<%BANNER%>

Partners in Time

Permanent Link: http://ufdc.ufl.edu/UFE0041929/00001

Material Information

Title: Partners in Time Evolutionary and Population Genetic Patterns of Coevolving Organisms
Physical Description: 1 online resource (103 p.)
Language: english
Creator: Allen, Julia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: anoplura, bacteria, coevolution, endosymbiont, gammaproteobacteria, lice, mammal, mutualist, mycetome, parastie, phylogenetics, primate, uganda
Biology -- Dissertations, Academic -- UF
Genre: Zoology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The way organisms evolve is thought to be due partly in response to their interactions with other organisms. An extreme form of interactions between organisms is relationships where one organism is completely dependent on another. My dissertation has focused on how organisms in these types of relationships have evolved over both long time scales (millions of years) and short ones (just a few generations). I have examined two groups of interacting organisms. First, sucking lice have mutualistic bacteria, which live inside the insect and provide nutrients to the louse. The bacteria are passed from mother to offspring to get incorporated into the next generation. I looked at 27 species of lice to see if they share a single genus of bacteria, which would suggest that the lice and endosymbionts have evolved together for millions of years. I found that closely related lice do share a single genus of bacteria but when you look at more distantly related lice they have unrelated lineages of bacterial endosymbionts. This work suggests that endosymbionts and lice might evolve together for some period of time but that over long time scales the bacteria possibly get replaced. Secondly, I have studied a group of Red Colobus monkeys and their parasitic lice from Kibale National Park in Uganda. Here I found that there is gene-flow between the monkey troops from across the park, suggesting there is contact between the host populations. This movement of the hosts likely affects the rate at which these parasites are able to move around the park.
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 Julia Allen.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Reed, David L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041929:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041929/00001

Material Information

Title: Partners in Time Evolutionary and Population Genetic Patterns of Coevolving Organisms
Physical Description: 1 online resource (103 p.)
Language: english
Creator: Allen, Julia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: anoplura, bacteria, coevolution, endosymbiont, gammaproteobacteria, lice, mammal, mutualist, mycetome, parastie, phylogenetics, primate, uganda
Biology -- Dissertations, Academic -- UF
Genre: Zoology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The way organisms evolve is thought to be due partly in response to their interactions with other organisms. An extreme form of interactions between organisms is relationships where one organism is completely dependent on another. My dissertation has focused on how organisms in these types of relationships have evolved over both long time scales (millions of years) and short ones (just a few generations). I have examined two groups of interacting organisms. First, sucking lice have mutualistic bacteria, which live inside the insect and provide nutrients to the louse. The bacteria are passed from mother to offspring to get incorporated into the next generation. I looked at 27 species of lice to see if they share a single genus of bacteria, which would suggest that the lice and endosymbionts have evolved together for millions of years. I found that closely related lice do share a single genus of bacteria but when you look at more distantly related lice they have unrelated lineages of bacterial endosymbionts. This work suggests that endosymbionts and lice might evolve together for some period of time but that over long time scales the bacteria possibly get replaced. Secondly, I have studied a group of Red Colobus monkeys and their parasitic lice from Kibale National Park in Uganda. Here I found that there is gene-flow between the monkey troops from across the park, suggesting there is contact between the host populations. This movement of the hosts likely affects the rate at which these parasites are able to move around the park.
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 Julia Allen.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Reed, David L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041929:00001


This item has the following downloads:


Full Text

PAGE 1

1 PARTNERS IN TIME: EVOLUTIONARY AND POPULATION GENETIC PATTERNS OF COEVOLVING ORGANISMS By JULIA M. ALLEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

PAGE 2

2 2010 Julia M. Allen

PAGE 3

3 To my fellow graduate students, my committee and my family for all of their support

PAGE 4

4 ACKNOWLEDGMENTS I would like to first thank all of the graduate students in the Biology Department for all of their support and encouragement, in particular those in my cohort. I would also like to thank my advisor David Reed for always listening and allowing me to have the freedom to develop my own ideas, but also challenging them, which made me think about things more critically all along the way. In particular, I would like to thank him for his seemingly unlimited amounts of time to discuss ideas. I would also like to thank Mike Miyamoto for spending hours with me explaining coalescent processes and population genetics, and always pushing me to be a better scientist. Marta Wayne for providing true mentorship not only to me, but to all of the graduate students in the department. I would like to thank Matt Gittendanner for his time helping me with the cluster, and Pam and Doug Soltis for giving me unlimited access to their lab, without which, many of the techniques I needed to learn would not have happened. I would also like to thank Rebecca Kimball and Edward Braun for giving me the time to discuss lab work and troubleshooting, usually in last minute frantic meetings for which they always found the time. Also, I would like to thank Gordon Burleigh for all of his work on the bacterial phylogenetics, he is an honorary member of my committee. Lastly, I would like to thank my family for all of their support along the way.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 8 LIST OF FIGURES .............................................................................................................. 9 ABSTRACT ........................................................................................................................ 10 CHAPTER 1 INTRODUCTION ........................................................................................................ 12 Overview ..................................................................................................................... 12 Part I: Lice and their Primary Endosymbionts ........................................................... 12 Part II: Sucking Lice and their Mammalian Hosts ...................................................... 13 2 EVOLU TIONARY RELATIONSHIPS OF CANDIDATUS RIESIA SPP. ENDOSYMBIOTIC ENTEROBACTERIACEAE LIVING WITHIN HEMATOPHAGOUS PRIMATE LICE ........................................................................ 15 Int roduction ................................................................................................................. 15 Methods ...................................................................................................................... 18 Specimen Preparation ......................................................................................... 18 Bacterial Diversity in Pediculus Humanus Capitis Using Molecular Methods ... 18 Targeted Sequencing of Endosymbionts from Additional Taxa of Lice ............. 19 Fluorescent InSitu Hybridization ........................................................................ 20 Phylogenetic Analysis .......................................................................................... 21 Results ........................................................................................................................ 22 Phylogenetic Analysis and Taxonomic Position of Louse Endosymbiont .......... 22 Fluorescent InSitu Hybridization ........................................................................ 23 Discussion ................................................................................................................... 24 Overview ............................................................................................................... 24 Nomenclature ....................................................................................................... 25 Coevolution and Rates of Substitution ................................................................ 26 3 MUTATIONAL MELTDOWN IN PRIMARY ENDOSYMBIONTS: SELECTION LIMITS MULLERS RATCHET ................................................................................... 31 Introduction ................................................................................................................. 31 Materials and Methods ............................................................................................... 35 Age of the Riesia/Louse Assemblage ................................................................. 35 Specimen collection and DNA sequencing .................................................. 35 Phylogenetic an alysis .................................................................................... 37 Absolute rates of nucleotide evolution in Riesia ........................................... 38

