................................................................................68Cow/Calf Relationships ...................................................................................................70Potential for Adoption .....................................................................................................70Grandmother Pedigrees ...................................................................................................72Conclusion .................................................................................................................... ..........724 MOLECULAR TOOLS FOR M ANATEE CONSERVATION ............................................89Sirenian Conservation .............................................................................................................89Why Study Manatees? ............................................................................................................91Genetic Markers ......................................................................................................................92Allozymes ..................................................................................................................... ...92Mitochondrial DNA .........................................................................................................93Microsatellites ............................................................................................................... ..93SNPs .......................................................................................................................... ......94Gene Expression ..............................................................................................................95Genomes ....................................................................................................................... ...96Implications for Manatee Conservation .................................................................................. 97Genetic Samples ..............................................................................................................97Molecular Tools ...............................................................................................................975 CONCLUSIONS AND FUTURE DIRECTIONS ............................................................... 101Recovery of an Endangered Species in Florida ....................................................................101Manatee Analysis using 454 GS-20TM Pyrosequencing Technology ............................ 103Future Directions ..................................................................................................................105Sequence the Manatee Transcriptome usi ng Massively Parallel Pyrosequencing ........105Genetically Identify Individual Crystal Rive r Manatees and Integrate Data with the Manatee Individual Photo-identification Sy stem for Capture-Recapture Studies ..... 108Examine the Effective Population Size ( Ne) of the Current and Past Florida Manatee Population ...................................................................................................109Examining the Evolution and Relatedness of Trichechids ............................................ 110Continue Collaboration on Wildlife Species with USGS Scientists and International Researchers to Further Genetic Capabilities .............................................................. 113Conclusions ...........................................................................................................................114

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9 APPENDIX: TAIL NOTCH PROTOCOL ..................................................................................123Protocol for Collection of Manatee Tissue Sa mples for Genetic Research March 2009 .. 123LIST OF REFERENCES .............................................................................................................128BIOGRAPHICAL SKETCH .......................................................................................................144

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10 LIST OF TABLES Table page 3-1 Manatee sample collection information. ............................................................................ 743-2 Characteristics of the 11 polymorphic micros atellite loci utilized for the analysis of Crystal River manatee ( T. m. latirostris ) samples. ............................................................ 805-1 List of di-, tri-, and tetra-nucleotides identified using FLX technology on manatee DNA with number of repeats for each sequence. ............................................................ 118

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11 LIST OF FIGURES Figure page 1-1 Timeline of genetic re search studies conducted on the Florida m anatee. .......................... 321-2 Pregnant Florida manatee at Crystal Rive r, Florida. (Photo by R.K. Bonde, USGS, Sirenia Project)...................................................................................................................331-3 Sirenian global distribution. Red is St ellers sea cow, pink is dugong, light blue is Amazonian manatee, dark blue is West Indian manatee, and green is West African manatee. ...................................................................................................................... .......342-1 Evolution of sirenians with comparisons of cranial morphology; adapted from Domning 2005 (Reep & Bonde 2006). .............................................................................. 482-2 Sirenian rostral deflection from least to greatest. For speci es designations, Ts = Trichechus senegalensis ; Ti = T. inunguis ; Tmm = T. manatus manatus ; Tml = T. m. latirostris ; and Dd = Dugong dugon .................................................................................492-3 High sea level. During a warming of climat e, water is released from sea ice resulting in an increase in sea level. The orange co lor is land mass at high sea level, outline is land mass, and water level is depicted in light blue. .......................................................... 502-4 Moderate sea level. This is the presen t day condition. The orange color is land mass at middle sea level and water leve l is depicted in light blue. ............................................. 512-5 Low sea level. During very cold clima tic conditions, ocean water is tied up in sea ice resulting in a lowering of sea level. The orange color is land mass at low sea level, outline is land mass today, and wate r level is depicted in light blue. ...................... 523-1 Map of Caribbean region depicti ng manatee haplotypes, from Vianna et al 2006. .........813-2 Notcher used to remove a small piece of skin from the tail margin of manatees. ............. 823-3 Probability of identity ( P(ID)) is illustrated for the manatee population calculated for sibling association (in red), for Hardy Weinberg equilibrium (in blue), and for values observed in this study (in green) with step-w ise addition for each allele in order of decreasing identity using GENALEX 6. ..............................................................................823-4 Neighbor-joining tree describing the re lationships among individuals from the Crystal River, Florida manatee population. Known family units are depicted by similar colors. Two distinct cl usters are apparent and labeled A and B with an outlier group C This neighbor-joining tree was rooted fo r clarity to help vi sualize clusters. ..... 83

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12 3-5 Summary plot of q estim ates generated by the seque ntial cluster analysis of the program STRUCTURE using a K = 3 value performed on th e Crystal River, Florida population. Individuals assigne d to neighbor-joining tree clus ters are indicated by A, B, or C. ...............................................................................................................................843-6 Crystal River, Florida manatees depi cted in a neighbor-joining tree using the program, PHYLIP and color-coded to assigned clusters generated from STRUCTURE using a K = 3 value with q estimate > 60. .......................................................................... 853-7 Summary plot of q estimates generated by the seque ntial cluster analysis of the program Structure using a K = 2 value performed on the Crystal River, Florida manatee population and compared to the rest of Florida. At top is the structure output generated by Pause 2007 for all four management areas of Florida. ...................... 863-8 Scatter plot of genetic distance versus re latedness of Crystal Ri ver manatee samples. As genetic distance decreases, relatedness increases (P < 0.001)......................................873-9 Crystal River, Florida manatees compared with PAST multivariate analyses using non-metric multidimensional scaling for rela tedness (on left) and genetic distance (on right). Each graph wa s color-coded with six known individual family groups observed over time with life history observ ations. Lines correspond to convex hulls, or polygons, containing all po ints of that color. ................................................................ 885-1 Population size as compared to genetic diversity and fu ture trends (adapted from Wahlund 1928). Population size and time are not scaled to speci fic events. How will the Florida manatee population respond with an anticipate d loss of allelic diversity? .................................................................................................................... ......1195-2 Current winter Florida ma natee subpopulation designations (shaded area). Blue stars are natural warm water sour ces, yellow stars are passive (alternate) warm water sources, and red stars are artifical sources of warm water. Four management units are represented by shaded areas; Northwest (pink), Southwest (blue), upper St. Johns River (green), and Atlantic coast (yellow). ......................................................................1205-3 Total number of di,tri-, and tetranucleotides identified using FLX technology on manatee DNA, of 346 nucleotide repeat s detected during one titration run. ...................1215-4 Number of putative microsatellites with di ,tri-, and tetra-nucleotides with at least 40 bp of flanking region at each end using FLX technology on manatee DNA. ............. 1215-5 Potential winter Florida manatee subpopul ation designations (shaded area) after the loss of artifical sources of warm water in winter. Blue stars ar e natural sources and yellow stars are passive (alternate) sources of warm water. Note the loss of artifical sources of warm water previously depicted by red stars and the anticipated shift in wintering population patterns. .........................................................................................122

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13 LIST OF ABBREVIATIONS Organizations ABI Applied Biosystems FWC Florida Fish and Wildlif e Conservation Commission FWRI Fish and Wildlife Research Institute SESC Southeast Ecological Science Center UF University of Florida UF ICBR University of Florida, Interdisci plinary Center for Biotechnology Research USFWS United States Fish and Wildlife Service USGS United States Geological Survey General Abbreviations Bp A single base pair CR Crystal River, Florida DPSs Distinct population segments ESA Endangered Species Act FLXTM Roche 454 standard technology GS-20 Roche 454 Genome Sequencer MUs Management units Mbp Million base pairs MIGS Manatee Individual Gene tic-identification System MIPS Manatee Individual Phot o-identification System MMPA Marine Mammal Protection Act mya Million years ago

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14 Laboratory BSA Bovine serum albumin DMSO Dimethyl sulfoxide EDTA Ethylenediaminetetraacetic acid EtOH Ethyl alcohol MgCl2 Magnesium chloride NaCl Sodium chloride PCR Polymerase chain reaction SDS Sodium dodecyl sulfate Tris-HCl Tris(hydroxymethyl )aminomethane hydrochloride Genetics cDNA Complementary DNA CO1 Cytochrome c oxidase 1 d-loop Displacement loop dNTP Deoxynucleotide triphosphate DNA Deoxyribonucleic acid mtDNA Mitochondrial DNA ncRNA Non-coding RNA PCR Polymerase chain reaction PSA Proportion of shared alleles RNA Ribonucleic acid SAA Serum amyloid-A SNP Single nucleotide polymorphism repeat

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15 SSRs Simple sequence repeats STRs Short tandem repeats VNTRs Variable number of tandem repeats Genetic Analyses FST Fixation index. The proportion of genetic variation distributed among subdivided populations. This is used as an index for estimating gene flow and population subdivision. HE Expected heterozygosity. A measur e of the expected proportion of heterozygotes in the population based on the princi ple of Hardy-Weinberg equilibrium. Ho Observed heterozygosity. The actua l proportion of heterozygotes in the population calculated from the dataset. HWE Hardy-Weinberg equilibrium. A la w stating an idealized population of diploid organisms that reproduces sexually in a random fashion with nonoverlapping generations. The mode l population of infinite size experiences no mutation, migration, and selection processes. In population genetic data analysis, this principle states that allele frequencies will remain the same, but genotype frequencies will change over time. Values for Ho and HE are used to test if allele frequencies meet HWE expectations. K Number of genetic population cl usters assigned by the program STRUCTURE. MCMC Markov Chain Monte Carlo. An an alog statistical technique used to estimate the probability of distributi on for all possible combinations of parameter values in equilibrium dist ribution (Excoffier & Heckel 2006). N Number of samples NA Number of alleles NE Effective number of alleles. The measur e of allelic diversity that takes into account the homozygosity as well as the number of alleles (Yeh & Boyle 1997). Ne Effective population size

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16 NJ Neighbor-joining tree P( ID ) Probability of Identity. Probability of choosing two individuals from a population that has identical genot ypes at the loci examined. P( ID )HW Standard probability of identity as suming sample is in Hardy-Weinberg equilibrium (Waits et al. 2001). P( ID )sib Probability of identity of siblings wh ich accounts for related individuals in the dataset (Waits et al. 2001). PR(X/K) Log probability of data q estimate Amount of admixture associated with assignment of an individual to a population cluster using the program STRUCTURE. Admixture of individuals within a population corresponds to mixed ancestry.

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17 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy POPULATION GENETICS AND CONSERVATION OF THE FLORIDA MANATEE: PAST, PRESENT, AND FUTURE By Robert Knudsen Bonde December 2009 Chair: Peter M. McGuire Major: Veterinary Medical Sciences Manatees have the ability to establish new populations within their subtropical range, as evidenced by their evolutionary history and genetic traits. Although vicariance separated the taxa over time, the vagility of this unique group of aquatic mammals enabled populations to disperse through deliberate migration or stocha stic events. Although nov el habitats are not always suitable, the trichechines exhibit adaptiv e plasticity, as shown by behavioral modification and distinct, subtle morphological characte ristics among populations (Domning 2005). The Florida manatee population was established recen tly, within the last 1215,000 years. Genetic profiling examining relatedness a nd genetic distance among family units of manatees in the Crystal River subpopulation provides clues of behavioral traits that support familial distribution and potential mate selection patt erns. Thus, within peninsular Florida, where populations are well established, distinct habitat t ypes require manatees to maintain different survival strategies that are passed on from generation to generation. Although there has been an in crease in the population of Fl orida manatees in recent decades, genetic evidence of prior founder events affecting the population is still evident. A reduction in manatee numbers due to anthropogenic loss occurred for centuries, and it will likely take many generations to resolve the genetic cons equences. Scarcity of warm water habitat in

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18 the future will have severe effects on the recove ry of the species. Knowledge of the genetic composition of the population will determine whet her, and to what ex tent, breeding between geographic areas is occurring. Li nking genetic tools with indivi dual photo-identification will assist in understanding the population structure by complementing efforts to model various manatee life history traits. There is low to average genetic variation in the Florida population. However, the genetic data suggest that even with adequate gene fl ow, there may be concerns regarding low allelic diversity within manatee population units due to inbreeding and bottleneck events. Employment of genetic tools will clarify st ochastic demography of the Florida population. The best remedy for this low genetic diversity in the population would be to encourage growth and address conservation practices to allo w breeding between population units throughout all regions of Florida.

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19 CHAPTER 1 INTRODUCTION Project Summary This project is divided into five primar y chapters: Chapter 1, Project Introduction with an introduction to manatee biology and conservatio n; Chapter 2, Trichechid Evolution, which details the evolution of the manatees; Chapte r 3, Crystal River Manat ee Fine-scale Genetic Analysis and how the Florida manatee persists in todays environmen t; Chapter 4, Molecular Tools for Manatee Conservation and where we as researchers are directing future genetic research efforts; and Chapter 5, Conclusions and Future Direc tions. More specifically, in Chapter 1 there is a discussion on the status and the unique biological capabilities of the manatee, and sirenians in general. In Chapte r 2, an evaluation of tr ichechid evolution is illustrated with possible scenarios to explain pres ent day distribution patterns. Chapter 3 covers the present day trends in the Florida manatee popul ation, as well as a review of genetic studies conducted to date, and presents new informati on on the fine-scale population structure of the manatee population in Crystal River, Florida. This information will interface with the existing Manatee Individual Photoidentification System (MIPS) databa se and thus provide information to support assumptions that predict rates and trends in the population. Chapter 4 is an examination of current molecular technologies, capabilities, and tool s suggested for better understanding manatee populations and conservation. Current information on manatee life history, coupled with 454 Next Generation se quencing technology, will direct future research efforts. Lastly, the final chapter assim ilates this information and makes concluding recommendations to help guide managements recovery efforts and improve the understanding of modern evolutionary synthesis.

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20 The Manatee Manatees are large aquatic mammals that can attain a maximum length of over 4 meters and weigh up to 2,000 kilograms (Figure 1-2). De tailed accounts of sirenian biology can be found in Manatees and Dugongs (Reynolds & Odell 1991), Manatees (Powell 2002), Mysterious Manatee (Glaser & Reynolds 2003), and The Fl orida Manatee: Biology and Conservation (Reep & Bonde 2006). A summary of life history characteristics is provided in Table 1-1. Global distributi on of all sirenians is por trayed in Figure 1-3. Sirenian Evolution The mammalian order Sirenia encompasses two unique families, the dugongids and the trichechids (Reep & Bonde 2006). The dugongids are marine adapted and composed of two species, the dugong ( Dugong dugon) and the recently extirpated Stellers sea cow ( Hydrodamalis gigas ). The trichechids on the other hand are aquatically ad apted and represented by the Amazonian manatee ( Trichechus inunguis ), the West African manatee ( Trichechus senegalensis ), and the West Indian manatee ( Trichechus manatus). There are two recognized subspecies of the West Indian manatee, the Antillean manatee ( T. m. manatus ) and the Florida manatee ( T. m. latirostris ). Sirenian distribution has been centered along th e warm equatorial band, with the exception of the Stellers sea cow that inhabited the Beri ng Sea off the Commander Islands of present day Russia. Due to hunting by Russian fur sealers for food in the mid 1700s, the Stellers sea cow was completely eradicated a short 27 years follo wing their discovery (Stejneger 1887). The Stellers sea cow was larger than present day sirenians, up to 8 meters long and weighed approximately 8-10 tons (Scheffer 1972). Their mass gave them a physiological advantage and provided insulation necessary for li fe in arctic conditions. Altern atively, the extant sirenians of semitropical habitats have had an evolutionary tract that met the challenges of removing excess

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21 body heat, not conserving it. It is this mech anism alone that predominantly controls the boundaries of their distribution. Based on phylogenetic analysis the sirenians evolved through a complex series of taxa that originated with the founders of the mammalian superorder Afrotheria, approximately 100 million years ago (mya). Afrotheria al so comprised the ancestral bridge s to the elephants, hyraxes, aardvarks, tenrecs, golden moles, and elephant sh rews. The clade Paenungulata branched from Afrotheria approximately 20 million years after the appearance of that superorder and includes the ancestors of extant sirenians, elephant s, and hyraxes. Protosirenians, such as Pezosiren were present 70 mya and still retained evidence of hind limbs. This genus provided the evolutionary link tying the dugongids and the trichechids togeth er, although these families split about 30 mya (Domning 2005). More recently, within the last 5 million years, trichechids started evolving into the present day manatee species (Domning 2005). Mu ch of the evolutionary adaptations evident today were influenced by glacial events that alte red habitat types, result ing in specialization of different taxa and species. Today many of the differences among these extraordinary mammals can be traced through unique morphological char acters, such as tooth development, bone processing, and more specifically, rostral deflection, all derived by adaptive behaviors and fixed through natural selecti on (Domning & Hayek 1986). Florida Manatee Biology Florida manatees are semitropical, herbivor ous, K-selected, aquatic mammals (Reep & Bonde 2006). Apart from r-selected animals, the Kselected manatees are generally large, have only one or two (~1.5%) offspring at birth, put a gr eat deal of energy into multiple pregnancies, spend long periods nurturing their young, reach sexual maturity late, and have long life expectancies. Manatee anatomy is quite differe nt compared to their most closely related terrestrial relatives and their phys iology restricts them to a life in warm tropical waters. Suited

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22 for a shallow-water existence, manatees generally spend much of their time in and around coastal communities, often bringing them into direct co ntact with humans. Along with other myriad threats associated with coexisting with humans, very often manatees are struck and killed by water vessels (Lightsey et al. 2006; OShea et al. 1985). As more humans and boats are introduced into the habitat, the compatibility between civilization a nd all living diversity, manatees included, is increasingly challenged. The Florida manatee is a unique aquatic mamm al living in diverse habitats throughout its distribution. Florida is at the northern limit of the species distri bution and this subspecies has been subjected to restrictions of population expa nsion primarily due to temperature barriers. Between glacial episodic events, manatees in th e southeastern United States have pulsed in and out of geographic and temperature limited boundari es. During glacial events, land formations were in closer proximity, and allowed trichechids the mobility to cross traditional vicariate barriers into suitable habitats. Behaviorally, manatees in Flor ida quickly adapted to utilize natural warm water springs to meet their therma l needs in winter. By the 1950s they also had adapted to utilize artificial warm water sources, such as power plant effluents, to survive cold winters, a major physiological chal lenge for this subspecies existi ng on the energetic fringe at their northern range limit. Being obligate, genera list herbivores much of their adaptability to occupy these areas has been aide d by the presence of abundant aquatic plants and expansive seagrass beds. They are not dependent on any on e type of food source, bu t rather to a host of species, both native and exotic. Their life history traits have mediated this ability to meet challenges afforded by the environment throughout their evolutionary history. The factors that make manatees so resilient lie in their biological and behavioral traits, whic h are discussed here in detail (Table 1-1).