PAGE 6

6 Substitution Rates Among Host/Endosymbiont Lineages .................................. 39 Results ........................................................................................................................ 40 Age of Riesia /Louse Association ......................................................................... 40 Absolute Rates ..................................................................................................... 41 Substitution Rates Among Host/Endosymbiont Lineages .................................. 42 Discussion ................................................................................................................... 42 Overview ............................................................................................................... 42 Endosymbiosis ..................................................................................................... 44 4 MULTIPLE LINEAGES OF BACTERIA IN ANOPLURA INDICATE A HIGH RATE OF BACTERIAL REPLACEMENT ON A SHORT EVOLUTIONARY TIME -SCALE .............................................................................................................. 53 Introduction ................................................................................................................. 53 Methods ...................................................................................................................... 55 Lice Sampling ....................................................................................................... 55 Bacterial Sampling Alignment and Tree Building ................................................ 56 Results ........................................................................................................................ 57 Endosymbionts ..................................................................................................... 57 Phylogenetic Analysis .......................................................................................... 58 Discussion ................................................................................................................... 59 Endosymbionts ..................................................................................................... 59 Taxonomy ............................................................................................................. 60 Bartonella as an Endosymbiont ........................................................................... 61 Number of lineages .............................................................................................. 61 Evolution of Sucking Lice and their Endosymbionts ........................................... 62 Blo od Feeding as a Source of Endosymbionts ................................................... 63 5 POPULATION GENETICS OF HABITAT SENSITIVE RED COLOBUS SUGGEST LONG -TERM STABILITY OF KIBALE NATIONAL PARK ..................... 70 Introduction ................................................................................................................. 70 Methods ...................................................................................................................... 72 DNA Collection and Storage ................................................................................ 72 DNA Extraction and Quantification ...................................................................... 72 Genotype analysis ................................................................................................ 73 Number of Populations ........................................................................................ 74 Analysis ................................................................................................................ 75 Estimates of Theta, Population Size and Bottlenecks ........................................ 75 Results ........................................................................................................................ 76 Genotyping Errors, Linkage and Hardy Weinberg Equilibrium ........................... 76 Number of Populations ........................................................................................ 77 Effective population size, Coalescent Point and Bottleneck .............................. 78 Discussion ................................................................................................................... 79 Number and size of populations .......................................................................... 79 History of Red Colobus in Kibale National Park ................................................. 80

PAGE 7

7 6 CONCLUSIONS .......................................................................................................... 87 Part I: Lice and their Primary Endosymbionts ........................................................... 87 Part II: Sucking Lice and their Mammalian Hosts ...................................................... 88 LIST OF REFERENCES ................................................................................................... 91 BIOGRAPHICAL SKETCH .............................................................................................. 103

PAGE 8

8 LIST OF TABLES Table page 2 -1 Pairwise differences for p endosymbionts from human head and body lice Pediculus humanus ............................................................................................... 28 3 -1 Percent sequence divergence and rate of nucleotide substitution of the 16S rDNA gene of Riesia. ............................................................................................. 47 3 -2 Bacteria taxa, their hosts, and GenBank accession numbers used in the phylogenetic analysis presented in Figure 31. ................................................... 48 4 -1 Louse taxa used in this study (arranged by family), host associations, and GenBank accession numbers. ............................................................................... 64 4 -2 Taxon sampling, and endosymbiont determination. ............................................. 66 5 -1 Microsatellite loci genotyped for Red Colobus ( Piliocolobus tephrosceles ) from Kibale National Park. ..................................................................................... 82 5 -2 Results from RSTCALC. ........................................................................................ 83

PAGE 9

9 LIST OF FIGURES Figure page 2 -1 Phylogenetic tree of endosymbiont from sucking lice (Anoplura) and other bacteria.. ................................................................................................................. 29 2 -2 Fluorescent in situ hybridization microphotograph of thorax and abdomen (ventral view) of a second instar nymph of a head louse ( Pediculus humanus capitis ).. .................................................................................................................. 30 3 -1 Maximum likelihood phylogram representing phylogenetic relationships of alll bacteria used in this study.. ................................................................................... 49 3 -2 Maximum likelihood phylogram representing phylogenetic relationships of Riesia p endosymbionts. ........................................................................................ 50 3 -3 Primary endosymbiont nucleotide substitution rates as a function of the age of insect/p endosymbiont association. .................................................................. 51 3 -4 Reduced major axis regression of the log transformed data from Figure 3 -3. ............................................................................................................................... 52 4 -1 Bacterial tree showing placement of Anopluran endosymbionts. ......................... 67 4 -2 Phylogenetic tree of Louse taxa from Light et al. (In Review). ............................. 68 4 -3 Maximum Likelihood tree of 279 Bartonella sequences ..................................... 69 5 -1 Map of Kibale National Park (KNP). ...................................................................... 84 5 -2 Log likelihood scores for output from Structure. ................................................... 85 5 -3 Log likelihood scores for the program Migrate.. .................................................... 86

PAGE 10

10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Do ctor of Philosophy PARTNERS IN TIME: EVOLUTIONARY AND POPULATION GENETIC PATTERNS OF COEVOLVING ORGANISMS By Julia M. Allen August 2010 Chair: David L. Reed Major: Zoology The way organisms evolve is thought to be due partly in response to their interactions with other organisms. An extreme form of interactions between organisms is relationships where one organism is completely dependent on another. My dissertation has focu sed on how organisms in these types of relationships have evolved over both long time scales (millions of years) and short ones (just a few generations). I have examined two groups of interacting organisms. First, sucking lice have mutualistic bacteria, which live inside the insect and provide nutrients to the louse. The bacteria are passed from mother to offspring to get incorporated into the next generation. I looked at 27 species of lice to see if they share a single genus of bacteria, which would suggest that the lice and endosymbionts have evolved together for millions of years. I found that closely related lice do share a single genus of bacteria but when you look at more distantly related lice they have unrelated lineages of bacterial endosymbiont s. This work suggests that endosymbionts and lice might evolve together for some period of time but that over long time scales the bacteria possibly get replaced. Secondly, I have studied a group of Red Colobus monkeys and their parasitic lice from Kiba le National Park in Uganda. Here I found that there is geneflow between the

PAGE 11

11 monkey troops from across the park, suggesting there is contact between the host populations. This movement of the hosts likely affects the rate at which these parasites are able to move around the park.