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23 Florida manatees have an uncanny ability to move over extended ar eas when necessary, although the motives may not be apparent. Their counterparts in the Caribbean probably have less migratory drive as these animals have thei r biological needs met w ithin their restricted tropical range, but would consequently be limited in mate selection and re productive strategies. On the other extreme, Florida manatees must trav el great distances to meet challenges related to temperature and food resources. Home ranges ev en vary among groups of individuals. Some may travel only a few kilometers during the year, whereas others will span greater distances. Manatees in Florida may travel up to several hundred kilometers during the summer months, and in some cases warm season sightings have even been in excess of over 1,000 kilometers from a known wintering site (Reep & B onde 2006). Much of this migr atory behavior is probably hardwired as instinctual behavi or or is reminiscent behavior taught to individuals by their mothers. Much like elephants, manatees have a very specialized ability to replicate distribution patterns taught to them during their period of dependency (Deutsch et al 2003). Cow/calf bonding is very strong and deliberate. Some calves will remain with their mothers for just one year, but most will stay two, three and rarely four years depending on the complexity of meeting challenges related to the habitat within their specific home range. For example, mothers in fresh water systems like th e St. Johns River may only need one year to bond with and teach their young, as there is ample fora ge material and adequate warm water available in the springs of that riverine system to meet their needs ye ar round. Manatees nurtured in conditions that are brackish to saline generally have a longer period of dependence to allow more time for training related to finding food, fresh wate r, and warm water ref ugia during winter. Mothers also teach their calves the migratory ro utes and sites that best meet their needs, regardless of the sex of the calf. Female natal philopatry is also common in elephants (Okello et

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24 al 2008a). However, female manatee calves are mo re likely to follow the mothers pattern than their male counterparts who tend to deviate from their taught site fidelity soon after weaning. This deviation amongst the young males is influenced by loose associat ion with other male counterparts, each possibly with different site fi delity and movement patterns imparted by their mothers. The ranges of young males may therefor e be influenced by hormonal activity and the motivation to breed. Males have adapted to fi nd receptive females for breeding, whereas weaned females are driven by nutritional requirements to meet the challeng es of pregnancy. General social organization in manatees is loose, except for the tight bond between the mother and her dependent calf. Single calves are common, while twinning is rare. Although many manatees are often sighted together, these groups are ge nerally associated with an environmental catalyst that makes the site suitab le for meeting basic biological needs, such as feeding, drinking fresh water, cavorting, breeding, resting, or thermoregulating. Through these interactions among cohorts, post-we aned manatees start to learn ot her patterns and strategies that have been passed on by their mothers. This information continues to build on their life experiences and adaptability so integral for su rvival in these ever-changing environments. People have also directly affected the hab itats, and manatees have, thus far, responded accordingly. Overview of Manatee Status, Research, and Management The trichechids have been resilient throughout their history, but th e ability of Florida manatees to survive perturbations due to habi tat changes and recent anthropogenic threats has made them an appropriate model for championi ng the success of the U.S. Endangered Species Act (ESA). Population expansion events have be en common in this familys 5 million years of evolution, despite the loss of some local populations and some trichechid species. This unique animal has been valued in Florida since early protections for the Florida manatee were

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25 implemented at the turn of the 19th century. Following the implementation of the ESA in the United States in 1967, manatees have been affo rded protections that have undoubtedly allowed growth in the population. Protections anal ogous to the ESA were recommended by the United Nations International Union for the Conservati on of Nature (IUCN) for manatee populations throughout their range. Hunting dur ing the last couple of centuries significan tly reduced the size of the Florida manatee population. However protection measures, coupled with the advent of artificial warm water sites used as cold weat her refugia, the accidental introduction of exotic vegetation used as a food source by manatees, and abundant natural vegetation, enabled the population to rebound (Hartman 1974; OShea 1988). The historic genetic diversity of the population is unknown, but anthropogenic deaths caused a severe decline in manatee numbers during the last century and affected their distribution. Protected by state and local laws in Florida as fa r back as 1893, manatees were recognized early on as a keystone species, bringing not only their p light to the forefront of the community consciousness, but extending that pr otection on to many other types of wildlife and habitats. This appreciation of natural, wild areas and diverse speciation was expressed via additional legislation through the decade s that followed (OShea 1988; OShea et al 2001). Public awareness of the environm ent during the times of Archie Carrs treatise The Sea Turtle: So Excellent a Fishe was growing, along with pub lic interest in wildli fe (Carr 1986). Most significantly, the advent of power plants allowed the manatee population to better survive winters and increase in the sout heastern United States from th e mid-1900s to present (Laist & Reynolds 2005). Long-term field observations, aided by photo-id entification and radio-tracking technology, have provided invaluable data on the life history of the Florida manatee. The first pioneering

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26 study resulting in baseline information on the biology of the manatee was begun in Florida during the late 1960s with the groundbreaking work of Daniel Woody Hartman. In his dissertation, Hartman outlined much of the biolog ical character of the manatees wintering in Crystal River, Florida (Hartman 1971), and late r published a book that remains a highly cited reference on manatee life history (Hartman 1979). Since that time, scie ntists with the USGS Sirenia Project have spent over 30 years studying Florida manatees and have taken the opportunity to examine in fine-sca le the genetic attributes of the Crystal River population. Since donning wet suit, mask, snorkel, and flippers and entering the underwater e nvironment, we have learned quite a bit about the Florida manatee. Understanding the popul ation genetics of the manatee will help us be better stewards and bett er grasp the fundamentals of recovery of this endangered species. Sustainable protection of ma natees requires a better understanding of the genetic background of the species. Early genetic studies of the manatee included allozyme analysis (McClenaghan & OShea 1988) and part ial sequencing of the cytochrome b gene (Bradley et al 1993). Results from the McClenaghan and OSh ea study indicated that the 59 manatee samples examined from the Florida population exhibite d evidence of population heterozygosity with ample gene flow among five selected geographi c areas. The first DNA-based study to follow by Bradley and colleagues provided m itochondrial DNA (mtDNA) cytochrome b sequence data for 225 base pairs to identify 75 amino acid codons. These were then compared to published genes from proboscideans. This study only examined three manatee samples, all sharing the same haplotype, but suggested intraand inter-spec ies evolutionary eviden ce linking sirenians to proboscideans. It was not until later when st udies on the mtDNA d-loop and microsatellites were

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27 initiated that scientists started to understand the genetic picture of the present day Florida manatee population. Detailed analysis of mtDNA d-loop sequences led to the conclusion that the Florida manatee may have little haplotype diversity (i.e. all individuals have the same haplotype), but permitted the definition of population structure throughout the range of Trichechus manatus Three distinct clusters were identified: (I) Fl orida to Puerto Rico and the Dominican Republic, (II) along the Gulf and Caribbean coasts of Mexico, and Central America, and (III) South America and the Atlantic coas t of Brazil to the area around the mouth of the Amazon River (Garcia-Rodriguez et al 1998; Vianna et al 2006). More precise delineation of manatee population structure within Florid a required the development of mi crosatellite fingerprinting for this species, which soon followed (Garcia-Rodriguez et al 2001; Nourisson 2005; Pause et al 2007; Tringali et al 2008b; Hunter et al. 2009b). To date several polymorphic loci have been used to identify individuals and examine populati on structure. Each of those studies observed little genetic diversity in Flor ida. Recently, however, distinct population structure has been detected among the populations in Florida, Puerto Rico and Belize (Kellogg 2008). This distinctness should be expected, as there is no known reprodu ctively effective movement occurring among any of these populations. More detailed studies are being employed now to look at finer scale population structure within the endangered manatee population in Florida (Figure 1-1). This research will allow studies of kinship groups within geographic populations and facilitate genetic analyses of inbreeding, ma ting success, and paternit y/maternity, as well as post-mortem identification of carcasses. Thes e genetic data will identify and determine the extent of family structures and allow modeling of demography based on field observations. They also may lead to better-informed mana gement decisions rega rding conservation of

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28 manatees and assist researchers with model de velopment to predict pot ential for long-term sustainability, longevity, and population growth of this endangered species. In 1974, Hartman predicted the manatee popula tion in Florida based on aerial survey counts to be between 750-800 individuals, with a possible calculated maximum of 1,000 and a conceivable minimum of 600. Soon after, Ir vine and Campbell in 1978 conducted additional aerial surveys and counted 738 manatees in Florida, and Brownell et al (1981) concurred with a range of 800 to 1,000 animals. By 1985 the estimated population number was 1,200, which appears to have increased ever since. Trends in positive population growth have been observed at intensively monitored sites, su ch as in Blue Spring and Crystal River, Florida, with the use of aerial surveys and ground censuses. The last synoptic statewide aerial survey conducted in January 2009 counted 3,807 manatees (FWC 200 9a; USFWS 2009a). This number is not an estimate of the population as it is dependent on observer conditions, but merely reflects the actual number of individuals recorded on one day of survey effort. Care should be taken when trying to predict estimates for population size or minimum population size as these are very difficult to justify statistical ly using current methodologies and are often misunderstood and quoted out of context by the lay person (OShea 1 988). Use of current genetic data coupled with other evaluation tools in understanding manat ee population demographics will give a clearer picture of manatee recovery. Effort s to calculate effective population (Ne) size in manatees using genetic samples holds great promise as long as the appropriate number of samples can be obtained and the potential for sampling bias is avoided. Recent protection measures, such as the ESA, the U.S. Marine Mammal Protection Act (MMPA) of 1972, and the Florida Manatee Sa nctuary Act of 1978, provided much needed regulatory authority for manatee protection to state and federal agencies (USFWS 1979). These

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29 protections not only afforded th e opportunity for the Florida ma natee population to reach levels of presumed recovery, but also recently influenc ed State managers to consider down-listing the species to threatened status. In 2007, the U.S. Fish and Wild life Service published the ESA mandated 5-Year Status Review (USFWS 2007) which also sugge sted down-listing the manatee to threatened. In 2007, a petition to down-list the manatee from e ndangered to threatened status on the States endangered species list was denied despite recent scientif ic findings. The outcry from public sources prompted the Florida Fish and Wildlife Conserva tion Commission (FWC) delegates to disregard scientif ic data and management opinion, likening the decision to the one made by the judge in the cl assic movie Miracle on 34th Street (wherein the judge claimed that there really is a Santa Claus) and rejected th e petition to down-list (Pittman 2007). Soon after the decision was rejected by the Commission, th e State of Florida embraced their recently completed Manatee Management Plan (FWC 2009b) focusing and redirecting their research efforts to obtain an accurate census to determin e the size of the population. Manatee population status in Florida is still not completely understoo d, and their fate lies not only in the commitment to anticipate and eliminate future threats, but also in the abilit y to look ahead and secure a place for them in the decades to come. Why Do We Need to Study Manatee Genetics? Humankind has the responsibility to be better stewards of our environment and resources. Manatees are an excellent ex ample of how we might effectively manage and conserve a population through strategic habitat c onservation efforts. The species resilience to perturbations and their short-term adaptability make them excellent candidates for recovery given that appropriate and adequate measures are in place to protect them and their ha bitat. Knowledge of their genetic make-up, coupled with understanding th e limitations of the environment, will assist

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30 managers in developing conservation strategies to ensure continued success and survival of this unique endangered species.

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31Table 1-1. List of life hi story characteristics for the Florida manatee (Modified from Reep & Bonde 2006). Population and Habitat Population size ~4000; listed as Endangered under ESA Marine and freshwater dist ribution, primarily coastal Availability of fresh water and warm water sources required Generalist obligate aquatic herbivore Abundant food resources No natural predators Adapted for tropics-subtropics; Florida at northern extreme of winter range Low metabolic rate; susceptibility to cold stress Seasonal migrations for thermoregulation Seasonal site fidelity Low genetic diversity Little genetic intermingling between East and West coast populations Evolved ~5 mya from Caribbean stock Anthropogenic causes account for ~30% of annual mortality Life History Sexual maturity at 3-5 years of age Mating herds and scramble polygyny Long gestation time ~12-14 months Single offspring, twinning rare (~1.5%) Newborns weigh 18-45 kg; are ~1.2 m long 1-4 year calf dependency period Calves suckle from two axillary teats Suckling underwater for 3-5 min every few hours Long period of early learning Maternal-calf bond main social unit No territoriality or aggression Vocalizations rare; chirps and squeaks 2.5-3.0 year calving interval Feeding Preferred water depth of 1-4 meters Snout deflection consistent with generalist feeding strategy Consume 4-10% of body weight/day from a wide variety of plants Feed for ~6 hours/day Prehensile use of perioral bristles during feeding Resting manatees breathe ~every 10 minutes Long gut transit time, ~7 days Hindgut fermentation like horses Continual molar tooth replacement Anatomy Large body size (adults usually 400-550 kg; 2.7-3.0 m long) Record body size (up to 2000 kg; 4.0 m long) Females tend to be larger and heavier Pachyostosis/osteosclerosis bone; may aid hydrostasis Two hemidiaphragms, oriented longitudinally Lungs elongated Internal testes Paddle shaped tail Fingernails (3-4) on pectoral flippers Skin gray-brown with numerous pits Skin is shed often Behavior and Brain Slow movements, 0 7 m/sec Low encephalization quotient Reduced visual, olfactory, and taste systems Expanded sensory hair system Tactile exploration using oral disk Kissing, nuzzling behavior Hearing best at 10-18 KHz

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32 Figure 1-1. Timeline of genetic research st udies conducted on the Florida manatee.

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33 Figure 1-2. Pregnant Florida mana tee at Crystal River, Florida. (Photo by R.K. Bonde, USGS, Sirenia Project).

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34 Figure 1-3. Sirenian glob al distribution. Red is Stellers sea cow, pink is dugong, light blue is Amazonian manatee, dark blue is West Indian manatee, and green is West African manatee.

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35 CHAPTER 2 TRICHECHID EVOLUTION Systematic Characters Phylogenetically, sirenians (manatees and dugongs ) are classified with the Paenungulata in Afrotheria (Mammalia) (Stanhope et al. 1996; Springer et al 1999; Parr 2000; Hedges 2001). This group of taxa places sirenians in a genetic relationship with the Proboscidea (elephants) and Hyracoidea (hyraxes) (Pardini et al 2007). Studies to detect thes e evolutionary associations explore gene similarities by employing chromoso me painting techniques with sophisticated cross-species fluorescent in situ hybridization (FISH) probe technology (Kellogg et al 2007; Pardini et al 2007). Much of the previous amino acid and gene identification in sirenians has been conducted on dugongid samples (De Jong et al 1981; Rainey et al 2984; Shosgani 1986; Wyss et al 1987; Ozawa et al 1997; Springer et al 1999; Novacek 2001; Gheerbrant et al 2003; Yang et al 2003; Redi et al 2007; Springer & Murphy 2007; Tabuce et al 2008), but recently researchers have hybridized chromosome segments in trichechid tissues with other species (Kellogg et al 2007; Pardini et al 2007). Additional scrutiny of the segments is useful to identify relationships of taxa within the P aenungulata and clarify th e processes encountered during evolution. It should be noted that early studies on morphology associate the sirenians within the Ungulata group in contrast to recent genetic evidence for clas sification. This discrepancy remains unresolved today, but as more genetic pieces to the puzzle are introduced, bridges between these camps are being crossed. Osteologic and dentitional evidence also associates sirenians with the extinct embrithopods, which we re rhinoceros-like herb ivorous mammals with plantigrade feet, and the desmostylians, whic h were hippopotamus-like amphibious creatures (Simpson 1945; Domning 2001a).

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36 Manatee Evolution According to the fossil record, the delineation of present-day sirenians can be traced back to the most primitive taxon Prorastomus (Figure 2-1; Savage et al 1994; Domning 2001a). Later, the Pezosiren was recognized as a primitive four-leg ged ancestral member of the Sirenia that appeared in the Eocene, about 40 mya. Pezosiren exhibited some of the characters observed in all sirenian species that followed (Domni ng 2001b). Certainly the present forms, both marine/aquatic (trichechids) and completely mari ne (dugongids), have lost their hind limbs, but other characters in common with Pezosiren such as their dentition, mandibular structure, and rostral deflection, are retained to day in extant species. The Pezosiren type specimen was recovered from a dig site in Jamaica and many of the other 35 known extinct species of sirenians that followed were also recovered from fossil sites in warm, tropical environments (Domning 2005). An intermediary taxon that evolved between Pezosiren and extant sirenians was Protosiren (Domning 2005). This genus occurred in the Middle Eocene. Two families of sirenians, the Dugongidae and the Trichechidae, comprise todays four surviving species (Hatt 1934; Domni ng & Hayek 1986; Domning 1994a): the dugong ( Dugong dugon), the Amazonian manatee ( Trichechus inunguis ), the West African manatee ( T. senegalensis ), and the West Indian manatee ( T. manatus ). The latter species is divided into two subspecies, with the vernacula r names Antillean manatee ( T. m. manatus ) and Florida manatee ( T. m. latirostris ). It is not certain when the transitional or intermediary steps between Protosiren and the two branches com posing the dugongids and trich echids actually took place, but it was probably sometime during the midEocene to Oligocene periods (~30-40 mya) (Domning 2005). At that time, ancestral lineag es for the present day dugongs and manatees separated into two distinct clades.

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37 The more recent evolution of the trichech ids can be directly traced back to Potamosiren during the Middle Miocene (~12-14 mya), a genus in which horiz ontal molar tooth replacement was not evident (Domning 2005). The horizonta l tooth replacement seen in all extant trichechines followed later (Domning 1983; 1994b; 1997). Trichechid evolution was further refined with the appearance of Ribodon from South America which followed in the Early Pliocene (~5 mya). Both Potamosiren and Ribodon exhibited apomorphic characters defining the trichechids (Domning 2005). The Amazonian manatee ( T. inunguis ) appears in some ways to be the most primitive present day species but still has the largest number of uniquely derived autapomorphic (a derived trait that is unique to a related group) char acters. These include (compared to other trichechids): smaller body si ze, smooth skin surface, fewer ribs (n = 14-16 pairs), no nails on flippers (hence the specific ep ithet), and more chromosomes (diploid n = 56) (Domning & Hayek 1986). Characters include mid-range rostral deflection of the premaxillary bones (25-41). On the other hand, the West African manatee ( T. senegalensis ) has the fewest derived characters and the leas t rostral deflection of the pr emaxillary bones (15-40), though very little is known about this species (Domning & Hayek 1986). Analysis of their morphological features places them as more primi tive than, but more closely related to, the West Indian manatee ( T. manatus ) than to the Amazonian manatee ( T. inunguis ). This divergence between West African and West Indian manatees must not have resulted from the opening of the Atlantic Ocean that occurred during the Mesozo ic period, but instead can be attributed to dispersal that occurred more recen tly. It is likely that at leas t one transatlantic dispersal event must have taken place sometime later, perhap s as recently as five mya (Domning 2005). The T. manatus autapomorphic characters include a larg er body mass, rougher skin surface, greatest

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38 rostral deflection of the premaxillary bones (29-52), and fewer chromosomes (diploid n = 48) (Domning & Hayek 1986; Gray et al 2002). Distinction between the subspecies of West Indian manatees includes craniometrical characters and slight but signifi cant differences between the rostra l deflection (Figure 2-2) of the premaxillary bones ( T. m. m. 29-48and T. m. l 30-52) as described by Hatt (1934). The type specimen of T. m. latirostris was described by Harlan (1824). Certain cranial morphological characters are diagnostic of the Antillean subspecies. In T. m. manatus the angle of rostral deflection of the premaxillary is comparatively less than measured in T. m. latirostris (Domning & Hayek 1986). With respect to classification, a subspecies has been defined by Mayr (1970) as an aggregate of phenotypically similar popula tions of a species, inhabiting a geographic subdivision of the range of a species, and differing taxonomically from other populations of the species. Additionally, the species should differ taxonomically by exhibiting distinct morphological characters and the su bspecies should not be a un it of evolution, except when it corresponds to a genetic isolat e. Domning and Hayek (1986) also caution that there are overlapping ranges of variation in morphological characters within the populations. The information on morphology, along with other cons iderations such as isolation between geographic populations and the low potential for reproductivel y effective movement, has substantiated the subs pecies designation. Recent studies have associated the actual sh ifting of populations with rapid climatic change (Carstens & Knowles 2007). Through the c ourse of time, changes in sea level have occurred ranging from high (Figure 2-3) making movement across seas more difficult, to the moderate (Figure 2-4) status of todays sea leve l, to low (Figure 2-5), more conducive for species to bridge vicariance barriers. Timing of these geological epochs has resulted in changes in the

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39 potential for manatees to make population expansion moves. Th ese climatic and stochastic events during the last 5 million years have re sulted in changes that allowed trichechine populations to pulse in and out of critical habitats (Domni ng 1994b). During a period 2-3 mya there was a severe ecological upheaval that infl uenced much of the flora and fauna in the Caribbean and Florida region (Bianucci et al 2008; Domning 1994b). During this period dugongs, which were well established in the region, were extirpated due to loss of old forms of abundant vegetation. Within a couple of million years, new forms of vegetation became established, favoring newly evol ved manatees to occupy the ra nge (Domning 2005). A similar phenomenon may have occurred with T. senegalensis even more recently. As sirenians did not exist until much later, it would have been impo ssible for the West African manatee to have taken advantage of the continental drift initiated in the Mesozoic. Instead, much more recently, individuals ancestral to T. senegalensis probably dispersed from the New World via ocean currents to establish a populati on in distant continental Africa A plausible possibility was proposed by Boekschoten and Best (1988): a pproximately 18,000 years ago a high-velocity current is thought to have developed in the Atla ntic Ocean between South America and Africa. Salinity gradients could have allowed a lens of fresh water to persist at the surface to provide drinking water for long-distance traveling manat ees. Generally, a physiologic need for fresh water (Ortiz et al 1999) has been one of the primary c onstraints on dispersal of manatees. Perhaps wayward manatees used that current a nd were cast into the nearshore habitats to colonize West Africa during the Pleistocene. The trip would obviously have been one-way west to east and not intentional, but afforded the opportunity for se ttlement into a new niche. It was the predecessors to present-day West African ma natees that were the best candidates for this dispersal event (Domning 2005). Having recently arrived into the Caribbean from South

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40 America, these manatees were well-suited for feeding on floating vegeta tion. It is therefore plausible that these individuals made the first translocation across the Atlantic Ocean, sustained during the crossing by rafting vegetation on a lens of fresh water (Doming 2005). Conversely, the remaining manatees in the Ca ribbean were free to develop the greater rostral deflection observed today which was better ad apted for surviving in more marine habitats when necessary, versus their relatives in the West African manatees (Figure 2-2). Domning (2005) reports another sister taxon, T. m. bakerorum, within the T. manatus group. T. m. bakerorum appears to have established itself in the southeastern United States within the last 125,000 years. Specimens of this subspecies have been discovered in North Carolina and Florida. It is likely that this subspecies was extirpated during glacial events when temperatures became so cold that these tropical descendents were not able to survive. A cold period exists in the geologic record approximately 110,000 years ago. The presence of this subspecies may exemplify how groups of manatees from the Cari bbean dispersed into peninsular Florida during interglacial warm periods. Such a dispersal ev ent may have resulted fr om the propensity that manatees have for colonization through natural p opulation expansion events. At present, we do not know much about the population structure of manatees in neighboring Cuba; studies on these manatees could shed some light on this hypothesis of dispersal from neighboring communities. As ecological (temperature, salinity, vegetation type) and physical (water, land) barriers would pose a challenge to dispersal, these events were more likely to have occurred during large-scale climatic or stochastic events (e.g., ice ages, c limatic upheavals, hurrican es). Sensitivity to climate-induced habitat change has affected several species of marine mammals (Laidre et al 2008).