PAGE 12

12 CHAPTER 1 INTRODUCTION Overview The evolutionary history of an organism is strongly affected by its interactions with other species in a shared environment (e.g. predator prey). Interactions with these organisms may affect the evolutionary trajectory of a lineage. An extreme case of suc h species interactions is in obligate relationships because one organism is com pletely dependent on the other. It is important to understand how the evolutionary processes of one partner affect s the evolutionary history of the other. Blood -sucking lice fo rm two strong obligate relationships; they are obligate parasites of mammals, and each louse harbors a mutualistic species of bacteria. These relationships make sucking lice ideal for examining the co evolutionary history of interacting lineages. Part I: L ice and their Primary E ndosymbionts Primary endosymbiotic bacteria (pendosymbionts) are thought to be partially responsible for the incredible diversification of insects. P endosymbionts are found in insects that specialize on nutrient poor diets, where t hey supplement their insect hosts diet with nutrients (Buchner 1965). It has been suggested that the acquisition of a bacterial lineage in insect ancestors has played a key role in the species diversification of some insect groups. Endosymbiotic bacteria cannot live outside of the insect hosts and are transmitted from mother to offspring through the eggs. Within insect groups with p endosymbionts, all individuals share the same lineage of bacteria. For example all aphids have the same genus of bacteria called Buchn era This suggests a long coevolutionary history between the insect -bacteria l partners.

PAGE 13

13 The primary endosymbiont in the human head louse was first discovered over three hundred years ago, (Hooke 1664) but before my research began had not been f ormally described and named In Chapter 2 I will describe the endosymbiont in Great Ape lice. In Chapter 3 I examine its rate of molecular evolution in light of Mullers Ratchet and the rate of evolution of other insect/endosymbionts. Then in Chapter 4 I describe endosymbionts in many species of sucking lice to determine a rate of endosymbiont replacement in this group. Part II: Sucking Lice and their Mammalian H osts Organisms involved in obligate relationships are affected on both a long-term species lev el time scale and short term population-level time scale (Clayton and Johnson 2003). For example, the size of a parasite population is strictly tied to the social structure, dispersal ability and population size of its host In the case of lice and mammal s, lice only move between hosts during direct host to host contact. Therefore, louse population dynamics are particularly constrained by the social structure of their hosts (e.g. which hosts come into contact and how often they come into contact). The effect of mammalian host population dynamics on louse population dynamics remains poorly understood. The R ed C olobus monkey is parasitized by lice in the genus Pedicinus and these monkeys breed and forage in stabl e social groups. For these primates, it is thought that only females migrate from troop to troop (Struhsaker 1975). Female migration may therefore provide the mechanism for lice to disperse between monkey troops. To examine the host population structure I have selected six troops of monkeys that resi de within Kibale National Park ( Uganda ). Using molecular markers, in Chapter 5 I calculate

PAGE 14

14 the number of populations of monkeys in the park and the migration rates of the hosts to understand more about louse movement

PAGE 15

15 CHAPTER 2 EVOLUTIONARY RELATIONSHIPS OF CANDIDATUS RIESIA SPP. ENDOSYMBIOTIC ENTEROBACTERIACEAE LIVING WITHIN HEMATOPHAGOUS PRIMATE LICE Introduction Insects in general are an incredibly successful and diverse group. Part of their success is undoubtedly due to mut ualistic primary endosymbiotic bacteria, which have enabled insects to radiate into niches that include nutrient poor diets (Buchner 1965, Douglas 1989, Perotti et al. 2006) such as eating wood ( e.g. termites), plant sap ( e.g. aphids) and blood ( e.g. su cking lice in the Anoplura). Primary endosymbiotic bacteria (as opposed to secondary endosymbionts[S endosymbionts]) are normally maintained inside specialized host cells called mycetomes (Douglass 1989, Perotti et al. 2006) usually exhibit nucleotide A+T bias greater than 50%, and show elevated sequence evolution with respect to their free living counterparts (Brynnel et al. 1998, Wernegreen and Moran 1999, Woolfit and Bromham 2003). These endosymbionts are required for host survival and they provide nutr ients that are not available in the insects specialized diet (Buchner 1965). Most primary endosymbionts leave their mycetomes to migrate to the ovaries so that they may be incorporated into developing eggs (transovarial transmission) and thus be passed onto the next host generation (Douglas 1989, Perotti et al. 2006) leading to long -term, shared coevolutionary histories between the insects and their symbionts It is estimated that there are 14,000 species of haematophagous insects (Adams 1999, Lehane 20 05) but only a few P endosymbionts have been described from these insects (e.g., Wigglesworthia). Because blood is a nutrient poor diet, all sucking lice likely have some form of endosymbiont and many are reported to have obligate primary

PAGE 16

16 endosymbionts ba sed on microscopic observation. The P endosymbiont in the human head and body louse ( Pediculus humanus ) was first seen over 340 years ago (Hooke 1665) with some of the very first microscopes It has a complex migration (Buchner 1965) that involves four dif ferent mycetomes and two extracellular migrations as it moves to the eggs in the adult female lice (Perotti et al. 2007). This migration was first observed by Ries (Ries 1931) and later shown by scanning and transmission electron microscopy by Eberle and Mclean (1983) Wolbachia is the only other bacterium that has been found among human lice (Perotti et al., 2004, Kyei -Poku et al. 2005,). The complex migration associated with transovarial transmission stands as potential evidence of the importance of the relationship between the P endosymbiont and the lice. If the mycetome is removed from a young female louse, she dies after only a few days and her eggs are deformed (Aschner and Ries 1933). Furthermore, if the bacteria are removed from the eggs directl y, the larvae only survive a few days (Aschner 1934). Puchta (1955) demonstrated that lice without P endosymbionts were able to survive if their diet was supplemented with nicotinic acid, pantothenic acid, and beta biotin, suggesting a basis for a mutuali stic long -term relationship. Humans have three types of lice, head and body lice ( Pediculus humanus ), which are currently classified as two distinct subspecies ( Pediculus humanus capitis and Pediculus humanus humanus ) and pubic lice ( Pthirus pubis ) (Durd en and Musser 1994). Body lice are known to transmit three diseases, louse borne epidemic typhus (LBET), relapsing fever, and trench fever (Buxton 1946). Although head lice can transmit LBET in a laboratory setting (Goldberger and Anderson 1912, Robinson et al. 2003), there has never been evidence of LBET transmission by head lice in nature.

PAGE 17

17 Currently, it is not known why one subspecies of P. humanus can transmit three deadly ba cterial agents while the other subspecies, epidemic in school children, effectively cannot. Although the secondary endosymbiont of Tsetse flies ( Sodalis glossinidius ) has been shown not to affect the ability of its host to transmit Trypanosoma congolense (Geiger et al. 2005), it is possible that there are differences in the endosymbiotic bacteria of head and body lice and that these differences may reinforce patterns of disease transmission in human lice. Primate lice show a history of cospeciation with t heir hosts, and have been used effectively to infer human evolutionary history (Kittler et al. 2003, Yong et al. 2003, Reed et al. 2004, Leo and Barker 2005). Most of this work has relied upon mtDNA from the lice and to a lesser extent on nuclear markers. If a single lineage of endosymbiont were found among primate lice, the endosymbiont could serve as still another marker of human and primate evolutionary history. In addition, this new three-tiered assemblage of primates, lice, and endosymbiotic bacteria would yield a new system in which to study relative and absolute rates of evolution in three disparate lineages (vertebrates, insects, and bacteria). In this study, we describe the molecular characterization of the P endosymbiont of primate lice including head and body lice ( Pediculus humanus ) characterized by Sasaki Fukatsu et al. (2006) and present new data from chimpanzee lice ( Pediculus schaeffi ) and human pubic lice ( Pthirus pubis ). We use the full -cycle rRNA approach including comparative 16S rRNA gene analysis and the detection of endosymbionts within the host cell by means of fluorescent in situ hybridization using specific 16S rRNA -targeted oligonucleotide probes. By sampling across a phylogenetically diverse assemblage of

PAGE 18

18 louse species, we expe ct to capture a greater percentage of the diversity in this lineage of P endosymbiont. Methods Specimen Preparation Specimens of human head lice ( Pediculus humanus capitis ) were collected from patients in West Palm Beach, Florida and from school children in La Rioja, Argentina (Perotti et al. 2004). Specimens of body lice ( Pediculus humanus humanus ) were acquired from the rabbit adapted strain held at the insect control and research lab in Maryland, USA and in Cambridge, United Kingdom. Chimpanzee lice were collected in Uganda, and samples of Pthirus pubis were collected in Utah. All lice were surfacesterilized using either a lysis -sodicum dodecyl sulfate buffer as described in Reed and Hafner (2002) or with 0.7 % sodium chloride, 0.05 % Triton X -100 f or 20 s in an ultrasonic bath, then rinsed with sterile distilled water to remove any surface contamination The lice were rehydrated in PBTA phosphate buffer with Triton X 100 plus sodium azide (PBTA) in three consecutive steps: 30, 60 and 100% PBTA. Whole mycetomes were dissected manually under a Leica stereoscope with a magnification of X100 using tungsten tips and special carbon steel blades A maximum dissecting resolution of 10 15 m was obtained. The dissected bodies were fixed inside 1.5 ml tubes for DNA extraction using the DNA extraction methods described in Reed and Hafner (2002). Bacterial Diversity in Pediculus Humanus Capitis Using Molecular Methods Universal bacterial 16S rDNA primer 27F (5 GAG TTT GAT CCT GGC TCA G -3) was used with ei ther 1492R (5 CAC GGA TCC TAC GGG TAC CTT GTT ACG ACT T -3) or 1525R (5 -AGA AAG GAG GTG ATC CAG CC -3) to amplify 16S rDNA

PAGE 19

19 sequences. PCR was performed on the isolated DNA using standard reaction conditions with 10 ng of template DNA, 300 nM of each primer, 200 M of each dideoxy nucleotide triphosphate, 2.5 mM MgCl2 and 0.02 U of Taq DNA polymerase per l of reaction mix. Cycling conditions consisted of an initial denaturation step (95C, 10 min) followed by 30 cycles of amplification involving denat uration (95C, 1 min), annealing (50C, 1 min) and extension (65C, 1 min), followed by a final extension step at 65C for 10 min. The PCR product was analyzed by gel electrophoresis. The 1.5-kbp 16S rDNA PCR product was purified with Exo-SAP-IT (USB Cor p) as prescribed by the manufacturer. The PCR product was then cloned into the pTOPO 4.0 vector (Invitrogen) according to the suppliers instructions to generate clone libraries. Recombinant clones were sequenced to completion at the University of Flori da sequencing facility and in the Bangor lab, United Kingdom, using vector -specific primers and internal sequencing primers that were designed as sequence information became available. The computer program Sequencher v. 4.1 (Gene Codes Corporation, Ann Arbor, MI) was used to join contiguous 16S rDNA fragments into a single consensus sequence. Targeted Sequencing of Endosymbionts from Additional Taxa of Lice The 1525bp 16S rRNA sequence for the endosymbiont of P. humanus capitis was aligned to other 16S sequences in the ARB database (Ludwig et al., 2004). A new PCR primer specific to the endosymbiont sequence was then created with the intention of excluding contaminant sequences coamplified with the endosymbiont target. The single specific primer, in co ncert with the general Eubacterial primer, preferentially amplifies the endosymbiont from whole insect preparations. The 1525R primer was paired with

PAGE 20

20 the specific primer 461F (5 -ACA GAA GAA GCA CCG GCT AA3 ) to produce a 1,200bp fragment of the 16S rRNA gene. These primers were used to amplify, clone, and sequence the endosymbiont from three additional taxa of body lice ( P. humanus humanus ), chimpanzee lice ( Pediculus schaeffi ), and human pubic lice ( Pthirus pubis ). Genbank accession numbers are (EF110569 EF110574). Fluorescent In-Situ Hybridization Specimens were washed as for dissections and selected under the microscope on a slide with a drop of water and immediately fixed with ethanol glacial acetic acid (3:1) for 2 h. Preparations were kept overnight in a 1:1 mixture of xylene and ethanol (1:1) at 4C, were then transferred to xylene:ethanol (1:2) for 30 minutes and to ethanol for 30 minutes, washed with 20C 80% acetone for 20 minutes and dehydrated in ethanol. Specimens were rehydrated using an ethanol/PBTA mixture with ratios of 2:1, 1:1, and 1:2 for 20 minutes each with a final rehydration of only PBTA for 30 minutes, where specimens remained at 4 incubated with PBTA -hydration buffe r (HB) at a ratio of 1:1 (Hybridization Buffer: TRIS HCl 0.02 M, sodium chloride 0.09 M, SDS 0.01%, formamide 35%, Denhardts solution 15%) for 20 minutes followed by only hybridization buffer (around 500l/tube), sonicated in an ultrasonic bath for 20 sec onds and then probes and corresponding helpers added (final concentration of 100 pmol each). Samples were incubated at 47 C in total darkness for 16 h, then washed for 1 h with the same HB without probe/helpers at 47C, then changed to HB:PBTA, 1:1, at r oom temperature and finally to PBTA. Samples were mounted with PBTA:glycerol mounting medium. In order to ensure that the 16S rRNA sequences that we retrieved were from the same mycetome-bound bacteria described by Ries (1931), we created a species -