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41 Manatees also have been detected in fossil records well up the Mississippi River and Ohio River basins (Williams & Domning 2004). These manatees were probably opportunistic wanderers during periods when their dispersa l was only limited by weather and not human hunting pressures. When the weather was good, mana tees would travel great distances to exploit available habitats in new frontiers. Recently, a free-ranging manatee was observed, but died, in the Mississippi River near Memphis, Tennessee (USGS Sirenia Project files). Radio-tagged manatees from Florida have traveled successful ly within a season along the entire Atlantic seaboard all the way up to R hode Island and back (Deutsch et al 2003), and sighting events outside of Florida during summer are now quite co mmon, with recent sightings from as far north as Cape Cod Bay, Massachusetts (U SGS Sirenia Project files). Population Expansion It is possible that some individuals, by natu re of their morphology and preferred feeding strategy, may be adapted to forage on floating vegetation as well as submerged vegetation, as was suspected for manatees that initially populated the peninsular Florida area. This could allow for differences in subpopulation di spersal patterns by some groups of manatees. Manatees have been observed rafting along with mats of floating vegetation in ma rine waters off Puerto Rico (USGS Sirenia Project files). If the vegetation flotsam is large and becomes displaced it could effectively get carried great distances by drif t currents. Once the vegetation is depleted, manatees may be left victims of the current. Su ch cases have been reported for manatees in Florida that have been in the Gulf Stream a nd essentially carried out to sea (USGS Sirenia Project files). In one case, a male manatee (Mo), was released with a radio tag in Crystal River, FL but was later rescued after he drifted 300 m iles south to the Dry Tortugas. Similarly, a female manatee (Xoshi), released in Miami with a radio tag, also required rescue some 30 miles

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42 off Merritt Island, Florida, before she was carried further out, irretr ievably, into deeper Atlantic Ocean waters. In the early 1990s a dependent female ma natee was photographed in the Homosassa River, Florida. In February 2000, a manatee was sighted in the Bahamas, well afield of natural manatee habitat. When a USGS researcher trav eled to the Bahamas to radio-tag the manatee it was positively identified as the manatee previously documented in the Homosassa River (near Crystal River) population (USGS files) At long-term study sites in Florida, there is a decrease in the estimated annual adult survival rates fo llowing extreme storm ev ents (Langtimm & Beck 2003). It is possible that this manatee was displaced from the Homosassa River area during a storm event, or was driven away by increased hu man disturbance or behavioral conflicts, and was entrained by the Gulf Current, and subse quently found a niche in the Bahamas (USGS Sirenia Project files). Continued monitoring in the area of her Bahamian sighting subsequently documented her with a calf. In 2006, scientists received a report of a lone calf in the surf off the Cayman Islands. Habitat in and around the Cayman Is lands is not suitable for meeti ng the needs of manatees. The 116 cm male newborn orphan calf was rescued and sent to Florida for rehabilitation. Genetic analysis of the manatees tissues determined that this animals parents may have originated from Antillean manatee stock. Could this manatee be the result of a translocation event of the pregnant mother? Drift pa tterns throughout the Caribbean could explain how a wayward manatee might end up on shores of several Ca ribbean islands, or on the coastal beaches of Mexico. Other events have shed an interesting light on the subject of manatee dispersal. Recently another manatee (CR131) from Crystal Rive r, Florida was photographed and positively

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43 identified in Cuba by her distin ct scar patterns (Alvarez-Aleman et al 2007). This manatee was a well-known adult female that had a more than 25 year sighting histor y in Florida with the Manatee Individual Photoidentification System (MIPS). Sh e had also been documented during several successful calving events in her monitoring history. Her last sighting in Florida was in July 2006, with a calf. In January 2007 she wa s sighted with a calf and positively identified by photographs taken in the Camilo Cienfuegos power plan t intake canal, just east of Havana, Cuba. Although the number of extra-limital reports is increasing annually, out-of-state sighting events have led to greater public awareness. It is possible that these events are the result of natural population expansion. Presently, there ar e many out-of-state sighti ngs along the Gulf and Atlantic seaboards ranging from Texas to Massachusetts (Fertl et al. 2005; USGS Sirenia Project files). In addition to the ma natee in 2006 that was observed we ll up the Mississippi River near Memphis, sightings have been documented in Massachusetts during the early fall of 2008 and 2009, and increasingly in the Carolinas, Chesapeake Bay, and Texas. Fossil and recent records have placed manatees in these areas during fluc tuating geological epoc hs and prior to human occupation (Barber 1982; Williams & Domning 2004; Domning 2005). Today, it is presumed that most of these animals continue to use these extra-limital habitats but return to overwinter in Florida, as did the well-documented manatee Chessie (TBC-42) on severa l occasions, but some fall victim to the temperature limitations encoun tered by all Florida mana tees found outside their expected winter range. Other interesting dispersal events have been taking place in the last couple of years as well and may be influenced by behavioral traits. In March 2009 a large well-known female manatee (RB050) with an extensive sighting history from Riviera Beach, Florida, was sighted off Rum Key in the eastern Bahamas. Researchers hypothesi ze that she was caught in the Gulf Stream off

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44 the coast of eastern Florida and drifted north. As she reached th e northern end of the Bahamas, she was carried around the north of the island chai n then southward into the eastern reaches of the islands some 400 miles southeast of Florid a. By August 2009 she was re-located further southwest off Long Key where she was rescued, but died a short time late r from dehydration and starvation imposed by lack of appropriate habitat and the st ress of the journey. Manatee distribution could be limited by the need for fresh water. Without an available freshwater source, manatees in marine habita ts are not likely able to adequate ly digest seagrasses consumed. A trend in the dispersal pattern of this manatee toward the west due to drift currents is also supported by island lizard distribution patterns in the Bahamas (Calbeek & Smith 2003). Molecular Status At present, Florida manatees exhibit low genetic diversit y, most likely due to a founder effect. This is not that surprising for such a recent population as we have in Florida and given their isolation from other mana tee populations by di stance and vicariance. If the Florida population has only been isolated for a short 12-15,000 years (moving here during the last low sea level conditions (Figure 2-5), then this current resident popul ation has not had adequate time to fix a large number of neutra l alleles. The Florida subspeci es does, however, have unique alleles that, when compared with the Puerto Ri co and Belize populations distinguish it from these Antillean manatees (Hunter et al 2009a; 2009c). This information, coupled with the morphological evidence, continues to support sub-speciation between th e two populations of T. manatus It is interesting that manatee populations thr oughout their range can become so small that they become difficult to locate and economically unfeasible to hunt. Many manatee populations have been hunted for long periods of time and numbers have been reduced (Lefebvre et al 2001). However, those remaining in reduced popul ations continue to have a unique ability to

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45 find each other and successfully breed. It may be through an ability to detect breeding partners that manatees in Florida are more apt or pre ssured to disperse. Alte rnatively, as populations increase, detection among individual s is even more likely to lead to excessive breeding activity by many individuals, which may tend to drive mana tees out of their esta blished normal home ranges. Recent observations involving aggressive pursuit by male s during mating has resulted in cases where the target adult female has died (deWit 2009 pers. comm.). Male elephants will adopt different behavioral and migr atory patterns in an attempt to avoid opportunities that lead to breeding with related individuals (Archie et al 2007). This elephant fission-fusion society enables random breeding by dominant males, resulting in gene flow among strongly aligned matrilineal groups (Archie et al 2008). Future studies examining fitness need to be implemented before we fully understand whether this phenomenon is affecting manatee dispersal. Extinction Processes Scientists agree that there has been great opportunity for inbreeding and a founder effect in manatees from Florida, especially if we agr ee with Domning and others on the likelihood of recent dispersal events. The genetic uniqueness of the Florida manatee, c oupled with specialized morphologic characters, has formed the framework for speciation. The possi bility arises that perhaps the manatees, through human inroads and perturbations, have altere d their abili ty to pass beneficial genes among the population successfu lly, while at the same time eliminating deleterious genes. Natu ral events have ensured the survival of manatees in the niche they now occupy, but human interaction has also tampered with the normal adaptive processes. Natural selection processes have been disrupted by recent historical events that have dictated manatee population decline as well as growth over relatively short time periods.

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46 Past fluctuations in population size have been common throughout the manatees range, primarily due to gradual climate change. Neve rtheless, anthropogenic effects have impacted population size during the last 10,000 years. The shift toward rapid population decreases due to hunting has forced issues related to population stabi lity. The result, outside of Florida, has been the survival of more stealthy manatees better equipped to coexist among humans undetected. We have already observed how hunting of a populat ion can lead to rapid extinction as observed with the Stellers sea cow (Ste jneger 1887). Recently, however, in Florida, this has changed with advent of protective measures and develo pment of artificial warm water sources, and manatees are gaining an unparalleled trust in humans. Are there dele terious implications associated with that trust? Seldom do species perform well with low allelic diversity. We have not ed that there is low genetic diversity in the Florida manatee. Nonetheless, some species like elephant seals (Hoelzel et al 1993; Slade et al 1998) and flightless cormorants (Duffie et al 2009) have maintained healthy attributes despite low di versity. However, other populati ons like those of the cheetah, Florida panther, whale species, and lions (OBrien et al 1985; OBrien 2003) have shown severe loss of fitness and even genetic anomalies due to reduced populat ion size, low genetic diversity, and inbreeding. Finding appropriate habitat for populations is important for securing their establishment and survival (Mayr 1954; Palumbi 1994; Rocha & Bowen 2008). Recent findings in surgeonfishes from the Hawaiian Islands show that certain individuals separated by currents show specialized adaptations that make them de pendent upon local habitats while at the same time unable to survive in an adjacent habitat o ccupied by very similar i ndividuals of the same species (Eble et al 2009).

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47 With regard to Florida manatees, we also see another conundrum. Ma natees have become dependent on artificial sources of warm water during co ld weather. This has influenced their winter distribution and dispersal, and to a certain degree limited thei r selection of mates. In the very near future these aging power plants will become obsolete, will close, and will no longer be available as a warm water habitat for manatees (Laist & Reynolds 2005). When that occurs, the existing population of manatees will once again u ndergo modification of their routines. Home ranges will invariably change as manatees l ook for other over-wintering options. Population numbers will be reduced, winter range will constrict, and possibly several smaller populations might be isolated, causing genetic harm to the manatees fitness through reduced gene flow. We can only hope that their previous adaptive resilie nce, honed over millions of years, will help them during this trying period.

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48 Figure 2-1. Evolution of sirenians with comp arisons of cranial morphology; adapted from Domning 2005 (Reep & Bonde 2006).

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49 Figure 2-2. Sirenian rostral deflection from l east to greatest. For species designations, Ts = Trichechus senegalensis ; Ti = T. inunguis ; Tmm = T. manatus manatus ; Tml = T. m. latirostris ; and Dd = Dugong dugon

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50 Figure 2-3. High sea level. During a warming of climate, water is released from sea ice resulting in an increase in sea level. The orange color is land mass at high sea level, outline is land mass, and water level is depicted in light blue.

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51 Figure 2-4. Moderate sea level. This is the pr esent day condition. The orange color is land mass at middle sea level and water leve l is depicted in light blue.

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52 Figure 2-5. Low sea level. During very cold cl imatic conditions, ocean water is tied up in sea ice resulting in a lowering of sea level. The orange color is land mass at low sea level, outline is land mass today, and wa ter level is depicted in light blue.

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53 CHAPTER 3 CRYSTAL RIVER MANATEE FINE-SCALE POPULATION ANALYSIS Introduction In 1978, a long-term research project was initiated by Departme nt of the Interior federal scientists to look at the life hi story parameters of the winter resident population of manatees ( Trichechus manatus latirostris ) in Crystal River (CR), Florida. It built upon pioneering studies by Daniel Hartman and James Powell (Hartman 1974) begun in CR in 1968, when the winter CR population estimate was a mere 63 manatees, and was the first research to lay the foundation for the present day Manatee Individu al Photo-identification System (MIPS) managed by the USGS, Sirenia Project. The MIPS relies on a compilatio n of photographs and ancillary information on manatee biology and reproduction collected on the da te of each sighting, obtained from staff swimming amongst manatees in their natural habitat. Individual manatees are identified from their photographic documentati on, allowing maternal pedigrees through association to be formulated from field notes. In 1989, the firs t manatee biopsy tissue samples were collected by USGS, Sirenia Project staff from free-ranging manatees (mostly ca lves) and archived for future genetic analysis. To date over 1,000 biopsy sample s have been collected from manatees in the CR population, forming the source of material enab ling the research of Peter McGuire, Angela Garcia-Rodriguez, Kimberly Pause Tucker, Marg aret Kellogg Hunter, my self, and others. This project was initiated to complement photo-identification efforts by utilizing genetic capabilities to improve data co llection techniques a nd reduce uncertainty in the estimation of vital rates. Presently manat ee vital rates ob tained from photographs have been employed in population models to estimate population growth rate (Runge et al 2004) and project population viability (Runge et al 2007b). Possible sources of error w ith photo-identification, which can be reduced with additional genetic data include, but are not limited to, (1) difference in survivability

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54 between sexes, (2) ability to re-identify individu ally scarred manatees accurately, (3) accuracy of visual observations to associate cow/calf pairs, and (4) reliance on visu al observation to assign sex (Langtimm et al 1998; Kendall et al 2004; Runge et al 2004). Furthermore, genetic information can provide additiona l information currently not possible with photo-id data for incorporation into more complex population models such as identification at various life stages, sex, age, age at first reproduction, mate selection and success, identification of unscarred individuals, and links fr om individuals to various mortality factors. Though future research efforts will address most of the points mentione d above, this study will establish criteria for examining a couple of those capabil ities, including identification of specific individuals, age, age at first reproduction, accuracy of assigned re latedness between cow/calf associations, and maternal lineages. This study seeks to identify genetically dist inct individuals of the CR manatee population, address the genetic or familial structure, match genotypes of samples taken from animals as calves and adults, confirm pedigr ees assigned by MIPS using micros atellite loci, and strengthen tools for determining survival estimates and mo rtality information. A panel of 11 markers was selected and analyzed against a subset of indivi duals with maternal associations, i.e., cows and their dependent calves. The fine-scale gene tic information on relatedness will enhance the understanding of the biology and reproductive capabil ities of the individual s within this primary manatee population center in Florida. Previous studies using cytochromeb (Bradley et al 1993) and d-loop mitochondrial DNA (Garcia-Rodriguez et al 1998; Vianna et al 2006), determined that the Florida manatee population had only one haplotype and provided no information on relatedness or gene flow. However, allozyme studies of 59 samples and 24 loci conducted in 1988 (McClenaghan &

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55 OShea) suggested normal levels of heterozygos ity and ample population mixing (implying gene flow) for all Florida manatees, prompting further research. The wave of genetic studies that followed focused on developing microsatellite prim ers to identify highly variable sites in the nuclear DNA. To date, there has been marked success in developing prim ers (Garcia-Rodriguez 2000; Garcia-Rodriguez et al 2001; Pause et al 2007; Pause 2007; Kellogg 2008; Tringali et al. 2008a; Hunter et al 2009b) useful for analysis of polymorphi c loci in the manatee genome. A molecular clock of separation (based on one mutation every 1.25 million years) was proposed by Garcia-Rodriguez et al (1998) wherein mitochondrial DNA (mtDNA) was examined among several populations of T. manatus throughout the species range. This information formulated the theory that manatees in Florida were more closely related to present day populations located in adjoining islands of th e Caribbean, while more distant to counterparts in Central and South America. It is likely th at these Caribbean manatees have found their way into Florida many times over the last 1.25 millio n years (Domning 2005). The last migration into peninsular Florida probably occurred a bout 15-20 thousand years ago during the last interglacial period and fits the molecula r clock mutation rate (Garcia-Rodriguez et al 1998; Laist & Reynolds 2005). The single d-loop haplotype identified in the Florida population (A01) is shared with the Mexico, Dominican Republic and Puerto Rico Antillean manatee populations in the Caribbean (Garcia-Rodriguez et al 1998). The low mtDNA diversity in the Florida manatee population is thought to be the result of a genetic bo ttleneck or founder event (Garcia-Rodriguez et al. 1998). After the work of Garcia-Rodriguez and co lleagues in 1998, additional mtDNA studies were incorporated into a study involving many more samples (Rodriguez-Lopez 2004; Cantanhede 2005; Vianna et al 2006), but still confirming the single haplotype, A01, in Florida. Vianna and

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56 colleagues also supported the Garcia-Rodriguez proposal of three distinct clusters of West Indian manatees: (I) the Caribbean and Florida, (II) co astal Central America, and (III) coastal South America (Figure 3-1). The identification of the mtDNA haplotype A01 in manatees occupying the Antilles resulted in some confusion with respect to the subspeci es designation between T. manatus manatus and T. manatus latirostris that had been based on morphological characters (Domning & Hayek 1986). Since the Florida population is recent relative to other T. manatus populations, and has not had time to undergo mutati on events for differentiation, the use of more informative tools such as microsatellites became necessary to advance res earch capabilities. Regarding the manatee genetic population stru cture within Florida, preliminary results using microsatellite primers (Pause 2007) identified a high degree of gene flow, in agreement with the allozyme study conducted in 1988. The lack of genetic separation of manatees within the Florida population provides evidence that there is enough movement for ample gene flow among individuals throughout management units or between discrete geographical regions. In an effort to examine the effectiveness of the 18 polymorphic primers on 400 manatees in the study by Tucker et al. (2009), Hunter et al (2009c) examined approximately 100 manatees from Puerto Rico and compared those data with the Fl orida data. The results to date indicate clear differentiation between these two populations, signifying geographic is olation between the Florida and the Puerto Rico populat ions of manatees. This certainly has been due to vicariance and limitations to dispersal and would support the subspecies designations based on unique morphology (Domning & Hayek 1986). The panmictic population structure also indicates a recent colonization to Florida, resulting in genetic founder effect influe nces. This period of 12-15,000 year s ago corresponds to the last ice age when the earths temperature was drama tically reduced and continental shelves were

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57 exposed as the water level receded. This lowered water level brought coastl ines closer together, making deep ocean barriers less significant and p opulation dispersal more likely. Natural springs (now many miles off shore) and shallow natural structures an d marshes like the present day River of Grass in the Everglades, might have helped supply solar-warmed water to assist manatees during cold periods, th ereby providing suitable habitat to allow establishment of a new population. These conditions additionally provided adequate access to and availability of food resources for manatees. This study incorporated tissue samples from 145 individual manatees. All of these adult individuals were known from scar patterns or we re calves of well-known, related mothers from the overwintering population in CR. The manatee population in CR is unique in that it has been extensively studied for over four decades and is undoubtedly the most thoroughly researched population of sirenians in the world. The data set represents a si gnificant portion of the suspected total overwintering popu lation using CR. Best estimates based on aerial surveys for this area are 480 manatees, with increases in th e counts each year since 1978 (CRNWR files). Additionally, there is a large amount of ancillary reproduction and cow-calf association data taken at the time of sample collection. This ancilla ry data can be compared to the genetic results and used to help strengthen th e understanding of the manatee popul ation and provide insight into the biological capabilities of the CR population. Th ese genetic data will tighten the assumptions of reproductive potential, benefit tracking of individuals through th e Manatee Individual Genetic-identification System (M IGS) and the MIPS, and result in better estimates of agespecific survival. Future research and new innovative modeling efforts are focusing on determining population structure based on sex, season, and range within the CR area and the entire Florida population using more informative markers (Bonde & Hunter in progress ).