PAGE 21

21 specif ic probe using the ARB database in combination with known probes and helpers of bright fluorescence (Fuchs et al. 1998). The endosymbiont -specific probe 2 -Sd -Cy5 (5' -Cy5 -GAG ATT GTT GCC TAG GTG -3') which does not match any published sequence; and the 3 h elpers, Helper 1 -Sd (5' ACC TCA CCT ACT AGC TAA TCT C-3'); Helper 2 -Sd ( 5' -GTA TGG GCT CAT CTA AAG -3') ; and Helper 3-Sd (5' -TTT AGG TAG ATY CCC ATA T -3') were based on the endosymbiont sequence obtained from human head louse endosymbiont. No probe and com petition suppression controls using excess unlabeled probes were performed. Fluorescent in situ hybridization was conducted on whole mount specimens and on 14 m thick serial sections of whole individuals using the endosymbiont -specific probe. Samples were analysed with a Zeiss LSM510 confocal microscope with Coherent Multiphoton laser. Phylogenetic Analysis Nearly complete 16S rDNA sequences were obtained and added to the ARB rRNA sequence database, which contained over 16,000 homologous small subunit rRNA primary structures. Multiple sequence alignment was achieved using the ARB automated alignment tool (program available at http://www.mikro.biologie.tumuenchen.de), with modification by eye. Comparative sequence analysis revealed that the 16S rRNA gen es of the endosymbionts were novel and showed highest sequence similarities with members of the Enterobacteriaceae. Therefore, we downloaded an aligned dataset of additional taxa from the ARB database and GenBank in order to perform more complete phylogen etic analyses (alignment available in TreeBase [www.trebase.org], no. SN3132). We used the computer program ModelTest (Posada and Crandall 1998) as a guide to determine the best -fit maximum likelihood (ML) model

PAGE 22

22 as described by Cunningham et al. (1998). ModelTest examines maximum likelihood models ranging from simple to complex. This method increases the number of parameters in the ML model incrementally until the addition of a new parameter no longer increases significantly the fit between the model and the data. ModelTest calculated likelihood scores for 56 nested ML models and used hierarchical likelihood ratio tests (LRTs) to determine the best -fit model. We incorporated the best -fit model of nucleotide evolution in ML heuristic searches in Paup* (Sw offord 2006) and in Bayesian searches in MrBayes (Hulsenbeck and Ronquist 2001) using the maximum likelihood optimality criterion. Multiple outgroups were chosen from phylogenetic studies of Enterobacteriaceae especially endosymbiotic taxa, and the phylogenetic tree was rooted on the most divergent outgroup taxon, the alpha proteobacterium Wolbachia pipientis MrBayes was run for the 43-taxon dataset for 10 million generations. Burnin was achieved within the first 100,000 generations; therefore to be conservative, our posterior probabilities are based on the last 9.8 million generations (phylogenetic trees available in TreeBase, no. M3012). Results Phylogenetic Analysis and Taxonomic Position of Louse Endosymbiont DNA amplification and sequencing with t he Eubacterial primers 27f, 1492r and 1525r led to the nearly complete sequencing of the 16S rRNA for two head lice and two body louse (ca. 1,520bp). The design of species -specific primers led to the sequencing of shorter fragments from the 16S rRNA gene ranging from 960bp to 1212bp for the remaining taxa. Pairwise sequence divergences among the four individuals of P. humanus ranged from 0.0% to 0.3% (Table 21). The two head lice were identical to each other in sequence as were the two body lice sequenc es. The head lice differed

PAGE 23

23 from the body lice by 0.3% (GTR+I+ model), resulting from five fixed differences between the two types of lice. BLAST searches of Genbank showed that the Pediculus humanus endosymbiont was nearly 100% identical to the sequence s of Candidatus Riesia pediculicola found by Sasaki -Fukatsu et al. (2006) BLAST searches also indicated that our sequences were very similar to those of the S endosymbiont Candidatus Arsenophonus insecticola, which was supported in the phylogenetic a nalysis as well (Figure 2 1). Phylogenetic analysis based on the 16S rDNA locus revealed that the symbionts from human head and body lice, chimpanzee lice, and human pubic lice were monophyletic with 100% support from the Bayesian posterior probabilities. The ML analysis produced a topology that agreed fully with the Bayesian analysis presented in Figure 21. The endosymbiont sequences obtained from P. humanus contain 139 substitutional differences when compared to Candidatus Arsenophonus insecticola (G enBank accession no. DQ115536), and they share 89% sequence identity over 1,260bp. The sequence from the P endosymbiont of all lice surveyed were greater than 50% A+T, which is typical of true primary endosymbionts (Woolfit and Bromham 2003; Table 21). The endosymbiont sequences from two genera and three species of primate lice form a monophyletic assemblage with an average percent sequence divergences of 4.8% within the clade (Table 21). Fluorescent In-Situ Hybridization The ARB database was used to generate a species -specific oligonucleotide probe, which was 100% identical to the endosymbiont from P. humanus but no other taxa in the database or Genbank. In situ hybridization confirmed the specificity of this probe in sec tioned and whole lice producing strong signal within the clearly visible stomach disc

PAGE 24

24 of P. humanus (Figure 2-2). The in situ hybridization confirmed that the bacterium from which we recovered 16S rRNA sequence is indeed the bacterium found in the mycetom es, which have been known and visualized since the 1,600s. Discussion Overview Bayesian phylogenetic analysis and BLAST searches demonstrated that this new lineage of Enterobacteriaceae is closely related to a lineage of S endosymbionts (Candidatus Arsenophonus spp. ). Candidatus Arsenophonus endosymbionts have been found living within Hippoboscid flies (Dale et al. 2006), commonly called louse flies. Louse flies parasitize a wide range of birds and mammals, and are known to physically carry lice as hitchhikers among vertebrate host individuals (called phoresy). The lice, which are particularly bad dispersers having no wings of their own, use the flies as a means of dispersal. While phoresy itself does not involve any humoral interaction between passenger (phoront) and carrier, in one case, a phoretic mite (Macrocheles subbadius ) has been shown to feed on the hemolymph of its transporting host ( Drosophila nigrospiracula) (Polak 1996). The close association between the endosymbionts of lice and l ouse flies might be explained by horizontal transmission, which appears to be more common among microorganisms than previously thought (e.g., Moran and Dunbar 2006, Saito and Bjornson 2006). One might presume that the Pendosymbiont in lice is derived from the S endosymbiont (i.e., increasing specialization over evolutionary time), however this presents a hypothesis that can be tested directly in future studies. Our phylogenetic analysis shows that the endosymbionts of primate lice are distinctly different from each other and their closest relatives, and yet they represent a strongly supported monophyletic clade.