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58 Materials and Methods Sample Collection and DNA Extraction Dermis tissue used in this study was collected from the tail margin of wild, free-ranging manatees by swimmers in the water using a cat tle ear notcher (Figure 3-2) (Appendix). All samples were stored in a high salt buffer solution (SED buffer, 0.24 M EDTA, pH 7.5, 2.99 M NaCl, 20% DMSO) at room temperat ure (Amos & Hoelzel 1991; Proebstel et al. 1993). Geographic location and date of sample coll ection, sex, and other basic information were obtained from biologists' field notes and are listed in Table 3-1. All samples used in this study were collected by the USGS, Sirenia Project and obtained from their tissue archive maintained under USFWS Wildlife Research Permit MA791721, issu ed to the USGS, Sirenia Project. From over 700 samples, 145 were selected for genetic analysis, greater than 25% of the current minimum CR population estimate of 480. DNA Isolation Florida manatee genomic DNA was isolated fr om these tissues using QIAGENs DNeasy Blood and Tissue kits (Valencia, California) fo r the 145 individuals, consisting of 47 well-known mothers and their 98 calves (54 males and 44 females). DNA was isolated from the skin samples using the QIAGEN DNeasy Tissue Kit protocol, eluting the DNA in 30 l AE (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0) in the final step (QIAGE N, Valencia, CA). All isolations were quantified using a spectrophotometer (Nanodrop, Th ermo Scientific, Wilmington, DE). Each DNA isolation was diluted to a final concentration of 10 ng/ l for use in polymerase chain reaction (PCR) amplification. Microsatellite Analysis The eleven most informative manatee microsatellite loci (Garcia-Rodriguez et al 2001; Pause et al 2007) were selected for use on the CR, Florida samples (Hunter et al 2009b). All

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59 microsatellites are di-nucleotides except for Tm aH13, which is a tetra-nucleotide. Isolated DNA was PCR amplified using: 14 ng DNA, 0.8 mM dN TPs, 1x Sigma PCR buffe r (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.001% gelatin), 0.04 units Sigma Jump Start Taq polymerase, 0.24 M of each primer and bovine serum albumin (B SA) where needed (Table 3-2). MgCl2 concentrations were 3 mM, except for TmaH13, TmaKb60, and TmaSC5, which required 2 mM. Amplifications were carried out on a PTC-200 (MJ Research, Waltham, MA) or a Touchgene Gradient thermocycler (Techne, USA) using th e following conditions: initi al denaturing at 95C for 5 min, 35 cycles at 94C for 30 s, anneali ng temp for 1 min, 72C for 1 min, final extension 10 min at 72C. Most samples were successfu lly amplified at 11 loci, however a few tissue samples were problematic and only partially amplif ied using the marker set. Fragment analysis was performed on an Applied Biosystems ABI 3130 XL Genetic Analyzer. GENEMARKER, version 1.75 (Soft Genetics, State College, PA), was used to analy ze the microsatellite fragment data. A Microsoft Access database (MIGS) was used to store allelic information for each individual. Statistical Analysis The level of polymorphism was estimated by the observed (Ho) and expected (HE) heterozygosity, polymorphic information content (PIC), and the number of alleles per locus (Table 3-2) calculated using GENALEX 6 (Peakall & Smouse 2006). Departures from the expected genotypic frequencies in Hardy-Weinberg equilibrium (HWE) were tested using the Markov chain method (dememorization 10,000, batche s of 100, iterations per batch 5,000) in GENEPOP 3.4 (Raymond & Rousset 1995). Additionall y, linkage disequilibrium was tested for non-random associations between alleles of different loci. The Markov chain method was implemented and the P-values were adjusted us ing Bonferroni sequential correction for multiple comparisons (Rice 1989). Confirmation of twin s related to their co ws included sighting

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60 corroboration within the MIPS database, analysis of genotypes, as well as relatedness tests using CERVUS (Marshall et al. 1998), ML-RELATE (Kalinowski et al. 2007), and an EXCEL spreadsheet developed by M. Tringali (2009 pers. comm.). CERVUS and ML-RELATE was used to confirm cow/calf rela tionships reported in this study. CERVUS and ML-RELATE are computer programs that use genetic markers for assignment of parents to their offspring. Both use maximu m likelihood, a well-establis hed statistical method employed to assign parentage. Milligan ( 2003) recently showed that maximum likelihood estimates of relatedness usually have a lower ro ot mean squared error than other estimators. CERVUS introduces two key enhancements to this process: (1) likelihood ratios are calculated allowing for the possibility that the genotypes of parents and offspring ma y be mistyped, and (2) simulation determines the level of confidence in the parentages it assigns. ML-RELATE makes compensation for null alleles, common in microsatellite data that can easily lead to errors in estimating relatedness or relationship if their potential presence is no t accommodated during calculations (Dakin & Avise 2004; Wagner et al. 2006). Neighbor-joining trees Neighbor-joining (NJ) trees based on individual genetic distan ces were used to visualize relationships among suspected related individuals. MICROSAT version 1.5d (Minch et al. 1997) estimated genetic distances based on pairwise Ca lvalli-Sforza and Edwards chord distance, DC. DC is based on allele frequencies and provides accurate microsatellite tree topology (Takezaki & Nei 1996). Trees were constructed by comparing individual genotypes without a priori partitioning. The distance matrix was analyzed with the Neighbor algorithm in the Phylogeny Inference Package (PHYLIP) version 3.6 (Felsens tein 2004) to produce the NJ tree. Genetic differentiation statistic (FST) was calculated from clusters identified in the NJ tree. Suspected

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61 related individuals were analy zed from known individual sighti ng records obtained from a MIPS dataset for the CR population. Analysis using STRUCTURE The program STRUCTURE version 2.3.1 was used to illustrate relationships between individuals within the CR population of manatees. STRUCTURE employs princi ples of HardyWeinberg equilibrium and linkage equilibrium to create subgroupings of unique genotypes that meet the equilibrium expectations. Multilocu s genotypes were used to identify the most probable number of genetic clusters in the CR dataset. The probability of the data, Pr( X|K ) was estimated and a value of K (number of clusters, 1-7 averaged over three runs) was chosen for the best fit approach. The probability was estimated using a burn-in of 10,000 iterations and 100,000 repetitions of a Markov chain Monte Carlo (MCMC) analysis. Po sterior probabilities, Pr( K|X), can be calculated following Bayes rule. The best fit value of K with the highest posterior probability based on ad hoc approxima tion is the most likely number of clusters (Pritchard & Wen 2004), however this can deviate from biological interpretation for selecting the appropriate K (Evanno et al. 2005; Pritchard et al. 2009). Prichard and colleagues suggest that researchers focus on selection of values of K that capture most of the structure in the data set, while still remaining biologically sensib le. A more detailed explanation of K value can be found in STRUCTURE help documentation (Pritchard et al. 2009). Individuals were assigned by proportion of membership ( q estimates) to the assigned STRUCTURE cluster when they had > 60% of that character. Multivariate analysis GENALEX was used to compare coefficients betw een relatedness in a Mantel test as determined by Lynch and Ritland (1999) analyses and the proportion of shared alleles (PSA) derived from MICROSAT. The program PAST (Paleontological Statistics) version 1.95 will be

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62 used for multivariate analyses to detect diffe rences based on relatedness and allelic distances (Hammer et al 2001). Principal coordinates, non-metric multi-dimensional scaling, correspondence, and detrended correspondence will be examined in PAST to determine significance. Results Of the 145 individual samples collected, 139 were suitable for compar ative analysis. Two of the samples did not provide adequate DNA for allelic detection. The remaining four were discarded since slight errors were detected th at could be explained by errors in field data documentation, accidental re-sampling of the same individual, or improper maternal association made through MIPS assignments. These errors were checked against the MIPS data and subsequent sighting information gathered after ti me of initial sample collection was corrected. Microsatellite Marker Analysis The 11 nuclear microsatellite markers (Table 3-2) had similar levels of polymorphism (HE = 0.5451 (ranged from 0.384-0.656); HO = 0.5313 (0.401-0.654); NA = 2.9 (2-5)), when compared to Florida manatee population analyses that used 11 loci (HE = 0.410 (0.186-0.645), NA = 4.1 (2-7)), detailed in Tucker et al. (2009). No evidence of heterozygote deficiency due to null alleles was detected. HardyWeinberg (HW) disequilibrium was observed at only one locus TmK01 (P < 0.05) using 55 pairwise comparisons in GENEPOP and a sequential Bonferroni correction. There was linkage observed after a Bonferroni correcti on between TmE1 and TmE14. Cluster Analysis Using Multi-locus Genotypes The GeneCap genetic P(ID) result for P(ID)sib was 1.618E-03 and translated to a probability match in a population of 618 individuals, whereas the P(ID)HW was 1.472E-06 and resulted in a population of 679,346 individuals that could be individually identified. Since the winter

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63 population in CR is approximately 480 individuals, with an upper estimate of 600 (based on MIPS identities and potential for errors in aerial survey techniques), both of these calculations afforded acceptable ranges for significant indi vidual identification. Furthermore, the P(ID)observed was calculated using GENALEX, which indicated that it was in closer approximation to the P(ID)HW calculations than to the P(ID)sib calculations (Figure 3-3). Th erefore, the upper end of the estimate would fall closer to 1 in 679,346. With this estimate, the ability to correctly differentiate individuals from this populat ion falls well within acceptable limits. Neighbor-joining trees The individual NJ tree identified two unique cl usters, labeled A and B, and a more distant group labeled C (Figure 3-4). This tree was rooted to visualiz e the genetic distance between potential groups in the population. In the tree, suspected lineages were found within and between each group. The genetic distance separa ting the two main groups was modest. When compared to the K = 3 cluster (Figure 3-5) in STRUCTURE, most of the individuals in the A cluster of the NJ tree were also found in the red cluster in STRUCTURE. This was also the case for the individuals in the B cluster of th e NJ tree and the blue cluster in STRUCTURE. The green cluster appeared to be the result of mixing of the two NJ clusters (A & B) and also contained many of the individuals that had the largest genetic distances from the two main clusters. This information is also presented in an un-rooted NJ tree labeled with STRUCTURE assigned clusters greater than 60% of the q estimate (Figure 3-6). Genetic differentiation statistics investigated the separation of the clusters (A & B) identified in the NJ tree. Genetic differentiat ion was observed in the CR data set after estimating FST, which was significant at 0.035. This finding suggests low differentiation but supports two significant groups within the population examined.

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64 Analysis using STRUCTURE The STRUCTURE Bayesian assignment test suggests K = 1 (ln(PD)AVE = -2604.3) was the appropriate cluster since the lowest number of K should be used with the highest posterior probability (Pritchard & Wen 2004). However three of the K -values (1-3) had similar ln(PD)AVE values. The ln(PD)AVE values for K = 2 (-2573.6) and K = 3 (-2557.3) were also investigated as these clusters appeared to cap ture biologically feasib le structure (Prichard et al. 2009). The clusters indicat ed by the NJ tree and the K = 3 cluster in STRUCTURE corresponded to one another as discussed a bove. From the 139 individuals examined, 48 (35 > 60% q estimate) were assigned to cluster A, 47 (28 > 60%) were assigned to cluster B, and 44 (32 > 60%) were assigned to cluster C. A data set consisting of 119 manatees from CR was compared to a set of 84 Florida manatees from outside the CR region. The program STRUCTURE identified K=2 (ln(PD)AVE = -4126.0), separating CR from the rest of Florida (Figure 3-7). Multivariate analysis The 139 CR individuals exhibite d a relatively strong correlation between genetic distance and relatedness. As genetic distance decrease d, relatedness increased (Figure 3-8). This was significant to an R2 value of 0.5791, with a probability of <0.001. However, multivariate analyses using relatedness and PSA showed simila r trends when compared. Color labels were added to the data set for six known family units for visual comparison and displayed using the non-metric multidimensional scaling analysis (Figure 3-9). Individual identity Of the resultant 139 individual samples, 45 were MIPS identified mothers and 94 were calves, of which 53 were male and 41 were female. Each of three calf genotypes matched three samples taken later from adults (CR458, CR485, and CR506; two of which were collected in 1995, one in 2001). Additionally, a set of susp ected twins of CR032 (CR032c4a & CR032c4b)

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65 appeared to be duplicate samples of the same indi vidual, later confirmed w ith sighting data in the MIPS archive. In the 94 cases of suspect co w/calf pairs, four pairs (CR130c2b, CR104c4, CR271c2a, CR506c1) were identified by genetics to be unrelated. It is in teresting to note that two of those cases involved one of the cal ves from a suspected set of twins. Cow/Calf Relationships Successful assignment of genetically fraternal twins was evident in one cow/twin set. CR205 was observed with two calves, a male (CR205c1a) and a female (CR205c1b). Both calves were about the same estimated length and weight and were sampled on the same day in 1996. Subsequently, they were re-sighted and recorded in the MIPS database with their biological mother on numerous occasions, validating the finding. In the 94 cases of suspect cow/calf pa irs, two cases (CR130c2a & CR130c2b and CR271c2a & CR271c2b) were suspected to be sets of twins but were not ge netically related. In these cases, it is presumed that the lactating mother was behaviorally willing to provide for a stray calf. One of the cow/calf pairs ( CR506 & CR506c1) was visu ally identified and convincingly associated by their obs erved behavior at the time of sample collection. However, the genetic samples did not relate the two, the moth er was not observed with a calf in subsequent sightings, and no identifying photographs of th e cow with the suspect calf exist. In some cases, individuals from one family unit (e.g., CR271, and her five calves) matched precisely to clusters iden tified by both NJ tree and STRUCTURE assignment. This cluster association pattern suggests fam ilial related breeding among individu als. Generally, most related offspring in this analysis were associated within the same cluster. This is, however, not always the case. One example to illustrate this exceptio n involves members of the largest related group (CR071-CR104, and their seven calves) who apparently bred with males outside their NJ tree and STRUCTURE clusters.

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66 Grandmother Pedigrees The data set supplied by MIPS also identified four maternal lineages that extend from grandmothers to calves and subsequently on to grandcalves, which we re not excluded by the genetic data. One individual, CR071 with eight related offspring and grandcalves, gave birth to three calves, one of which in 1978 was a fema le (CR104) who successfully reached sexual maturity and gave birth to five calves during the sampling period. A si xth calf (CR104c4) was assigned to CR104 by MIPS but wa s genetically proven not to be hers, through mismatching alleles and relatedness analyses in CERVUS and ML-RELATE. Evidence of apparent calf adoption has been observed in manatees in Belize as well (Hunter & Bonde unpublished data). Other genetically feasible grandparent lineages included: CR046 and two of her four calves, one of which was a female CR506 who birthed two calve s; CR070 who is the known mother of CR130 who in turn gave birth to three calves; a nd CR321 who had four calves of which one, CR458, successfully gave birth to a calf. Discussion Population Biology Minimal genetic difference was detected with in CR, indicating no barriers to gene flow within the subpopulation and that breeding is occurring throughout the region. Biologically this is quite possible and is not surpri sing. NJ trees indicated two primary clusters (Figure 3-4) that reflected the division shown by STRUCTURE in the K = 3 scenario (Figure 3-5). The individuals outside of the two main clusters with the largest genetic distances from the others in the NJ trees may have been due to missing data or parental breeding success with partners outside of their assigned cluster. These observations could be explained by pair br eeding between the two groups, resulting in an offspring with one parent from each group. However, after review of the data it appears that the preponderance of breedi ng takes place between rela ted individuals within

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67 their assigned group. This is contradictory to re sults of previous eviden ce that has suggested very slight population clustering (Pause 2007; Kellogg 2008), but coul d be explained by the uniqueness of the CR population vers us the remainder of Florida. Other populations share this type of low genetic variability as seen in flightless cormorants (Duffy et al. 2009), Mediterranean monk seals (Pastor et al. 2004), and Guadeloupe fur seals (Weber et al. 2004). The STRUCTURE program identified one primary cluster. This finding also is not surprising, as strong site-fidelity is suspected with in this region of Florida and has been well documented by the MIPS for over 30 years. When CR was compared to areas throughout Florida, a K = 2 value was suggested, supporti ng differentiation between the two populations (Figure 3-7). However, it also implies that i ndividuals from both groups were reproductively mixing. Adult female manatees pass on their migr atory and site fidelity habits through a strong association with their calves during the long period of dependency. After weaning, young manatees may adopt similar site fidelity patterns and use site s in common with their moms (Deutsch et al. 2003). Through these familial traits, th e likelihood of breeding within ones assigned group is high. The STRUCTURE and NJ tree analyses sometimes placed strong association between family groups and clusters This familial cluster association pattern suggests that breeding among indivi duals is probably influenced by both manatee behavior and site fidelity patterns. Maternal migratory patterns, strong winter site fidelity, and routine repeated habitat usage most likely places related manatees in proximity during potential breeding periods thereby increasing the probability of mating between these more closely related individuals. STRUCTURE also suggested two other K-values, 2 and 3, which displayed similar ln(PD) results as K = 1. When examined between individuals, the K = 3 model was concordant within

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68 the two separate groups (A&B) assigned during NJ tree analysis. Support of genetic distance with the allelic relatedness strengthens the asso ciation between relatedness and possible family units or clans. This was addi tionally supported by correlation an alyses where individuals were linked by association of relatedness over values obtained from ge netic distance alone in 58% of the time (Figure 3-8). In an attempt to explain the other 42%, suspected known related individuals through field observati on were compared using multivariate analyses and there was no evidence that individuals were more closely associated eith er by relatedness or by genetic distance (Figure 3-9). This can be explained by the low genetic divers ity in the population and by breeding with genetically unique males outside of the family unit. Use of these genetic data w ill help in the development of more robust survival rate estimation models. Strengtheni ng of biological assumptions, such as sex determination, age class assignment, age at first re production, calving rate and interv al, and genetic identification of specific individuals not dependent on visual scar pattern acqui sition will improve capture-markrecapture analyses of Florida manatee populations. Similar mark-recapture models (Besbeas et al. 2004; McKelvey & Schwartz 2004a; 2004b) have been employed for populations of grizzly bears (Boulanger et al. 2004; Kendall et al. 2008; Kendall et al. 2009) and whales (Baker et al. 2007). Individual Identity Informative markers were selected for these analyses based on the Hunter et al. 2009 study. Examination of these samples with the se nsitive marker panel provided support for the individuals identified in the MIPS data set. In a Florida manatee populatio n, the selected 11 loci used the fewest number of markers necessary for positive identification and accurate genotyping of individuals, thereby decr easing cost and improving time effectiveness while achieving satisfactory PID values (Hunter et al. 2009b). There was sufficient power in the panel to

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69 determine match probabilities confidently, while taking into account the bias of known relatedness and current population size. One manatee (CR485) was uniquely scarred by propeller lacerati ons at a young age and entered into the MIPS database while still a depend ent calf. She also was genetically sampled as a calf in 2001, then re-sampled as an adult in 2008. The comparison of the genetic samples collected in 2001 and 2008, along with the identify ing photographs, verified the correct MIPS assignment of this individual, illustrating the utility of merg ing MIPS visual techniques with genotyping. Under conservative guidelines, the pr obability of a genetic mismatch is low when allowing for the small size of the population and th e reduced diversity of the loci used in this study. Another individual (CR458) was genetically matched to a sample collected when she was a scarred calf (CR321c1) of MIPS manatee CR3 21 in 1995. Matching of this genotype and MIPS sighting data yielded a great deal of information on the animals long-term reproductive contributions. CR458 was documented with a calf in 2000. Todays age of CR458 is estimated at 14 years and therefore the age of first known re production for this indivi dual is estimated at 5 years, supporting the estimated age. In another case, adult CR506 wa s linked to an indistinct calf of MIPS manatee CR046 also sampled in 1995 (CR 046c1). This confirmed the age of that animal as well, but also allowed researchers to make inference about her relatedness to her mother and siblings in the data set. Notes on her visual sightings indicated she also had her first calf at age 5. All these examples helped validate MIPS assumptions based on samples collected from unscarred calves and later matched to distinct scarred adults, thus providing additional data for survivability estimates. The genetic matching of individu als also identified mistakes th at were made during sample collection or handling. Inadvertently, one samp le appeared to be mislabeled. The match

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70 revealed that one of the samples labeled for manatee CR049 was actually a sample from CR046. Genetic mismatch of suspected mother and calves ve rified the error. Vali dation of this mistaken genotype will be conducted in the lab to determine if the sample was mislabeled at time of collection, or if an error occurr ed during handling on the bench. Similar mistakes in recording of field data from CR using mol ecular tools were recently published by Lanyon and colleagues (2009). Cow/Calf Relationships This study illustrates the importa nce of genetic data to validat e field data collected for the MIPS. Especially when twins are suspected, genetics can confirm field observation assumptions obtained by short-term sighting asso ciations between cow/calf pairs. At present, much of the discrepancy between cow/calf pairin g with the genetic data can be confirmed by analysis of the long-term archived files residing in the MIPS da tabase. Better field collection protocols and continued effort to document a nd monitor individuals may help to avoid this discrepancy. Manatees have unique behavioral traits that al low the skilled biologist to record cow-calf bonding with fairly good accuracy (Hartman 1979; Reynolds 1977). However, observed cases in the field (Hartman 1974; Bonde 2009 pers. obs.; Curtin 2009 pers. comm..; Hartley 2009 pers. comm.) have documented that some adult female manatees have been known to accept orphaned or stray calves, which needs to be factored in as a possible reason for mistaken associations in the field. Although adoption is probably a rare occu rrence, a healthy lacta ting mother, even with her natural calf, could supply necessary milk and nourishment to the adopted calf, thus reinforcing pair bonding with the mother. Potential for Adoption From these data there is genetic evidence using ML-RELATE (Kalinowski et al. 2007), NJ trees, and mismatched alleles to suggest that adoption is possible betw een receptive, lactating

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71 females and stray orphaned calves. Even adult ma natees in confined quarters may try to suckle from receptive females, as has been observed by this author on many occasions. In captivity, subadult and adult manatees, and even biological mothers of lactati ng adult offspring, may routinely nurse from each other for extended periods of time. This nurturing seems ingrained in manatee behavior, but acceptance is not always the norm. Body condition and nutrition can affect the ability of a female to produce ample m ilk to provide for her biological calf, let alone another individual. Field observations have been made of lact ating cows with non-biological, but related strays, orphan calves, or recently-weaned juveniles associating closely with and nursing from the cow. For example, BS196 (Georgia), nursed both her dependent calf, BS213 (Peaches) and another known male calf, BS200 (John) for severa l months. John had been previously weaned by his mother, BS092 (June) and was nutritionall y independent at the time. Small calves attempting to nurse with multiple lactating fema les also have been observed. On one such occasion in CR, a small stray calf was seen nur sing within 30 minutes from two adult female manatees, each with her own biologi cal calf. In one instance both the stray and biological calf were simultaneously feeding from the target female. The stray calf also was rejected numerous times by other females in and around the warm-water resting site in CR as it tried to initiate bouts of nursing. Another interesting cas e involved a radio-tagged manatee, BC288 (Ruth) whose small twomonth-old calf was observed suckling from a differ ent female while still in the company of Ruth. Within a week, the manatee Ruth died from a massive, chronic infection of her uterus presumed to result from a complication during parturition. It was probably due to this considerable internal infection that the moribund mana tee stopped producing milk a nd her calf started looking

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72 elsewhere for nourishment. After Ruths carcass was recovered, field crews monitored reports of stray live or dead calves from the area. None was reported, and it was presumed that Ruths dependent calf was successful in finding an ac cepting female to meet her nursing demands. It should be noted that larger calves or j uveniles are less behavi orally and nutritionally dependent on their mothers. Lactating females also have been observed rejecting attempts at nursing by either related or unr elated individuals. There has not been any documented aggression displayed in these manatees towards nursing attempts, only annoyance resulting in retreat being the most common negative response observed. Notably, in the examples mentioned above, field observers would likely assume re latedness between a cow and calf when nursing was observed, although that may not necessarily be the case. Genetics will be an effective tool to validate these processes and assumptions. Grandmother Pedigrees Individuals belonging to the f our documented maternal lineag es identified in this study were not excluded through genotype comparis ons, validating the MIPS assumptions. Through these lineages, we are able to acquire better es timates of age and obtai n accurate age at stage events, like first reproduction in young females. Coupled with size, behavior, and reproductive history, this information could yi eld potentially important insight about fitness and nutritional state. Complete examination of the genotype da ta with plans to proce ss 700 additional samples from CR and another 2,000 samples from carcasses collected statewide will allow researchers to relate these known individuals in mark-recapture models. Conclusion Since the Florida population was recently f ounded and experienced a genetic bottleneck, the use of highly variable tools such as microsatellites became necessary for a better understanding population biology in the Florida manatee. However, the lack of variation and

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73 genetic relatedness of manatees using these microsatellite tools limits the ability to use pedigree programs with any certainty. With more primers and integration of more variable markers such as single nucleotide polymorphisms (SNPs), we hope to initiate pedigree studies in the near future. Continuation of genetic screening of archived manatee samples from the CR population coupled with predictive modeling efforts and MIPS data will illuminate issu es related to basic manatee biology. Future research efforts s hould focus on utilizing SNPs, microarrays, and functional gene sequencing in the arsenal for fi ne-scale population relate dness studies. These molecular tools, coupled with more robust mode ling efforts, will permit better interpretation of recovery efforts for this uni que endangered species.

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74Table 3-1. Manatee sample collection information. Gen Process Number Gen Field Sample Date Mom IDNUM Calf Sequence Calf IDNUM Size(cm) Sex CR01 06-318-6 14-Nov-06 CR026 CR026 Mom 300 F CR107 05-362-1 28-Dec-05 CR026 CR026c1 05-362-1 230 M CR02 06-326-15 22-Nov-06 CR027 CR027 Mom 300 F CR60 97-336-3 02-Dec-97 CR027 CR027c1 CR027c 230 F CR70 99-320-3 16-Nov-99 CR027 CR027c2 99-320-3 175 F CR103 02-330-3 26-Nov-02 CR027 CR027c3 CR027e 220 M CR117 07-332-3 28-Nov-07 CR032 CR032 Mom 350 F CR118 92-043-3 12-Feb-92 CR032 CR032c1 92-043-3 220 F CR119 93-335-2 1-Dec-93 CR032 CR032c2 93-335-2 230 F CR120 97-344-2 10-Dec-97 CR032 CR032c3 97-344-2 180 F CR126 99-362-2 28-Dec-99 CR 032 CR032c4a 99-362-2 170 M CR127 99-363-3 29-Dec-99 CR032 CR032c4b 99-363-3 170 M CR03 07-015-2 15-Jan-07 CR045 CR045 Mom 340 F CR04 06-326-14 22-Nov-06 CR046 CR046 Mom 300 F CR50 95-032-4 01-Feb-95 CR046 CR046c1 CR046d 180 F CR61 97-344-1 10-Dec-97 CR046 CR046c2 CR046e 230 F CR05 06-281-3 8-Oct-06 CR049 CR049 Mom 300 F CR74 00-333-2 28-Nov-00 CR049 CR049c1 CR049f 210 M CR132 06-340-1 6-Dec-06 CR054 CR054 Mom 340 F CR133 92-043-2 12-Feb-92 CR054 CR054c1 92-043-2 230 F CR134 96-341-5 6-Dec-96 CR054 CR054c2 96-341-5 220 M CR135 02-350-4 16-Dec-02 CR054 CR054c3 02-350-4 190 F CR06 06-320-5 16-Nov-06 CR060 CR060 Mom 320 F CR68 99-055-6 24-Feb-99 CR060 CR060c1 CR060d 160 M CR82 01-303-2 30-Oct-01 CR060 CR060c2 CR060h 210 M Gen Process Number laboratory identification number, Gen F ield Sample number assigned to the sample in the field, D ate date collected, Mom ID suspected MIPS number of the mom, Calf Sequence sequence number of calves for suspected mom, Calf IDNUM MIPS ID number for the calf, Size total estimate length, and Sex sex of the animal. Shaded areas represent samples not utilized in the analysis.

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75Table 3-1. Continued. Gen Process Number Gen Field Sample Date Mom IDNUM Calf Sequence Calf IDNUM Size(cm) Sex CR07 06-320-6 16-Nov-06 CR060 CR060c3 06-320-6 160 F CR08 06-320-3 16-Nov-06 CR061 CR061 Mom 290 F CR80 01-011-2 11-Jan-01 CR061 CR061c1 CR061e 220 F CR09 07-029-7 29-Jan-07 CR070 CR070 Mom 320 F CR10 07-108-3 18-Apr-07 CR 071 CR071 Mom LA F CR53 96-068-1 8-Mar-96 CR071 CR071c1 96-068-1 230 F CR88 03-008-1 08-Jan-03 CR071 CR071c2 CR071i 180 M CR114 07-318-5 14-Nov-07 CR093 CR093 Mom 320 F CR102 02-323-3 19-Nov-02 CR093 CR093c1 02-323-3 230 M CR11 07-015-1 15-Jan-07 CR104 CR104 Mom 320 F CR46 93-020-2 20-Jan-93 CR104 CR104c1 CR104d 240 M CR57 97-017-1 17-Jan-97 CR104 CR104c2 CR104f 230 M CR66 98-114-1 24-Apr-98 CR104 CR104c3 CR104g 160 M CR71 00-035-1 04-Feb-00 CR104 CR104c4 CR104h 220 M CR79 00-325-3 20-Nov-00 CR104 CR104c5 CR104i 180 F CR85 02-017-1 17-Jan-02 CR104 CR104c6 CR104k 180 M CR12 07-029-10 29-Jan-07 CR123 CR123 Mom 320 F CR45 92-043-1 12-Feb-92 CR123 CR123c1 92-043-1 240 M CR54 96-330-1 25-Nov-96 CR123 CR123c2 CR123d 235 F CR69 99-055-4 24-Feb-99 CR123 CR123c3 CR123e 200 F CR72 00-325-2 20-Nov-00 CR123 CR123c4 CR123f 200 M CR13 06-320-2 16-Nov-06 CR125 CR125 Mom 280 F CR49 95-031-4 31-Jan-95 CR125 CR125c1 CR125e 210 M CR58 97-308-1 04-Nov-97 CR125 CR125c2 97-308-1 230 F CR76 00-335-4 01-Dec-00 CR125 CR125c3 CR125h 200 M CR14 06-281-4 8-Oct-06 CR130 CR130 Mom 300 F CR78 98-012-1 12-Jan-98 CR130 CR130c1 98-012-1 220 M CR89 03-008-2 08-Jan-03 CR 130 CR130c2a CR130g 230 M

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76Table 3-1. Continued. Gen Process Number Gen Field Sample Date Mom IDNUM Calf Sequence Calf IDNUM Size(cm) Sex CR90 03-017-3 17-Jan-03 CR 130 CR130c2b CR130h 210 F CR15 06-308-5 4-Nov-06 CR133 CR133 Mom 300 F CR63 97-356-1 22-Dec-97 CR133 CR133c1 CR133c 200 F CR95 00-336-6 01-Dec-00 CR133 CR133c2 CR133d 160 M CR16 06-308-4 4-Nov-06 CR157 CR157 Mom 340 F CR100 01-311-2 07-Nov-01 CR157 CR157c1 CR157b 160 F CR17 06-308-8 4-Nov-06 CR164 CR164 Mom 290 F CR64 97-363-3 29-Dec-97 CR164 CR164c1 CR164b 170 F CR84 01-317-1 13-Nov-01 CR164 CR164c2 CR164c 190 M CR116 07-332-2 28-Nov-07 CR171 CR171 Mom 320 F CR124 99-055-2 24-Feb-99 CR171 CR171c1 99-055-2 180 M CR115 07-332-1 28-Nov-07 CR171 CR171c2 07-332-1 170 F CR18 07-038-5 7-Feb-07 CR205 CR205 Mom 290 F CR55 96-341-3 06-Dec-96 CR205 CR205c1a CR205c 140 M CR56 96-341-4 06-Dec-96 CR 205 CR205c1b CR205d 140 F CR92 05-022-6 22-Jan-05 CR205 CR205c2 05-022-6 220 M CR98 01-005-1 05-Jan-01 CR205 CR205c3 CR205g 200 F CR113 07-318-3 14-Nov-07 CR235 CR235 Mom 290 F CR122 98-050-1 19Feb98 CR235 CR235c1 98-050-1 190 M CR105 03-317-1 13-Nov-03 CR235 CR235c2 03-317-1 210 F CR19 06-308-2 4-Nov-06 CR251 CR251 Mom 300 F CR65 98-040-1 9-Feb-98 CR251 CR251c1 98-040-1 190 M CR37 06-326-4 4-Nov-06 CR251 CR251c2 06-326-4 220 F CR20 07-031-10 31-Jan-07 CR263 CR263 Mom 280 F CR83 01-310-2 06-Nov-01 CR263 CR263c1 CR263c 210 F CR142 08-017-2 17-Jan-08 CR266 CR266 Mom 300 F CR143 95-032-1 1-Feb-95 CR266 CR266c1 95-032-1 200 F CR144 98-014-2 14-Jan-98 CR266 CR266c2 98-014-2 160 M

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77Table 3-1. Continued. Gen Process Number Gen Field Sample Date Mom IDNUM Calf Sequence Calf IDNUM Size(cm) Sex CR145 03-022-1 22-Jan-03 CR266 CR266c3 03-022-1 180 F CR21 06-326-2 22-Nov-06 CR271 CR271 Mom 300 F CR44 92-041-2 10-Feb-92 CR271 CR271c1 92-041-2 140 M CR73 00-326-2 21-Nov-00 CR 271 CR271c2a CR271b 180 F CR75 00-335-3 01-Dec-00 CR 271 CR271c2b 00-335-3 200 M CR87 02-364-1 30-Dec-02 CR271 CR271c3 CR271c 170 M CR38 06-326-7 22-Nov-06 CR271 CR271c4 06-326-7 180 M CR22 06-309-5 5-Nov-06 CR272 CR272 Mom 300 F CR121 98-040-2 09-Feb-98 CR272 CR272c1 CR272c 240 M CR106 04-357-1 22-Dec-04 CR272 CR272c2 CR272b 190 M CR23 06-308-1 4-Nov-06 CR277 CR277 Mom 340 F CR52 96-023-4 23-Jan-96 CR277 CR277c1 96-023-4 180 M CR91 03-315-1 11-Nov-03 CR277 CR277c2 03-315-1 190 M CR24 06-326-10 22-Nov-06 CR278 CR278 Mom 290 F CR47 93-035-1 4-Feb-93 CR278 CR278c1 93-035-1 180 F CR39 06-326-11 22-Nov-06 CR278 CR278c2 06-326-11 200 M CR25 06-318-2 14-Nov-06 CR321 CR321 Mom 300 F CR48 95-027-1 27-Jan-95 CR321 CR321c1 95-027-1 230 F CR59 97-312-9 08-Nov-97 CR321 CR321c2 CR321b 200 F CR81 01-302-4 29-Oct-01 CR321 CR321c3 CR321c 200 M CR93 05-022-2 22-Jan-05 CR321 CR321c4 05-022-1 190 M CR109 07-309-1 05-Nov-07 CR321 CR321c5 07-309-1 210 M CR36 06-281-2 08-Oct-06 CR333 CR333 Mom 290 F CR40 06-281-1 08-Oct-06 CR333 CR333c1 06-261-1 190 M CR26 06-326-18 22-Nov-06 CR339 CR339 Mom 280 F CR86 02-330-1 26-Nov-02 CR339 CR339c1 CR339a 220 M CR27 07-029-5 29-Jan-07 CR341 CR341 Mom 300 F CR51 95-052-1 21-Feb-95 CR341 CR341c1 CR341a 195 F

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78Table 3-1. Continued. Gen Process Number Gen Field Sample Date Mom IDNUM Calf Sequence Calf IDNUM Size(cm) Sex CR67 98-352-1 18-Dec-98 CR341 CR341c2 CR341b 200 M CR28 06-309-3 5-Nov-06 CR354 CR354 Mom 300 F CR99 01-008-2 08-Jan-01 CR354 CR354c1 CR354a 200 M CR101 02-016-1 16-Jan-02 CR354 CR354c2 CR354b 220 M CR41 06-309-4 5-Nov-06 CR354 CR354c3 06-309-4 180 F CR29 06-308-7 4-Nov-06 CR358 CR358 Mom 290 F CR62 97-344-5 10-Dec-97 CR358 CR358c1 CR358a 160 F CR77 01-010-1 10-Jan-01 CR358 CR358c2 CR358c 190 F CR42 06-308-6 4-Nov-06 CR358 CR358c3 06-308-6 160 F CR137 07-064-2 5-Mar-07 CR360 CR360 Mom 280 F CR139 99-004-1 4-Jan-99 CR360 CR360c1 99-004-1 200 M CR138 07-064-3 5-Mar-07 CR360 CR360c2 07-064-3 180 M CR128 07-065-1 6-Mar-07 CR363 CR363 Mom 320 F CR129 97-016-1 16-Jan-97 CR363 CR363c1 97-016-1 180 F CR130 00-039-1 8-Feb-00 CR363 CR363c2 00-039-1 180 F CR131 01-310-4 6-Nov-01 CR363 CR363c3 01-310-4 210 M CR30 06-308-3 4-Nov-06 CR385 CR385 Mom 300 F CR123 99-007-2 07-Jan-99 CR385 CR385c1 CR385b 200 M CR136 06-298-1 25-Oct-06 CR385 CR385c2 06-299-1 170 F CR31 06-309-7 5-Nov-06 CR401 CR401 Mom 300 F CR125 99-320-1 16-Nov-99 CR401 CR401c1 CR401b 190 F CR112 07-315-3 11-Nov-07 CR413 CR413 Mom 320 F CR104 02-336-6 2-Dec-02 CR413 CR413c1 02-336-6 220 F CR110 07-313-1 9-Nov-07 CR443 CR443 Mom 340 F CR111 07-313-4 9-Nov-07 CR443 CR443c1 07-313-4 160 M CR32 07-031-14 31-Jan-07 CR458 CR458 Mom 280 F CR94 05-022-1 22-Jan-05 CR458 CR458c1 05-022-1 180 F CR33 06-326-3 22-Nov-06 CR474 CR474 Mom 300 F

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79Table 3-1. Continued. Gen Process Number Gen Field Sample Date Mom IDNUM Calf Sequence Calf IDNUM Size(cm) Sex CR97 00-342-4 07-Dec-00 CR474 CR474c1 CR474b 180 M CR140 01-016-1 16-Jan-01 CR485 CR485 Mom 230 F CR141 08-017-1 17-Jan-08 CR485 CR485 Mom 280 F CR34 06-318-3 14-Nov-06 CR506 CR506 Mom 280 F CR96 00-336-7 01-Dec-00 CR506 CR506c1 CR506a 200 M CR108 06-067-2 08-Mar-06 CR506 CR506c2 06-067-2 220 F CR35 06-326-12 22-Nov-06 CR509 CR509 Mom 270 F CR43 06-326-13 22-Nov-06 CR509 CR509c1 06-326-13 170 M Gen Process Number laboratory identification number, Gen F ield Sample number assigned to the sample in the field, D ate date collected, Mom ID suspected MIPS number of the mom, Calf Sequence sequence number of calves for suspected mom, Calf IDNUM MIPS ID number for the calf, Size total estimate length, and Sex sex of the animal. Shaded areas represent samples not utilized in the analysis.