PAGE 25

25 Nomenclature These endosymbiotic bacteria have not been completely characterized or grown in pure culture, although they have been known and visu alized since the early 1600s (Hooke 1665, Perotti et al. 2007). Our endosymbiont sequences from Pediculus humanus were nearly identical to sequences from Candidatus Riesia pediculicola, but the additional primate louse species had closely related but di stinctly different endosymbionts. The endosymbionts from P. humanus were more than 3% divergent from those from the chimpanzee louse P. schaeffi and endosymbionts from the human pubic louse, Pthirus pubis were more than 10% divergent from both P. humanu s and P. schaeffi Therefore, following the recommendations for uncultured microorganisms (Murray and Schleifer 1994, Murray and Stackerbrandt 1995), we suggest that endosymbionts of P. schaeffi and Pthirus pubis be recognized as distinct species within t he genus Candidatus Riesia. The current taxonomic name Candidatus Riesia pediculicola is ambiguous as to the specific host species with which it is associated. The specific epithet of Candidatus Riesia pediculicola refers only to the genus of lous e host, and yet there are two species of Pediculus (P. schaeffi and P. humanus ) which have closely related P endosymbionts. Therefore in order to reduce further confusion, we propose to use the concatenated scientific names of the louse host for the speci fic epithet of the endosymbiont. We propose the name Candidatus Riesia pediculischaeffi for the primary endosymbiont of the chimpanzee louse ( Pediculus schaeffi ) Similarly, we propose the name Candidatus Riesia pthiripubis for the primary endosymbiont of the human pubic louse (Pthirus pubis ). By latinizing the full binomial, we can accommodate additional species within a genus, such the endosymbiont from the gorilla louse, Pthirus gorillae, when and

PAGE 26

26 if it is characterized. Although the names are verbose, we find this nomenclature appropriate and beneficial. Coevolution and Rates of Substitution The two 16S sequences from the endosymbionts of human head lice differ from the two endosymbiont sequences from body lice by five fixed differences (sites 218, 492, 877, 950, and 1203) in our aligned matrix (available in TreeBase). Although this appears to suggest distinction between the endosymbionts of head and body lice, recent additional sequence data acquired in the lab of DLR suggests this is an arti fact of sampling and is not indicative of real phylogenetic differences between the endosymbionts of head and body lice. The head and body lice of modern humans have been reasonably well sampled, and their mtDNA can be divided into three distantly related haplotypes (Kittler et al. 2003, Leo and Barker 2005, Yong et al. 2003). The first mtDNA haplotype (Type A) is worldwide in distribution, and is the most common in both the head and body louse morphotypes. The second mtDNA haplotype (Type B) has been found in the New World, Europe, and Australia and exists only in head lice. The third mtDNA haplotype (Type C) has only been found among head lice from Nepal and Ethiopia. Unpublished data from our lab suggest that the endosymbionts of P. humanus mirror th e host mtDNA haplotypes for P. humanus This is perhaps not surprising given that both the endosymbionts and mtDNA are maternally inherited in these lice. The phylogenetic relationships of the endosymbionts of primate lice (both species of Pediculus plus Pthirus ) are identical in topology to the phylogenies of their hosts (Reed et al. 2004) suggesting a longterm coevolutionary history between primate lice and their endosymbionts Reed et al. (2004) demonstrated that P. humanus and P. schaeffi diverged fr om one another ca. 5.6 million years ago based on mtDNA, which

PAGE 27

27 coincides precisely with the estimated divergence of their human and chimpanzee hosts based on mtDNA. The average percent sequence divergence (model corrected) between the endosymbionts of P. h umanus and P. schaeffi is 3.75% (Table 21). We can use the 5.6 million year divergence date as a calibration point to determine the rate of nucleotide substitution in these endosymbionts, which equates to an absolute rate of 0.67% per million years. Thi s rate is 15 to 30 times faster than the rates of 16S rRNA evolution estimated previously for Buchnera, the P endosymbiont in Aphids (1 to 2% per 50 million years) (Moran et al. 1995). A more thorough comparison of the Candidatus Riesia lineage with Buc hnera will determine whether our rate calculations are indeed correct. It is conceivable that the same mechanisms that cause more mtDNA substitutions in lice than in aphids (Johnson et al. 2003) may have the same effect on the endosymbionts as well. Becau se endosymbionts evolve more quickly than their hosts (Moran et al. 1995), they may record more nucleotide substitutions during recent (or rapid) evolutionary events than do their hosts. Because lice have strictly coevolved with their primate hosts, and provide clear resolution of both recent and rapid evolutionary events (e.g., the population expansion of humans out of Africa is also evident in louse mtDNA) they have been used as a fast evolving marker to examine different events in human evolutionary his tory (Kittler et al. 2003, Yong et al. 2003, Reed et al. 2004, Leo and Barker 2005). The evidence for coevolution between Candidatus Riesia spp. and their primate louse hosts, along with the estimate of faster evolutionary rates in Candidatus Riesia, leads us to conclude that this new lineage of endosymbiotic bacteria shows promise as another independent evolutionary marker of primate and human

PAGE 28

28 evolutionary history. If faster evolving markers can be found within the endosymbionts (markers much faster than the 16S rRNA), then very recent events in human evolutionary history (e.g., Peopling of the Americas) might be studied from the perspective of the endosymbiont of a human parasite. This new three-tiered assemblage of host, parasite, and endosymbiont i s among the first tripartite assemblages of this type and will permit many new tests relating to their shared coevolutionary history. Table 2 1. Pairwise differences for pendosymbionts from human head and body lice Pediculus humanus Abbreviations P. h.h. Pediculus humanus humanus, human body lice and P. h. h. Pediculus humanus capitis the human head louse. Sequence divergences were caluclated using a GTR +I+G model of molecular evolution and compared to the most closely related taxon from Genbank ( Ca ndidatus Arsenophonus insecticola, [GenBank accession no. DQ115536]). P. h. h. (USA) P. h. h. (Wales) P. h. c. (USA) P. h. c. (Wales) P. schaeffi Pthirus pubis P. h. humanus (USA, 50.9%) P. h. humanus (UK, 50.9%) 0.0000 P. h. capitis (USA, 50.7%) 0.0031 0.0033 P. h. capitis (Arg., 50.8%) 0.0032 0.0033 0.0000 P. schaeffi (50.4%) 0.0395 0.0378 0.0359 0.0366 Pthirus pubis (52.5%) 0.1239 0.1127 0.1085 0.1110 0.1055 Arsenophonus (52.2%) 0.1315 0.1557 0.1533 0.1540 0.1174 0.1342

PAGE 29

29 Figure 2 1. Phylogenetic tree of endosymbiont from sucking lice (Anoplura) and other bacteria. Tree is based on Bayesian phylogenetic analysis of 10 million generations using MrBayes. Posterior probabilities above 0.50 are shown above nodes. Endosymbionts of primate lice are monophyletic with a posterior probability of 1.00. GenBank accession numbers are given in parenthesies.

PAGE 30

30 Figure 22. Fluorescent in situ hybridization microphotograph of thorax and abdomen (ventral view) of a second instar nymph of a h ead louse ( Pediculus humanus capitis ). This whole -insect mount was probed with the species -specific probe designed from the 16S rRNA gene endosymbiont sequence obtained from another individual of P. humanus capitis Note the bacteria inside the mycetome in yellow (scale bar = 50 m).