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80 Table 3-2. Characteristics of the 11 polym orphic microsatellite loci utilized for th e analysis of Crystal River manatee ( T. m. latirostris ) samples. Locus N Tm( C) BSA NA NE PIC Ho HE TmaE1 136 55 + 4.000 2.907 1.181 0.551 0.495 TmaE02 135 62 2.000 1.969 0.685 0.401 0.384 TmaE7 136 56 + 3.000 2.338 0.954 0.552 0.496 TmaE08 137 60 3.000 1.978 0.706 0.401 0.391 TmaE11 136 58 5.000 2.824 1.154 0.474 0.481 TmaE14 134 56 + 4.000 2.582 1.020 0.618 0.613 TmaH13 137 60 3.000 1.624 0.672 0.562 0.572 TmaJ02 137 62 2.000 1.642 0.580 0.632 0.646 TmaKb60 137 62 2.000 1.985 0.689 0.654 0.656 TmaK01 138 58 3.000 1.926 0.840 0.630 0.619 TmaSC5 136 58 3.000 2.622 1.018 0.518 0.492 Locus name ( Locus ), number of samples (N ), Annealing temperature ( Tm( C ) ), BSA requirement (0.4 mg/mL), number of alleles (NA), effective number of alleles ( NE), polymorphic information content ( PIC ), and observed and expected heterozygosity (Ho and HE) for the Crystal River T. m. latirostris population.

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81 Figure 3-1. Map of Caribbean region depi cting manatee haplotypes, from Vianna et al 2006.

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82 Figure 3-2. Notcher used to remove a small piec e of skin from the tail margin of manatees. Figure 3-3. Probability of identity ( P(ID)) is illustrated for the manatee population calculated for sibling association (in red), for Hardy Weinberg equilibrium (in blue), and for values observed in this study (in green) with step-w ise addition for each allele in order of decreasing identity using GENALEX 6.

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83 Figure 3-4. Neighbor-joining tr ee describing the relationships among individuals from the Crystal River, Florida manatee population. Known family units are depicted by similar colors. Two distinct cl usters are apparent and labeled A and B with an outlier group C This neighbor-joining tree was rooted for clarity to help visualize clusters.

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84 Figure 3-5. Summ ary plot of q estimates generated by the sequential cluster analysis of the program STRUCTURE using a K = 3 value performed on the Crystal River, Florida population. Individu als assigned to neighbor-joining tree clusters are indicated by A B or C

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85 CR363c3 CR485 CR125 CR205c3 CR354 CR054c2 CR271c1 CR277c2 CR321 CR509 CR104c6 CR054 CR054c1 CR054c3 CR321c3 CR458c1 CR123c3 CR321c5 CR133 CR125c3 CR474 CR474c1 CR333c1 CR354c1 CR354c2 CR271c4 CR032c3 CR032 CR271 CR271c3 CR032c1 CR032c2 CR354c3 CR339 CR339c1 CR133c1 CR363c2 CR321c2 CR171c2 CR171 CR458 CR321c1 CR251c2 CR321c4 CR027 CR045 CR130c2b CR123 CR123c1 CR027c1 CR104c1 CR027c3 CR071 CR205 CR071c1 CR104 CR027c2 CR104c3 CR104c5 CR164 CR401 CR413 CR401c1 CR164c2 CR205c1a CR360 CR360c1 CR205c1b CR385 CR385c1 CR263c1 CR385c2 CR104c2 CR060 CR205c2 CR360c2 CR277 CR277c1 CR413c1 CR272c2 CR272c1 CR032c4a CR032c4b CR060c3 CR070 CR266c2 CR133c2 CR443c1 CR130c1 CR130 CR071c2 CR333 CR263 CR123c4 CR251c1 CR251 CR157c1 CR341c2 CR049c1 CR272 CR235c2 CR235c1 CR266c1 CR104c4 CR363 CR363c1 CR506c2 CR271c2b CR278c2 CR171c1 CR341 CR341c1 CR506c1 CR443 CR046c2 CR506 CR046c1 CR026c1 CR358c3 CR358 CR358c2 CR130c2a CR093c1 CR125c1 CR093 CR061 CR235 CR278 CR278c1 CR358c1 CR060c2 CR060c1 CR266 CR271c2a CR061c1 CR157 CR046 CR049 CR123c2 CR164c1 CR509c1 CR125c2 CR026 Figure 3-6. Crystal River, Florid a manatees depicted in a neighbor-joining tree using the program, PHYLIP and color-coded to assigned clusters generated from STRUCTURE using a K = 3 value with q estimate > 60.

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86 Figure 3-7. Summ ary plot of q estimates generated by the seque ntial cluster analysis of the program Structure using a K = 2 value performed on the Crystal River, Florida manatee population and compared to the rest of Florida. At top is the structure output generated by Pause 2007 for all four management areas of Florida.

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87 Figure 3-8. Scatter plot of genetic distance versus relatedness of Crystal River manatee samples. As genetic distance decreases, re latedness increases (P < 0.001).

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88 Figure 3-9. Crystal River, Flor ida manatees compared with PAST multivariate analyses using non-metric multidimensional scaling for relatedness (on left) and genetic distance (on right). Each graph was color-coded with six known individual family groups observed over time with life history ob servations. Lines correspond to convex hulls, or polygons, containing all points of that color.

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89 CHAPTER 4 MOLECULAR TOOLS FOR MANATEE CONSERVATION Sirenian Conservation Management and conservation of West Indian manatee populations would benefit from multiple methods of genetic population analyses Manatee behavior is unique and not well understood, and genetic data could provide insights into stochast ic demography. A variety of tools is available for investigating popul ation genetics. Mitochondrial DNA (mtDNA) sequencing, multi-locus analyses utilizing micros atellites and single nucleotide polymorphisms (SNPs), gene expression analysis, genomic sequencing, and identification of protein variants are the major techniques employed. Utilizing multiple genetic tools has strengthened the position for implementation of conservation measures in other threatened and endangered species (Fallon 2007). In the Florida manatee polymorphic microsatelli te loci are difficult to identify in manatee samples. Currently 36 microsatellite loci have been developed for manatee genotyping, however only a limited number of alleles have been identified (Garcia-Rodriguez et al 2001; Pause et al 2007; Tringali et al 2008b). This finding of low allelic diversity in the Florida manatee and other manatee populations decreases the accuracy of parental assignments, and the identification of more diverse microsatellite sites may allow for pedigree finge rprinting studies that are more precise. An additional 17 primers, designed originally for dugong populations hold potential for manatee population applications as well (Hunter et al 2009b). Examination of neutral and functional markers will enhance genetic research by providing information on whole genomic assemblies, indivi dual population distinctiveness, identification of specific genes and their regulation, and the organisms response to environmental change. Next Generation sequencing technology can id entify genes for molecular systematics and

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90 functional genomic analysis, as well as marker development. Darwins On the Origin of Species (1859) identif ied basic rules of evolution that proceed randomly during natural selection. Conservation genetics can instruct us on how to preserve species that are successful at meeting adaptive challenges to environmental change (Frankham et al 2002). Acting upon those random events are contemporary anthropoge nic processes. The effects of human-caused environmental changes as well as societys effo rts to manage a population may conflict with the natural evolutionary processes of a wildlife population (Munoz-Vinas 2004). Society often must make decisions regarding wildlif e populations based upon the best available science. More rigorous and detailed stud ies will assist managers in making policy decisions that could have long-term consequences. The protection of w ildlife may require delineation of management units (MUs), distinct population segments (DPSs), subspecies a nd species designations that are compatible with laws to preserve biologically intact communities (Haig et al 2006; USFWS 2007). The Manatee Core Biological Model (Runge et al 2007a) is a useful tool for assessing manatee population status based upon current unders tanding of annual variab ility in survival and reproductive rates, demographic stochasticity (including changes in effective population size), effects of changes in warm-water capacity, and catastrophes. A reducti on in effective population size will result in loss of genetic diversity (Frankham et al 2002; Allendorf & Luikart 2007). This loss of diversity will likely result in loss of the ability to adapt to environmental changes (Frankham 1995; Franklin & Frankham 1998). Theref ore, greater efforts should be maintained to improve effective population size in the manatee (Runge et al 2007b). As the human population continues to grow, its impact on th e environment and resources becomes even greater, creating a need for rigor ous scientific information on affected wildlife species (Reep & Bonde 2006).

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91 Why Study Manatees? Migration has played an important role in manatees ability to adapt and respond to survival challenges, and accounts for much of th eir geographic distributio n. Migration also is necessary for gene flow and can increase the ge netic diversity. Manatees have persisted in environments where other fragile wildlife populati ons like the Florida panther, Florida red wolf, pallid beach mouse, and Caribbean monk seal have not thrived or have been completely extirpated (IUCN 2009; USFWS 200 9b). Florida manatees have adapted recently to survival among human populations, whereas their Antillean manatee counterparts are much more secretive due to direct threats related to historical and continued hunting (OShea 1988; Deutsch et al 2007; Quintana-Rizzo & Reynolds 2007). Typically, manatee populations ha ve undergone significant fluctuations that have resulted in low allelic diversity when compared to other wildlife pop ulations (OBrien et al 1985; OBrien 2003). The observed low genetic diversity might have placed limits on their ability to cope with some environmental and anthropogenic changes, but those limitations appear not to have affected fecundity and survival of the Florid a manatee. The Florida manatees resilience to environmental pressures, coupled with the sepa ration of populations by distance and vicariance, has lead to interesting biological responses on the organismal level. For example, the patterns of winter site fidelity of manatees in response to cold temperatur es have lead to large numbers utilizing warm water sites. Manatees in large aggregations spend time in close proximity to one another, increasing the potential for disease transmission, through dire ct contact and through coprophagy. Understanding the relationship among manatee gene tics and manatee behavioral responses and adaptive capabilities will help ma nagers direct the recovery of the species. The consensus among researchers is that the Florida manatee population has grown in numbers in recent decades. The initial reacti on of managers is that the Florida manatee

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92 population is doing well. However, evidence of limited genetic diversity suggests that the manatee population may not be as healthy as pred icted based on just the number of individuals (Hunter 2009 pers. comm.). Lack of allelic diversity could have deleterious effects. Still, some wildlife populations, such as the Northern elephant seal, have been able to thrive despite severe historical bottlenecks and low allelic diversity (Hoelzel et al 1993; Slade et al 1998). Whether this will be the case for manatees has yet to be determined. Genetic data have been instrumental in the determination of listing criteria for several endangered species (Fallon 2007). A recent study on the genetic tools used to assess threatened and endangered populations of animals aptly illust rated that when multiple sources of genetic data were used in concert, there was a higher probability for the organisms to receive protection (Fallon 2007). The data from genetics, coupled with other demographic sources of information, are necessary for determining which population un its or stocks will be nefit optimally with applied management (Dizon et al 1992). Genetic Markers A host of genetic markers and tools are availa ble to researchers to aid in determining population status. The selection of the appropria te marker depends on several variables and on the animal under investigation. Considerations for selection criteria includ e: (1) Is the selected marker appropriate to answer the questions at ha nd? (2) Are there limitations related to sample size and method of preservation? and, (3) What are the budget restrictions if any? Many analyses are expensive, but with recent technological advances the cost has been reduced. Several techniques are discusse d in more detail below. Allozymes Historically, allozyme analysis was perf ormed on the Florida manatee to examine geographic distribution patterns in five areas of Florida co rresponding to carcass recovery

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93 locations (McClenaghan & OShea 1988). Hi gh rates of gene flow accounted for the homozygosity observed across all five geograp hic regions analyzed in the study. Mitochondrial DNA Matrilineal haplotypes from sequenced mitochondrial DNA can yield information on phylogenetics and evolutionary processes. The mitochondrial displacement loop (d-loop) has been used consistently on manatee p opulations to date (Garcia-Rodriguez et al 1998; Vianna et al 2006). A 410 bp segment has been most widely used. Cytochromeb analysis also has been implemented, and illustrated little variation in the Florida population, but the sample size was small (Bradley et al 1993). Sequencing other regions of the mitochondrial genome, such as the cytochrome c oxidase 1 (CO1) gene, has been proposed. The CO1 gene has displayed typical variation in many animal systems and is used in DNA barcode identification of species (Stoeckle 2003; Herbert et al 2004). New sequencing technology will make mapping of the entire Florida manatee mitogenome possible in the near future which will provide additional characters for determining genetic variation among populations Complete mapping of genomic mtDNA in killer whales ( Orcinus orca ), for example, has provided information on variations among populations that has lead to the discrimination of three ecotypes within the species (Morin et al 2009). The Antillean manatee mitogenome was se quenced recently and will have value in comparative research (Arnason et al 2008). Microsatellites Microsatellites, also known as simple se quence repeats (SSRs), short tandem repeats (STRs), or variable number of tande m repeats (VNTRs), are sequences of di, tri, tetra, or larger tandem nucleotides and are an excellent tool fo r determining relatedness among individuals and populations. These sites can vary in length at each locus among indi viduals. Microsatellites are neutral markers passed down from both pare nts that provide contemporary information on

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94 relatedness and population structur e. They also can be used to identify individuals in fingerprinting studies. This trai t has been particularly benefici al in mark-recapture studies, where genetic information from individuals is ma tched to life-history obse rvations and additional samples that are acquired through time. This techni que has been applied in the cradle to grave tracking concept, modeling survivability and cause of death, wherein the analysis can identify individuals genetic diversity in relation to health status cla ssification and mortality events (Tringali 2009 pers. comm.). Micr osatellites are helpful in co mparing allelic diversity among populations, determining effective population size, and calculating the size of an original founder population (Frankham et al. 2002; Allendorf & Luikart 2007). Microsatellite studies have been conducte d extensively on the Florida manatee population to determine structure (GarciaRodriguez 2000; Pause 2007). Micr osatellites also have been used to compare Florida manatees with other manatee populations (Cantanhede et al 2005; Vianna et al 2006; Kellogg 2008). Microsatellite studies of other threatened West Indian manatee populations are needed. SNPs Single nucleotide polymorphisms (SNPs) are nucleotide varia tions at a single base which also can aid in population genetic studies. SNPs are bi-parentally inherited and can be expressed in both intron (noncoding) and exon (coding) regions. The presence and uniqueness of polymorphic loci can yield information on taxon omy and individual identification, and have proven useful in identifying populations with low genetic diversity (Vignal et al 2002). Furthermore, Restriction-site Associated DNA (RAD) studies can identify gene sequences and large numbers of individual SNP sites (Baird et al 2008). Identification of SNPs from genes could provide functional inform ation in addition to detecti ng subpopulations for demographic analysis, enabling managers to implement meaningful conservation measures.

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95 Gene Expression Examination of gene expression and regulation in manatees will provide insight to health, reproductive fitness, and early disease detection. SAGE (Serial Analysis of Gene Expression) is an approach for analyzing the observed variation in gene expression among individuals and populations (Velculescu et al 1995). Understanding manat ee immunology, susceptibility to disease, wound healing, bone reso rption, dental or other adaptati ons, and response to cold stress or red tide will inform researchers and managers. Also, examination of major histocompatibility complex (MHC) genes can increase our unders tanding of population differences, genetic diversity, disease defense, mate selection, a nd reproductive fitness (B ernatchez & Landry 2003; Piertney & Oliver 2006). The variations in gene expression among individuals and populations also can provide information on demographics and adaptive responses. Behavioral responses to density-dependent pressures may affect breeding success, reproduction, dispersal, and disease susceptibility. These tools can he lp us gauge the cumulative impacts of climate change (Thomas et al 2004). This advanced form of health assess ment will allow scientists to develop predictive modeling programs and study target animal resp onse, which will enable a more informed approach for management. The development of manatee specific assays can provide information on the levels of gene expression of manatees encounteri ng various environmental stressors such as red tide (Bossart et al 1998), cold stress (Bossart et al 2003), and exposure to pollutants (Stockwell et al 2002). Such transcriptome or gene expres sion analyses can be used to de velop on-site tests to evaluate the health status of injured or im paired manatees. Research to de tect gene expression can utilize special microarray chip sets with unique seque nces. These chips are coated with several thousand DNA probes and when linked with a samp le will generate a signal if hybridization occurs. Gene expression research becomes ev en more pressing as the manatee population in

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96 Florida responds to lower carryi ng capacity due to a reduction in available warm water sites for artificial winter refugia with the imminent closing of power plants (Laist & Reynolds 2005; Runge et al 2007b). It is uncertain when artificial sources of warm water currently available for Florida manatee use will disappear, but all agree th at this will happen with in the next couple of decades. Use of field tests for wild manatees could be nefit diagnostic studies during manatee health assessments. For example, a manatee caught in 2004 in Port of the Is lands, Florida suffered from a massive internal infection undetected at the time of capture. Tools have been designed clinically to detect inflammation in manatees us ing levels of serum amyloid-A (SAA), but assays are expensive and must be perf ormed later in the lab (Harr et al 2006). The manatee captured in 2004 was released with a radio ta g but, unfortunately, died a month later. Had real-time information on the acute level of SAA been availa ble during the processing of the manatee, that manatee could have been taken to a rehabilitatio n facility for monitoring and treatment (Rember et al 2009). Genomes The Genome 10K Project provides whole-ge nome sequences from multiple vertebrate species for comparative purposes (Haussler et al 2009). To date, 32 species of mammals have been genotyped and entered into a comparative database (Haussler et al 2009). Proposed by the Genome 10K Project, manatees have not been in cluded in the species sequenced to date, but having total genome coverage would be extremely beneficial for the species and collaborative research.

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97 Implications for Manatee Conservation Genetic Samples Current genetic analyses of many manatee populations ar e incomplete because of inadequate sample size. Manatee sample colle ction has evolved from opportunistic carcass collection to live animal biopsy. Biopsy techniques have improved from cattle ear notchers applied to tail margins (Appendix 1), to dermal skin scrapers (Carney et al 2007), to biopsy needles (Davis et al 2009). The collection of fecal samp les also has led to innovative genetic applications in very remote areas of sirenian distribution where sample collection is extremely difficult (Tikel et al 1994; Muschett et al 2009). However, use of fecal material has limitations because the possibility of contamination and collection of multiple samples from the same individual may bias results. Additional samples obtained from remote areas throughout the range of manatees and analyzed with current genetic methods for assessing population structure also will be useful. Once a cache of samples is available, a host of options for genetic analyses exists. Molecular Tools Care should be exercised when selecting the appropriate set of markers. Studies have demonstrated that a more robust assessment is obtained when more markers are employed (Latta 2006). The discreteness (amount) and significance (typ e) of genetic analysis must be appropriate for the questions to be addressed (Fallon 2007). Microsatellites have been useful for delimiting population differences in many species (Quellar et al 1993; Jarne & Lagoda 1996; Goldstein & Pollock 1997). Species that have been separa ted for long periods of time generally require smaller data sets for analysis, whereas recent sepa ration of species (such as the Florida manatee) require more samples and genetic characters (F allon 2007). Generally, multiple genetic markers versus a single type of marker are favored. Neutral markers, versus genes that code for adaptive

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98 variation under selection in the environment, ch ange more quickly, making them good indicators of population uniqueness (Merila & Crnokrak 2001; McKay & Latta 2002; Hoekstra et al 2004). Care should be taken to make sure that the gene tic data are used in conjunction with ecologic, geographic, morphologic, or lif e history data sets (Haig et al 2006). Technologies and tools for molecular analyses continue to evolve and many currently are applied to understand the Florid a manatee population. Allozymes, mtDNA, and microsatellites demonstrated little to no genetic differences among the four existing MUs (McClenaghan & OShea 1988; Bradley et al 1993; Garcia-Rodriguez et al 1998; Vianna et al 2006; Pause 2007), however recent analyses using new statis tical techniques are suggesting significant differences among the four Florid a MUs and the populations inhabi ting the east and west coasts (Tucker et al 2009; Tringali 2009 pers. comm.). This also was supported when the Crystal River manatee population was compared to the rest of Florida and cluster analysis identified two distinct groupings. When samples were compared between the Florida and Puerto Rico manatee populations, very distinct st ructure was observed (Hunter et al 2009c), as expected, since the two groups represent sub-species. However, th e Endangered Species Act only recognizes the West Indian manatee throughout its range as one all-inclusive group for legal protection (USFWS 2007). Thus, one benefit of genetic scrutiny may be the development of separate management strategies for each subspecies. Genetic information currently is used when se lecting release sites for captive manatees, to ensure that manatees from one area of Florid a are not introduced into another. Presently, manatees typically are not moved intentiona lly from one coast to another, although through misguided efforts in the past this has occurred. Generally, captive manatees are released to

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99 populations that, to the best of our knowledge, have similar genetic characteristics, with the expectation of preserving be neficial local adaptations. Recent genetic findings have suggested that th ere is low allelic diversity in the manatee population in Florida (Pause 2007), likely the resu lt of a founder event in the Florida manatees history. Additionally, Florida manat ees have a low effective population ( Ne) size (Hunter 2009 pers. comm.). This raises concerns for the we ll-being of the manatee population as a whole in Florida, as many policies regarding their prot ection are based primarily on predicted population size. Additional studies are n eeded to examine population fitness and the impacts of inbreeding in the Florida manatee. Studies have show n that once a population drops below inbreeding thresholds, detrimental effects can occu r during stochastic change (Frankham et al 1995). Examination of genetic markers wo uld be very useful in determining the resiliency of manatee populations to perturbations lead ing to dramatic population fluctu ations. Genetic markers also can be utilized to determine evolutionary lin eages with predictions of founder population size and subsequent coalescent time calculation dating back to the event (Bea umont 2003). Currently, no standards or guidelines are in pl ace to assist researchers and managers with the appropriate selection of gene tic tools to study the status of wild populations (Fallon 2007). As each wildlife population is different and warran ts different considerations, standardization among species is problematic. The information gained through additional genetic analyses, however, would greatly enhance th e possibilities for preservation of the populations of West Indian manatees throughout their range. Thes e tools would be useful for studies of other trichechids and dugongids as well. The future direction of genetic research will utilize sequencing modalities, implementation of large volu mes of data, and will design tools to gauge

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100 the health and fitness of indivi duals within populations of sireni ans. New information will be provided to managers for implementati on in manatee prot ection strategies.

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101 CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS Recovery of an Endangered Species in Florida Research has paved the way for a better unders tanding of the Florida manatee. Research legacies have formed the cornerstones for much of the past, continuing, and proposed research directions. Several long-term data sets are proving to be very valuable, not only for interpreting the species biology, but also for designing tools to predict future scenarios through trend analysis. Genetics is one of those tools. Coupled with traditi onal field applications, molecular science is providing another discipline to enhance the knowledge of manatee biology. Manatees have low genetic diversity, a long generation time, a low rate of reproductive replacement, and in Florida, as elsewhere, th ere are significant anth ropogenic threats. The challenges confronting the manatee make rec overy of the species even more tenuous. Population viability trends are easily influenced by slight changes in life history parameters. The lack of diversity in the Florida manatee popula tion suggests that manatees have gone through a bottleneck in recent evolutionary time (Soule 1985; Amos and Harwood 1998; Waldick et al 2002), and likely reflects the recent colonization of Florida from stocks established in the Caribbean Islands, as suggested by Domning (2001a). One consequence of population reduction is loss of genetic diversity (F igure 5-1). Several generations of severe inbreeding in a small subpopulation, or repeat ed crashes of the numbers of animals in a subgroup, will result in a depletion of most of the genetic variation originally present in the initially larger populat ion (Wahlund 1928; Soule 1985; Okello et al 2008b). It is generally recognized that genetic variability is necessary for both adaptation to changing environments and long-term surv ival of the species (Ralls et al 1979; Frankel & Soule 1981; Frankham et al 2002; Allendorf & Luikart 2007). Strategies to preserve or increase genetic

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102 diversity require knowledge of the distribution of variation within subpopulations, levels of gene flow between these segments of the population, and mitigation of threats to the population. West Indian manatee mitochondrial DNA (mtD NA) haplotypes indicate patterns of evolution and phylogeography on a grand scale. Through mutations, molecular clocks give inference as to the length of time populations ha ve been separated from each other. However, mtDNA cannot answer the finer-scale questions re garding the diversity and movements of the Florida manatee population (Garcia-Rodriguez et al 1998; Garcia-Rodriguez 2000). Furthermore, no population structure among geographi c locations was detected in Florida using mtDNA, as only one haplotype has b een identified (Garcia-Rodriguez et al 1998; Vianna et al 2006; Hunter et al 2009c). Additionally, mtDNA is maternally inherited, representing only the female lineage, and therefore does not yield all the evidence to piece together sex-biased dispersal influences. Microsatellite nuclear DNA (nDNA) analyses reveal higher individual resolution in genetic diversity and population structure as compared to mtDNA analyses. Microsatellite markers are biparental and provide a contemporary vs. historic perspec tive of the population. The allelic variability at microsatellite lo ci is generally high enough to distinguish genetic relationships among closely-related populations (Allendorf & Luikart 2007). Micr osatellite analyses can also ascertain paternity and reproductive success, de termine pedigrees, mate selection, levels of inbreeding, effective population size ( Ne), local gene flow, and aid in forensic analyses (Budowle et al 1991; Edwards et al 1992; Bruford & Wayne 1993; Queller et al 1993; Morin et al 1994; Allen et al 1995; McConnell et al 1995; Blouin 2003). Building on previous work initiated by the USGS Sirenia Project, a panel of 18 primers (Garcia-Rodriguez et al. 2001; Pause et al. 2007) has been utilized to conduct fine-scale genetic

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103 structure and relatedness studies on the manatee populations in Florida (Tucker et al. 2009), Belize (Hunter et al. 2009a), Mexico (Nourisson et al. 2009), and Puerto Rico (Hunter et al. 2009c). The fine-scale manat ee population studies found low leve ls of genetic diversity and little sub-structuring among the four management un its in Florida (Figure 5-2). The Puerto Rico study (Hunter et al. 2009c) compared the Puerto Rico popul ation to the Florida population, since the populations are listed and managed together under the U.S. Endangered Species Act (ESA) by the U.S. Fish and Wildlife Service (USFWS 1982). The 2007 ESA Status Review suggested that the West Indian manatee be downlisted fr om endangered to threat ened, primarily due to successful recovery efforts in Florida which have resulted in a recent increase in population size. However, a highly significant differentiation was identified between Florida and Puerto Rico, suggesting little, if any, breeding and/or migr ation between the populations. This study by Hunter and colleages indicated that the Puerto Rico population would benefit from a separate management plan that also considered the unique threats, habitats, a nd significantly smaller number of individuals. In conjunction with the primers mentioned above, future studie s will utilize an additional 18 primers (Tringali et al. 2008b) developed for Florida manatees ( Trichechus manatus latirostris ) and 17 primers (Broderick et al. 2007) developed for dugongs ( Dugong dugon) which were determined to be polymorphic in the Florida manatee (Hunter et al. 2009b). These panels of microsatellites will be used to perform in-depth population and conservation genetic studies detailed below. Manatee Analysis using 454 GS-20TM Pyrosequencing Technology To illustrate the utility of new molecular technology, a sample of manatee blood was collected from a 34 year-old captive-born female manatee, Lorelei, from Homosassa Springs Wildlife State Park, Florida. Lorelei was born in 1975, conceived by two long-term captive

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104 manatees, Romeo and Juliet, at Miami Seaquarium. Her blood sample was centrifuged and the buffy coat containing white blood cells was isolat ed and placed into blood lysis buffer solution (100 mM Tris-HCl, 100 mM EDTA, pH 8.0, 10 mM NaCl, 1% SDS) at room temperature for storage (White and Densmore 1992). Geno mic DNA was isolated and submitted to the University of Florida, Interdisciplinary Center for Biotechnology Research (UF, ICBR), to be prepped for pyrosequencing with the Roch e 454 Genome Sequencer (GS-20) using FLXTM standard technology (Roche 2009). Titration sequence data produced segments of approximately 250 bases in length, which were generated and stored in massive text files. These data were run through a based algorithm bi oinformatics program, PERL SCRIPT (Temnykh et al 2001; modified by W. Farmerie, University of Florida, ICBR). The PERL SCRIPT program identifies segments of nucleotide repeats in the sequence data based on parameters set by the operator. Further data manipulation analysis allows investigators to so rt the data and identify unique sequences with adjoining flanking regions. Using this stochastic approach in the GS-F LX titration analysis, over 700,000 bases of the manatee genome were identified. PERL SCRIPT examined 2,200 reads of approximately 250 bases in length recording 347 di-, tri-, or tetr a-nucleotide repeat se quences (Figure 5-3). Screening only of available nucleotide data identi fied 120 di(5-14 repeats) 71 tri(4-7 repeats), and 12 tetra(4-6 repeats) nucleo tide sequences that contained at least 40 bp of flanking region at both the 5 and 3 ends (Figure 5-4; Table 5-1). In follow-up studies, flanking primers can then be designed for the putative loci and used with representative samples from a manatee population to detect the level of polymorphism. In one 10hour run, this capability amassed more potential polymorphic microsatellite loci than researchers have produced during th e last 10 years using enriched libraries, conven tional cloning, and capillary electrophoresis-based se quencing tools.

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105 Future Directions The aims presented here are di rectly related to longstanding e fforts by federal, state, and private agencies to ensure the continued health a nd survival of the endangered Florida manatee. These future research endeavors are supported by the collaborative efforts outlined in the stated goals of the Cooperative Florida Manatee Gene tics Working Group. This group guides genetic research efforts to address concerns and prioriti es outlined in the Florida Manatee Recovery Plan (USFWS 2001) by avoiding duplication of res ources and efforts and provides a forum to facilitate excha nge of ideas. By establishing the degree and pattern of ge netic variation in th e Florida population, informed judgments can be made ab out the ability of manatees to withstand periodic catastrophic losses, which have occurred or may be expected to occur due to assorted stresses, including prolonged cold weather, red tide events, or viral diseases. Manatee use of Florida habitat by the current population in winter a nd their dependence on warm-water refugia (Figure 5-2) are expected to change in the near future as arti ficial sources are reduced and natural springs and passive thermal refugia are utilized (Figure 5-5). Until now, manageme nt decisions, including establishment of habitat sanctuaries (USFWS 197 9), have been based largely on increasing the population, but future recovery plans could be guided by data on the effective population size, the fitness of individuals and populations, the ge netic diversity present in each geographic area, and the level of gene flow between areas. The studies detailed here will be conducted by the USGS, Southeast Ecological Sc ience Center (SESC) Conserva tion Genetics Laboratory. Sequence the Manatee Transcriptome usin g Massively Parallel Pyrosequencing The microsatellite studies conduc ted to date have revealed reduced genetic differentiation and poor resolution of structur e in the West Indian manat ee populations. Therefore, management and conservation of manatee popu lations would greatly benefit from the

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106 comprehensive information provided by massively parallel pyrosequencing technologies. Most wildlife species, including ma natees, do not have genomic or expressed gene sequences available. Massively parallel Roche 454 pyrosequencing is a highl y useful technique to identify expressed genes in non-model species like the ma natee. Gene sequences and markers gleaned from 454 pyrosequencing can be used in a vari ety of high-resolution studies, including the taxonomic classification of species and subspeci es and determining the genetic structure of populations or geographically di stributed groups. Comprehensiv e gene expression profiles can be used to address the overall he alth and fitness of i ndividuals within a popul ation and the impact of environmental stressors. West Indian manatees lack genetic divers ity, both at mitochondrial and nuclear loci, making fine-scale population studies difficult. In fact, all T. manatus populations inve stigated to date have lower microsatellit e diversity than the average demographically challenged mammalian population (HE = 0.5 to 0.6 and Aave = 6.9) affected by hist orical or long-term harvesting, fragmentation, or pollution (DiBattist a 2007). In comparison, large, undisturbed and healthy mammalian populations have hi gher average diversity values (HE = 0.6 to 0.7 and Aave = 8.8) (DiBattista 2007; Garner et al. 2005). Therefore, more variable and informative tools are needed to address population structure, divers ity, and fitness. As discussed, parallel pyrosequencing will be used to develop a transcriptome (or identify actively expressed nuclear genes) for the West Indian mana tee to allow for the identification of large-scale functional genomic tools sensitive to adaptive population differences. This technique also can be used to study the genetics of the dugong and other ma natee species (Tikel 1997; Cantanhede et al 2005; McDonald 2005; Vianna et al 2006). Pyrosequencing, or se quencing-by-synthesis, using a Roche 454 pyrosequencer, is a new technique dete cting the release of pyrophosphate during the

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107 nucleotide incorporation rather than traditional Sanger chain-terminator-based sequencing. This method increases DNA sequence depth and transcri ptome coverage, while decreasing costs, labor, and time. The development of a manatee transcriptom e-based analysis on mass gene expression would facilitate functional genomic studies (re lating genetic sequence and biological function) and gene expression profiling. Comparative gene expression profiles can identify adaptive differences between recently di verged populations, candidate gene s associated with the current states of fitness, or differences in transcriptome expression in re sponse to physical stressors such as cold temperatures, brevetoxi n poisoning, debilitation, or infec tion due to traumatic injury. From this information, on-site health test s could quantify the degree of physical or immunological stress the animal is experiencing in order to tria ge sick animals effectively. Transcriptome-based information would enable molecular phylogeneti c studies and single nucleotide polymorphism (SNP) marker develo pment. SNPs are often based on functional genes, sensitive to fitness conditions or adapti ve differences that can identify phylogenetic or evolutionary divergence of groups. SNP markers are especially useful as they are less expensive and easier to standardize than microsatellites, en abling more markers to be used and resulting in greater resolution of diversity. Improved population differentiation will perm it evaluation of the relative importance and validity of regions used for demographic benchm ark criteria in the Florida Manatee Recovery Plan (USFWS 2001). Additionally functional markers with improved resolution will enable biologists to determine the factors which affect gene flow in the population and the possible impacts of inbreeding. Health-related issues with regional significance could be identified.

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108 Understanding these factors will al low us to ascertain how differential mortality could affect population structure and genetic diversity. Genetically Identify Individual Crystal Riv er Manatees and Integrate Data with the Manatee Individual Photo-identification System for Capture-Recapture Studies The integration of genotyping with the Manat ee Individual Photo-id entification System (MIPS) data can improve the tools to examin e manatee population parameters. The genetic signature from microsatellite markers and SNPs can complement the MIPS (Beck & Reid 1995) by improving the ability to estimate adult surv ival and reproductive rates and to assess population growth rates and trends in models. Specifically, multiple identification photographs and genetic samples collected from individual mana tees during different lif e stages and at death will benefit the program. Genetic identifica tion of manatees without physical identifying features, such as scars, particularly for young calves, will allow capture-recapture analysis through subsequent sample collection opportunities. This also will enable determination of the identity of unscarred manatees, d ecomposed carcasses, and to a lesse r extent manatees with scar patterns that have changed since initial documenta tion to the extent that a photographic match is difficult or impossible. Genetic s can also be used to estimat e an error rate for MIPS-based identifications. As determined by our current acceptable estimates of probability of identity ( P(ID)sib = 1.618E-03, P(ID)HW = 1.472E-06), researchers will ha ve the ability to identify, confidently and accurately, individual manatees in the Florida population using 18 microsatellite markers (Hunter et al 2009b). Sex of the animal if unknow n also can be determined with specially designed probes (McHale et al 2008; Tringali et al 2008b; Lanyon et al 2009). Working closely with researchers at the US GS, Sirenia Project, a conceptual capturerecapture model developed by William Kendall (USGS) will jointly analyze the MIPS and genetic data. This novel effort builds on th e manatee stage-based matrix model (Runge et al

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109 2004) and provides the ability to tr ack individuals from their first identification as a calf, through the life stages, and terminating ultimately as a recovered carcass. The model is capable of estimating survival rates at each life stage. It will also be capable of determining individual breeding rates for females. Analysis will also allow for the partitioning of cause of death of individuals into relative sources of mortality. Additionally, by integrating genetics, pedigree studies can address factors such as the freque ncy of twins, whether twins are fraternal or identical, the frequency of adop tion, and the potential paternal co mponent. Selected samples, such as those from rehabilitated animals schedu led for release or animals rescued from areas outside of Florida, will be subjecte d to microsatellite studies to iden tify their location of origin. Since the manatee genetics sample collecting e ffort in Crystal River, Florida was begun by the U. S. Geological Survey (USGS) Sirenia Project in 1989, more than 1,000 genetic samples from free-ranging manatees (mostly calves, but mo re recently adults) have been collected and maintained in archives (Appendix). To date, 300 samples have been genetically identified, and two calf samples have been successfully linked by genetics to known MIPS individuals. Genetic data collected from carcasses provided by the Fi sh and Wildlife Research Institute (FWRI) in Florida are also availabl e for future matching. Examine the Effective Population Size ( Ne) of the Current and Past Florida Manatee Population Genetic diversity is critical to maintaining e volutionary potential a nd individual fitness. The loss of genetic diversity can result in reduced adaptability and decreased population persistence. Health or viabil ity of the population or long-term persistence of the species is quickly decreased in populations with low de nsities, limited genetic diversity, or high anthropogenic impact. Indeed, many T. manatus populations have been determined to have low mitochondrial DNA variation, and Flor ida has no variation (Vianna et al 2006). Similarly, a

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110 survey of 18 nuclear microsatellite loci by Hunter and colleagues ( 2009a; 2009c) has identified limited diversity within Florid a, Belize, and Puerto Rico (expected heterozygosity, HE = 0.41, 0.46, 0.45 and average number of alleles, Aave = 4.1, 3.4, and 3.9, respectively). Another indicator of genetic divers ity is the effec tive population size ( Ne). Ne was proposed by Sewall Wright who helped to define theoretical population genetics and the current paradigm of evolutionary biology. Ne is defined as the number of breeding individuals in an idealized population that would show the same amount of dispersion of allele frequencies under random genetic drift or the same amount of inbreeding as the population under consideration (Wright 1931). Ne can be calculated by the variance in alle le frequencies or by fluctuations in the inbreeding coefficient; the two are closel y related. Many populat ion parameters reduce Ne, including variations in population size, unequal sex ratios and overlapping generations. Although the Florida manatee census size has had a recent positive trend, the low haplotype diversity and nuclear diversity values, A and HE, indicate that the population was founded by only a few individuals, or has recently undergone a bottl eneck (a severe reduction in population size). It is therefor e important to calculate the Ne to determine the overall genetic health and long-term sustainability of the population. The Florida manatee Ne will be compared to the Belize and Puerto Rico manatee populations to provide a referenc e point that may be a more appropriate (i.e., biologically meaningful) nu mber for managers to strive to maintain or increase through management actions. Examining the Evolution and Relatedness of Trichechids The range of the West Indian manatee extends throughout tropical habi tats where there is unobstructed access to fresh water s ources. Generally this is from Brazil through the Caribbean and Central America to Florida. Throughout this range, manat ees are negatively impacted by habitat destruction and anthropogenically induced mortality. Effective management of imperiled