PAGE 31

31 CHAPTER 3 MUTATIONAL MELTDOWN IN PRIMARY ENDOSYMBIONTS: SELECTION LIMITS MULLERS RATCHET Introduction Primary endosymbiotic bacteria (pendosymbionts) are thought to have enabled insects to become ecologically diverse by facilitating radiations into niches with nutrient poor diets such as plant sap, wood, and vertebrate blood. P endosymbionts live within specialized host organs called mycetomes and are transmitted transovarially (vertically) from mother to offsp ring (Douglass 1989) Some pendosymbionts are required for host reproduction (Dedeine et al. 2003, Perotti et al., 2006,) whereas others provide essential services for their hosts such as light emission, or synthesis of amino acids, cofactors, and vitamins that are lacking in the hosts specialized diet (Buchner 1965). Because of their endosymbiotic lifestyle and strict vertical transmissi on, all pendosymbionts share many characteristics such as small populations, reduced genomes, and AT bias (Moran 1996, Clark et al., 1999, Lutzoni and Pagel 1997, Gill and Moya 2004). P endosymbionts also accrue slightly deleterious mutations at a faster rate than free living bacteria (Moran 1996). This is thought to be due to genetic drift acting on already small populations that go through population bottlenecks at each host generation (Ofallon 2008). Furthermore, because pendosymbionts are maternall y transmitted, it is thought that recombination cannot occur between different strains (Moran 2007) T he steady accumulation of these deleterious mutations is a process called Mullers ratchet (Muller 1964, Felsenstein 1974, Moran 1996). Mullers ratchet states that in small populations, due to genetic drift, there is a chance that individuals with the fewest mutations will fail to reproduce (Muller 1964, Lynch and Gabriel 1990) When this happens, the ratchet clicks (Felsenstein 1974)

PAGE 32

32 irreversibly increasing the overall mutational load of the population. As mutational load increases, the relative fitness decreases through reduced reproductive rate or reduced survivorship (Haldane 1937, Wallace 1987) If deleterious mutations continually get fixed over t ime, the pendosymbiont may experience a mutational meltdown ultimately resulting in extinction (Lynch and Gabriel 1990). It is thought that once a pendosymbiont has deteriorated to the point of being nonfunctional, it may be replaced by another bacterium (Andersson and Kurland 1998). Evidence for p endosymbiont replacement, however, is scarce, having only been found in a few species of aphids (Moran and Baumann 1994, Perez Brocal et al. 2006) weevils (Lefevre et al., 2004) and more recently in sucking lice (Hypsa and Krizek 2007, Perotti et al. 2009) In contrast, s ome insect/ p endosymbiont assemblages have existed for hundreds of millions of years without evidence of p endosymbiont replacement, suggesting that Mullers ratchet may slow or stop over t ime. Several mechanisms have been proposed to explain how Mullers ratchet might slow or stop over time. These mechanisms include back mutations (Atwood et al., 1951) compensatory responses (Hurst and Mcvean 1996), and selection. The probability of a bac k mutation correcting each slightly deleterious mutation is so minimal compared to the probability of a forward mutation that it has been ignored in models of Mullers ratchet (Atwood et al. 1951). Compensatory responses have been suggested in the case of the GroEL protein. The GroEL protein mediates the folding of polypeptides, and it is found to be highly expressed in Buchnera and other pendosymbionts. Up -regulation of the GroEL protein may reduce the effects of other slightly deleterious mutations t hat may change the folding of important proteins (Moran

PAGE 33

33 1996) However, little is known about the overall effect of compensatory responses on Mullers ratchet. Selection may be acting to slow or stop Mullers ratchet through long term bottlenecks which cause the variance in fitness to be increased among hosts for selection to act upon (Bergstrom and Pritchard 1998), and through epistatic interactions between slightly deleterious mutations (Charlesworth et al. 1993, Kondraschov 1994) where the effect on fitn ess of the p endosymbiont does not increase linearly with each mutation that becomes fixed in the population. Epistatic interactions may make the ratchet slow down but not necessarily stop (Butcher 1995) In this study, we are specifically interested in d etermining if selection is acting to reduce the number of slightly deleterious mutations that become fixed in the population, thus slowing the process of Mullers ratchet in some insect/pendosymbiont assemblages Early studies of non -synonymous nucleotid e substitution rates suggested that selection was weak in pendosymbionts (Moran 1996, Moran and Baumann 2000). Recent studies of the genomes of the p endosymbiont of aphids ( Buchnera), however, show that selection may play a role in slowing genome degradation (i.e., gene loss) and AT bias. For example, Tamas et al. (2002) found long -term genomic stasis in two Buchnera genomes that diverged around 50 70 Mya. They concluded that gene l oss must have occurred early in the association between Buchnera and its host, only to stabilize later due to selective constraints. Early and rapid gene loss was also found in another lineage of Buchnera with divergence dates of 80-150 My (van Ham et al. 2003, Klasson and Anderson 2004). Clark et al. (1999) suggested that selection might also reduce AT bias over time in Buchnera, and therefore slow the speed of Mullers ratchet.

PAGE 34

34 Selection could decrease the rate at which slightly deleterious mutations be come fixed in the population, especially as the p endosymbiont/insect association ages. As the mutational load increases in pendosymbionts, selection may act to remove individuals with the highest mutational loads (i.e., the least functional individuals) This would then slow the rate of fixation of slightly deleterious mutations, which would result in a reduction in the overall nucleotide substitution rate. Early estimates of nucleotide substitution rate in pendosymbionts consistently averaged 1-2% per 50 My (Ochman et al. 1999). However, recent studies have documented much faster evolving pendosymbionts (Degnan et al. 2004, Allen et al. 2007). The fastest rate reported to date is 33.5% per 50 My for Candidatus Riesia (hereafter Riesia ), the pendosy mbiont of primate sucking lice (Anoplura: Pediculidae and Pthiridae) (Allen et al. 2007). The pendosymbionts with the highest nucleotide substitution rates appear to be among the youngest insect/pendosymbiont associations, which suggest that rates may v ary in relation to the age of the association. However, the age of the association of Riesia with its host is unknown. Therefore, we first determine the age of the association of Riesia with its host and calculate more rigorously the rate of molecular evolution for Riesia Additionally we estimate the nucleotide substitution rates from the 16S ribosomal DNA gene ( 16S rDNA ) for a diverse assemblage of pendosymbionts to test the prediction that selection reduces the effect of Mullers ratchet over time. I f selection slows Mullers ratchet over time, then we should observe an inverse relationship between nucleotide substitution rates and the age of the insect/pendosymbiont assemblage. A decline in substitution rates would be consistent with an increase in