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111 species requires detailed geneti c and taxonomic information to identify species, subspecies, and distinct populations. A study looking at mitochondrial DNA disp ersal and relatedness in the West Indian manatee identified three e volutionarily related clades (Vianna et al. 2006); however, information addressing contemporary movement patte rns is needed in order to promote healthy ecological and evolutionary processes. Conservation practices that pr otect and maximize the existing genetic diversity are needed for manatee populations to recove r. The Florida, Belize, and Puerto Rico populations have reduced genetic diversity (Aave = 4.1, 3.4, and 3.9, respectively) as compared to the average hunted or fragmented mammalian population (Hunter et al 2009a; 2009c). Presumably, other manatee populations, which are smaller and have unsustainable conservation practices, possess even lower diversity. The data generated in th is study can help to identify imperiled manatee populations in need of enhan ced conservation efforts. A proposed genetic study will genetically investigate West Indian manatee samples from collaborators in at leas t 16 countries throughout the species range and compare their relatedness and genetic diversity to existing data sets established from the Florida, Belize, Mexico, and Puerto Rico populations. Effort is being employed to gather new samples and utilize the existing 200 archived samples held at the USGS Conserva tion Genetics Laboratory. Samples currently in the USGS archive to include in the study are from the Bahamas, Brazil, Cayman Islands, Colombia, Costa Rica, Dominican Republic, Fr ench Guiana, Guatemala, Guyana, Honduras, Panama, and Venezuela. DNA extracted from f ecal samples can also be used for genetic analysis (Tikel et al 1994; Muschett 2008; Muschett 2009 ). Microsatel lite nDNA and mtDNA will identify unique populations and genotype anal yses will explore the genetic diversity and identify regions in need of protection. Informa tion also will be provide d on the relationships of

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112 populations, phylogeographical dist ributions, and taxonomic status for the identification of appropriate stocks or management units. By examining the connectivity of threatened West Indian manatees we will better understand the connectivity of popula tions throughout the species range. This information will greatly assist in determining the proper mana gement units for improve d manatee conservation and recovery efforts. For example, the Beli ze manatee population is th e largest in the Wider Caribbean and may serve as a source for repopula tion of other Caribbean countries. However, increased anthropogenic threats, such as pollution and developmen t, could cause the population to decline, limiting the number of individuals able to migrate to other countries. Investigations in southern Belize, near the Gu atemalan border, have detected diseased manatees and a severe loss of sea grass beds (a primary food source), possibly attributed to the intensifying shrimp farming industry along the coastal habitat utiliz ed by manatees. With recent evidence of manatee poaching in Guatemala, informati on on local movements could launch additional protections for populations in both southern Be lize and Guatemala (Qui ntana-Rizzo & Reynolds 2007). Previous genetic studies have assisted in identifying management units for the conservation of manatee populat ions. With the Florida ( T. m. latirostris ) and Puerto Rico (T. m. manatus ) manatees currently listed together under th e U.S. Endangered Species Act, actions are being implemented to identify unique characters between these two populati ons that will lead to different management recovery plans (USFWS 2009a). A survey of microsatellite DNA identified highly significant di fferentiation between the Florida and Puerto Rico populations (FST = 0.16, P < 0.001), indicating that each population should be considered a unique unit of management (Hunter et al 2009c). This information has lead to consideration by the USFWS to

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113 treat the Florida and Puerto Rico manatee popu lations as two separa te distinct population segments when evaluating strate gic management actions (USFWS 2009a). Similar analyses will be utilized to identify the correct unit of ma nagement and identification of stocks for other T. manatus populations. Continue Collaboration on Wildlife Species with USGS Scientists and International Researchers to Further Genetic Capabilities Our genetics capabilities pr ovide the opportunity for colla borations among researchers within the USGS and with institutions outside of the Southeast Ecol ogical Science Center (SESC). In addition to the manatee component the SESC Conservation Genetics Laboratory is working on other related projects in conjunction with fellow scien tists. Two invasive species, the black carp and Burmese python, currently are be ing characterized genetically to determine relatedness and define the populatio n structures of these species. Important questions, such as source and sink population dynamics and potential release sites will be addressed. These findings have significant mana gement implications. Currently, two graduate students are worki ng with USGS staff on the population dynamics of manatees; one on the West African manatee ( Trichechus senegalensis ) and another on West Indian ( T. m. manatus ) and Amazonian ( T. inunguis ) manatees. Additional collaborations with international sirenian researcher s will provide much needed data to advance our understanding of sirenian biology. These collaborators include Drs. Janet Lanyon from the University of Queensland, Australia; Helene Marsh, James C ook University, Australia; Benjamin Morales, ECOSUR, Mexico; and Miriam Marmontel, In stituto de Desenvolvimento Sustentvel Mamirau, Brazil.

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114 Conclusions An increase in habitat loss and high mortality are factors which threaten the future of the Florida manatee. A low intrinsic reproductive rate, low natural populatio n density, and high use of urbanized habitats make this species particularly vulnerable to human perturbations. One consequence of population reduction is loss of gene tic diversity, and the reduced diversity leads to more reduction in population size. These f actors combined synergistically will hasten extinction processes. Several generations of severe inbreeding in a sma ll population or repeated crashes of the population leaving a small number of individuals can deplete most of the genetic variation from an initia lly larger population. It is generally recognized that genetic vari ability is necessary for both adaptation to changing environments and long-term survival of a species. Strategies to preserve genetic diversity require knowledge of the distribution of variation within populations and among species. As the existing manatee population in Flor ida changes, allelic diversity is more difficult to predict (Figure 5-1). What lies in the future for the Florida manatee? Manatee numbers were reduced due to anthropogenic lo ss for centuries, and it will likely take many generations to resolve the genetic consequences. Scarcity of warm water habitat in the future may have severe consequences for the recovery of the species. As artificial sources of warm water are eliminated in the next few generations, local manatee distribu tion will be affected (L aist & Reynolds 2006). The individual manatee will likely respond by adapti ng to smaller winter s ite fidelity clusters (Figure 5-5). These tighter aggregations will be separated by greater distances, making breeding between individuals from each area less likely th an it is today. Ensuring open travel corridors among these areas and providing ample habitat for ma natees will help facilitate adequate gene flow.

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115 The genetic tools described here will provide information on the frequency and success of different lineages or family units in each geogr aphic area, as well as th e level of gene flow among those areas. The studies described here w ill also establish a basis for examining disease susceptibility among manatees. Knowledge of hea lth and fitness of the manatee and associated biological systems will help predict outcomes from exposure to anthropogenic sources, habitat alterations, and climate change. Definition of the Florida manatee populati on structure and reproductive parameters will have conservation implications. Management po licies in-place and adopted for the future will benefit from recognition of family relationshi ps within geographic or wintering groups as expressed by management units. For exampl e, should depleted ma natee populations be supplemented by translocations from other areas? Should a rehabilitated ma natee be released in any appropriate location without regard to genetic consequences ? Misdirected translocations could compromise the integrity of genetic differe nces which have accumulated over evolutionary time, however they can also be nefit local populations. The definition of family units based on these genetic tools will help to avoid future inbreeding events, should animals be placed in captivity for long periods of time before they are returned into the wild. The data gathered on manatee genetics over th e last two decades are already being made available to managers for use in assessing future manatee recovery efforts. This molecular information is also helping reconstruct phylogenetic assumptions based on previous morphological characters and acquired traits. Sim ilarities in elephant behavior and biology are also helping the field scientist better understand manatees and their abilit ies to respond to various challenges. Debates regarding th e consequences of behavior (Bengtson 1981) and scramble promiscuity (Anderson 2002) may yield information on extra-limital movements and changes in site fidelity and home range boundaries. Recent evidence of over mating has occurred in Florida

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116 manatees resulting in death of the target female. This possible density dependent event may have influences on manatee social structure and migration. Misdirected captive releases and translocations of manatees may compromise th e integrity of genetic differences established between some areas. Certain depleted populations may require direct intervention through supplementation and reintroduction programs to aid th e fitness of the local gene pools, especially in the Caribbean and Central and South America. Misdirected translocation without regard for maintenance of genetic diversity will have c onsequences and be detrimental to the local population. These activities should be closely monitored. Is there a population separation between the east and west coasts of Florida and are they distinct evolutionary units? Da ta indicate that the amount of gene flow between the coasts is keeping populations from diverg ing into distinct evolutionary units. However, differences suggested by FST, and allelic frequencies show eviden ce of subtle population differentiation between the east and west coasts of Florid a (Garcia-Rodriguez 2000; Pause 2007; Tucker et al 2009), but more information is needed. A reduction in effective population size will over time affect the long-term maintenance of genetic diversity resulting in lower reproduction output and survival rates (Frankham et al 2002). Even with the resilien ce and plasticit y of manatees, without management based intervention a low Ne will eventually lead to inbreeding and extinction. Intervention will require human manageme nt responses to help ensure survival. This may include removal of individual s to protect them from threats and potential disease contact, thereby improving reproduction and survival. Some of these factors are currently being employed, but with better understand ing of complicated interactions between biological systems and the environment, greater genetic diversity can be achieved with the reduction of threats and translocation of individuals.

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117 Manatee populations have evolved over geologic time and have persisted. Recent conditions and the anthropogenic pressures have changed some natural processes, thereby threatening the manatee. Stringent conserva tion measures require our continued commitment and diligent attention. Especially, with th eir dependence on artificial warm water sources, Florida manatees will need to alter their alre ady compromised adaptive behaviors in order to persist into the next century.

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118 Table 5-1. List of di-, tri, and tetra-nucleotides identifie d using FLX technology on manatee DNA with number of repeats for each sequence. Number of sequences Seque nce Number of repeats 23 13 05 14 01 09 13 08 08 06 20 13 03 02 01 01 02 04 03 01 02 01 01 04 01 01 02 04 03 01 01 05 01 01 01 01 04 01 02 04 05 01 01 01 01 01 02 AC AG AT CA CG CT GA GT TA TC TG AAC AAT AGA AGC ATC ATT CAA CAC CCG CCT CGC CTA CTC CTT GAA GCC GGA GGT GTA GTG GTT TAA TAT TCC TGC TGT TTA TTC TTG AAAC ATCC CAAA CATC CTTC TTAT TTTG 5-14 5-8 5 5-10 6 5-7 5-7 5-8 5-8 5-7 5-9 4-6 4-5 4 4 4 4 4-5 4-5 4 4 4 4 4 4 4 4 4-7 4 4 5 4-6 4 4 4 6 4-6 6 4-5 4 4-5 6 4 4 5 4 4

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119 Figure 5-1. Population size as comp ared to genetic diversity and future trends (adapted from Wahlund 1928). Population size and time are not scaled to specific events. How will the Florida manatee population respond with an anticipated loss of allelic diversity?

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120 Figure 5-2. Current winter Flor ida manatee subpopulation designa tions (shaded area). Blue stars are natural warm water sources, yello w stars are passive (alternate) warm water sources, and red stars are artifical sources of warm water. Four management units are represented by shaded areas; Northwest (p ink), Southwest (blue), upper St. Johns River (green), and Atlantic coast (yellow).

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121 Figure 5-3. Total number of di,tri-, and te tra-nucleotides identifie d using FLX technology on manatee DNA, of 346 nucleotide repeat s detected during one titration run. Figure 5-4. Number of putative microsatellites with di,tri-, and tetra-nuc leotides with at least 40 bp of flanking region at each end using FLX technology on manatee DNA. 0 50 100 150 200 250 di tri tetra Number of sequences Number of sequences containing repeats (n=346) 0 20 40 60 80 100 120 140 di tri tetra Number of sequences Number of sequences containing repeats with flanking regions (n=203)

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122 Figure 5-5. Potential winter Fl orida manatee subpopulation designa tions (shaded area) after the loss of artifical sources of warm water in winter. Blue stars ar e natural sources and yellow stars are passive (alternate) sources of warm water. Note the loss of artifical sources of warm water previously depicted by red stars and the anticipated shift in wintering population patterns.

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123 APPENDIX TAIL NOTCH PROTOCOL Protocol for Collection of Manatee Tissue Samples for Genetic Research March 2009 Protocol used by research field staff to collect and preserve tissue samples while swimming with free-ranging manatees at Crystal River, Florida. Priorities are provided to rank the selection of target indivi duals to fit sampling design.

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124 Protocol for Collection of Ma natee Tissue Samples for Genetic Research March 2009 Objectives 1) Provide support for the Manatee Individua l Photo-identification System (MIPS) 2) Establish and maintain genotyping fi ngerprints for individual manatees 3) Document calves at early life stage for better age determination estimates 4) Provide basis for cradle to grave tracking 5) Evaluate age at first reproduction 6) Facilitate cow/calf dependency period through visible monitoring 7) Determine relatedness between cows and suspected calves 8) Assess male reproductive contri bution through pedigree lines 9) Verify sex determination with molecular techniques Animal Selection Priority for the collection of tissue samples from the tail margin of manatees is as follows: 1) Calves (helpful if sex of calf and MIPS ID of mother are determined) see figure for site placement of cookie on calves 2) Adult manatees with scar features (MIPS) that have an old cookie site is reserved 3) Adult manatees with scar features (MIPS) that do not have an old cookie site is reserved 4) Adult female manatees with calf, with old cookie, and without scar features site should correspond to location in figure 5) Adult female manatees with calf, without cookie and without scar features site s hould correspond to location in figure General rule is that it is always best to have samples from both calves and scarred cows when po ssible. Be discrete when collecting samples in areas populated with other divers. Though our efforts are research permitted, it is best to avoid situations where we would need to e xplain our actions under field scenarios.

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125 Methodology All manatees should be completely photo-documente d before collection of the sample. A cattle ear notcher, 20mm U-shaped, de vice should be used. Samples should be collected from the margin of the tail at a predet ermined location (see figure Ta il Notch Locations above). If possible, a photograph of the tail should be taken after the samp le has been collected. Once collected, the sample (cookie) should be remove d from the notcher and taken to a dry place for processing (see figure 1). Remove a 4-5mm wedge of the sample from the inside edge of the cookie with a sterile blade, and pl ace in a pencil labeled cryovial ; be certain this piece includes white connective tissue (see figures 2&3). The sample should be kept on ice until it can be frozen. The remainder of the sample should be cu t into two pieces and both halves placed into a labeled 20ml plastic vial containi ng prepared tissue buffer solution. That sample should be kept at a cool temperature until stored for long-term at room temperature. During any handling of samples it is best to wear gloves and take ever y precaution to ensure that you are not cross contaminating any tissues (see figure 4). Clea n up any processing areas with diluted bleach solution after pr ocessing. 1) Clean preparation area 2) Slicing manatee tissue 3) Sliced manatee tissue 4) Placing sample in vial

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126 Labeling Labels for any vials containing genetic material must consist of the sample ID number (2 digit year, 3 digit Julian date, and the sequential number). An example would be 09-055-1 for the first sample coll ected on 24 February 2009. Additional information on the size of the manatee, sex, site location abbreviation, and collector can also be on the label. More detailed notes must be written on the divers underwater slate, where information on the date and site location, cookie ID number, size of animal, sex, associated photographic frame numbers taken, and behaviors are recorded. To avoid confusion ge nerated at different collection sites on the same day, when assigning sequential nu mbers make sure you coordinate with any ot her researchers before samples are finally labeled. Duplication of field ID numbers will complicate tracking identity of the sample. Sampling Field Kits A field kit for sample collection should include the following supplies: a notcher, razor blades, sharps container, exam gloves, 1.0ml cryovials, 20ml tissue vials, pencil, permanent marker, cooler with ice, and diluted cleaner containing bleach. A copy of our federal research permit (MA791721) should be in the possession of each researcher. Any collected fecal samples should be placed in 100% EtOH (200 proof ethanol) in a labeled 50ml centrifuge tube for storage. Additional labeled 15ml centrifuge tubes are useful for freezing fecal samples as well. Kits should be well maintained and stored in environmentally stable conditions.

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127 Successful Application Below are examples and scenarios for selection of application sites. The first picture is of a recently collected sample from a calf. The second is from a calf that has had a sample collected previously (note how the open area stretches out as the tail margin grows). The third example is of a MIPS distinguishable adult where the sample was previously collected from the reserved area. And the final image is of a distinct MIPS adult that had been sampled as a calf and has also more recently been sampled in the reserve area. Other notches in the tail evident in pictures 2 and 3 are unrelated to genetic sampling. 1) New sample from a calf 2) Mana tee sampled previously as a calf 3) MIPS adult sampled in the reserved area 4) MIPS adult previously sampled as a calf (upper left margin) and sampled again from the reserve area

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128 LIST OF REFERENCES Allen PJ, Amos W, Pomeroy PP, Twiss SD ( 1995) Microsatellite variation in grey seals ( Halichoerus grypus ) shows evidence of genetic differentiation between two British breeding colonies. Molecular Ecology 4, 653-662. Alvarez-Aleman A, Powell JA, Beck CA (2007) First report of a Florida manatee documented on the North Coast of Cuba. SireNews (Newsletter of th e IUCN/SSC Sirenia Specialist Group) 47, 9-10. Allendorf FW, Luikart G (2007) Conservation and Genetics of Populations. Blackwell Publishing, Malden, Massachusetts. 642 pp. Amos W, Harwood J (1998) Factors affecting levels of genetic di versity in natural populations. Philosophical Transactions of the Royal Society of London B 353, 179-186. Amos B, Hoelzel AR (1991) Long-term pres ervation of whale skin for DNA analysis. Report of the International Whalin g Commission Special Issue 13, 99-103. Anderson PK (2002) Habitat, niche, and evolution of sirenian mating systems. Journal of Mammalian Evolution 9 55-98. Arnason U, Adegoke JA, Gullberg A et al (2008) Mitogenomic rela tionships of placental mammals and molecular estimates of their divergences. Gene 421, 37-51. Archie EA, Hollister-Smith JA, Poole JH (2007) Behavioural inbreeding avoidance in wild African elephants. Molecular Ecology 16, 4138-4148. Archie EA, Maldonado JE, Hollister-Smith JA ( 2008) Fine-scale population genetic structure in a fission-fusion society. Molecular Ecology 17, 2666-2679. Baird NA, Etter PD, Atwood TS et al (2008) Rapid SNP discovery and genetic mapping using sequenced RAD markers. Public Library of Science One 3, e3376. Baker CS, Cooke JG, Lavery S et al (2007) Estimating the number of whales entering trade using DNA profiling and capture-recaptu re analysis of market products. Molecular Ecology, 16, 2617-2626. Barber EA (1982) Mound pipes. The American Naturalist 16 265-281. Beaum ont MA (2003) Estimation of population growth or decline in genetically monitored populations. Genetics, 164 1139-1160. Beck CA, Reid JP (1995) An automated photoidentification catalog fo r studies of the life history of the Florida manatee. In: Population biology of the Florida manatee. (eds. OShea TJ, Ackerman BB, Percival HF), pp. 120-134. National Biological Service Information and Technology Report 1. Washington, D. C.

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144 BIOGRAPHICAL SKETCH Robert Bonde was born in Hawthorne, Califor nia in 1953. Bob earned his BA in History in 1977 and a Certificate in Environmental Studi es in 1978 at California State University Long Beach. Bob grew up in southern California befo re moving to Gainesville, Florida with his wife, Cathy in 1978 where he was employed by the U.S. Fish and Wildlife Service as part of the Sirenia Project. He continues to work for the Sirenia Project, now under the U.S. Geological Survey, where he has been studying manatee biology for more than 31 years. Bob continues to participate in studies of the life history of the manatee population in Crystal River, Florida. He consults with the NOAA-Fisheries Working Group for Unusual Marine Mammal Mortality Events on issues related to necropsy assessment of the stranded marine mammals and participates in the USFW S Manatee Rescue, Rehabilitation, and Release Program. He stays involved in field radio telemetry and tr acking studies, manatee genetic studies and biomedical health a ssessments, and international research projects and study design. Bob chairs the Florida Manatee Genetics Rese arch Working Group. In addition to over 70 technical and semi-technical sc ientific publications, Bob coauthored a book in 2006 with Dr. Roger Reep, entitled, The Florida Mana tee: Biology and Conservation. Bob entered graduate school in 2004 under the mentorship of Dr. Peter McGuire with the College of Medicine. Enrolled in the College of Veterinary Medicine (CVM), Department of Physiological Sciences, he has been an active member of the CVM Aquatic Animal Program and serves through a courtesy faculty appointment. Bob is always happy to share his knowledge of the manatee and to get into the field to discove r many new and interesting things about manatees and their intertwined connection with our planet. When chicken wings are not available, he enjoys breakfast, lunch, a nd dinner at Taco Bell.