PAGE 35

35 selection over time. The mutational meltdown model predicts that given no opposing force, p endosymbionts should steadily accrue slightly deleterious mutations until extinction. An increase in selection over time might allow pendosymbionts to stave off extinction, which would explain the existence of ancient insect/pendosymbiont associations Materials and Methods Age of the Riesia/Louse Assemblage The oldest split found within Riesia dates to 12.95 Ma and occurs between p endosymbionts associated wit h the louse genera Pediculus and Pthirus (Allen et al. 2007) However, some of the oldest divergences among related lice (those of the genus Pedicinus ; Anoplura: Pedicinidae) date back to the split between their Anthropoid and Cercopithecoid primate hosts ca. 25 -30 Ma. At present it is not known whether lice of the genus Pedicinus carry the Riesia lineage of pendosymbionts. Therefore, we have molecularly characterized the endosymbiont of Pedicinus to evaluate the age of the Riesia /louse association. If the pendosymbiont in the louse genus Pedicinus does not belong to the Riesia lineage, then the Riesia /louse association is between 12.95 and 25 My. Specimen collection and DNA sequencing To determine the age of the Riesia /louse as sociation, specimens of Pedicinus badii were collected from Red Colobus monkeys ( Procolobus rufomitratus ) from Kibale National Park in Uganda. Three human head louse specimens ( Pediculus humanus capitis ) and a single body louse specimen ( Pediculus humanus humanus ) were collected from individuals in West Palm Beach, Florida, USA, and the rabbit adapted strain held at the Insect Control and Research Lab in Maryland, USA, respectively, to

PAGE 36

36 determine the absolute rate of nucleotide substitution in Riesia Whole lice were washed twice with 400l saline EDTA, 15l of 20% SDS and 5l lysozyme to remove any external bacteria. The sample then was crushed and genomic DNA was isolated using the DNeasy Tissue Kit (QIAGEN Inc., Valencia, California). PCR amplification of the endosymbiont 16S rDNA gene (1.5 -kbp) was performed in 25l reactions with primers 27F (5 AGA GTT TGA TCC TGG CTC AG 3) and 1392R (5 CAC GGA TCC ACG GGC GGT GTG TRC 3) for the Pedicinus endosymbiont, and the Riesia specific primer 461F ( 5 ACA GAA GAA GCA CCG GCT AA 3) and general reverse primer 1525R (5 AGA AAG GAG GTG ATC CAG CC 3) for Pediculus endosymbionts. Each amplification was performed using standard reaction conditions with 10ng of template DNA, 300nM of each primer, 200M of each dNTP, 2.5mM MgCl2 and 0.02 U of Taq DNA polymerase (Promega, Madison, Wisc.) per l of reaction mix. Cycling conditions consisted of an initial denaturation step (94C, 10 min), 30 cycles of amplification involving denaturation (94C, 1 min) annealing (5052C, 1 min) and extension (65C, 1 min), and a final extension step at 65C for 10 min. The 16S rDNA PCR product was purified with ExoSAP -IT (USB Corporation) and then cloned into the pTOPO 4.0 vector (Invitrogen). Recombinant clones were sequenced in both directions at the University of Florida sequencing facility using vector -specific primers and internal sequencing primers as in Reed and Hafner (2002) Sequences were edited using Sequencher Version 4.1 (Gene Codes Corporation, Ann Arbor, Michigan) and deposited in GenBank (Accession numbers: EU827259EU827263 Reisia pediculicola from Pediculus humanus humanus three sequences of Riesia pediculicola from Pediculus humanus capitis and the primary endosymbiont from Pedicinus badii respectively ).

PAGE 37

37 Phylogenetic a nalysis Phylogenetic analyses were used to determine the placement of the Pedicinus p endosymbiont with respect to other known bacteria, and to estimate the age of the Riesia /louse association. The 16S rDNA sequence of the Ped icinus p endosymbiont obtained above was compared to 32 bacterial 16S rDNA sequences downloaded from GenBank which included louse pendosymbionts, insect pendosymbionts, free living Escherichia coli and sequences with the highest sequence similarity to t he Pedicinus and other louse pendosymbionts obtained form GenBank BLAST searches (Table 3-2 ). All sequences were aligned using Clustal X (Thompson et al. 1997) then manually adjusted by eye. Modeltest v. 3.7 (Posada and Crandall 1998) was used to deter mine a model of nucleotide evolution according to an Akaike Information Criterion (GTR+I+G; (Hulsenbeck and Rannala 1997, Posada and Buckley 2004). This best -fit model was used in Maximum Likelihood (ML) and Bayesian phylogenetic analyses performed in PAU P*4.0b10 and MrBayes 3.12 (Hulsenbeck and Ronquist 2001, Swofford 2002) respectively. For the ML analyses, full heuristic ML and bootstrap (200 pseudoreplicates) searches were conducted with 10 random addition replicates and tree bisectionreconnection branch swapping using the best -fit model in PAUP* 4.0b10 (Swofford 2002) In the Bayesian analyses, model parameters were treated as unknown variables with uniform priors and were estimated as part of the analysis. Bayesian analyses were initiated with random starting trees, run with four incrementally heated chains (Metropolis -coupled Markov chain Monte Carlo; (Hulsenbeck and Ronquist 2001) for 10 million generations, and sampled at intervals of 1000 generations. Two independent

PAGE 38

38 Bayesian analyses were ru n to avoid entrapment on local optima. Stationarity was assessed by plotting the loglikelihood scores of sample points against generation, and a conservative burn-in period of 25% was discarded. The retained equilibrium samples were used to generate a 5 0% majority rule consensus tree with the percentage of samples recovering any particular clade representing that clades posterior probability (Huelsenbeck and Ronquist 2001) Alternative phylogenetic hypotheses were compared statistically using the Kishi no Hasegawa (KH) and the Shimodaira-Hasegawa (SH) tests as implemented in PAUP*4.0b10 (MP and ML analyses using RELL optimization and 1,000 bootstrap replicates; (Kishino and Hasegawa 1989, Shimodaira and Hasegawa 1999, Goldman et al. 2000). Suboptimal tr ees from the Bayesian analyses also were examined to assess alternative phylogenetic hypotheses. The frequency of the Markov chain Monte Carlo trees in agreement with an alternative hypothesis equals the probability of that alternative hypothesis being correct (Ihlen and Ekman 2002) The probability of trees agreeing with alternative subfamily hypotheses was calculated by applying constraint based filter trees implemented in PAUP*4.0b10 (Ihlen and Ekman 2002, Swofford 2002,) Absolute rates of nucleotide evolution in Riesia To determine the absolute rate of nucleotide substitution in Riesia t he 16S rDNA Riesia sequences obtained above were aligned with pendosymbiont sequences of human head lice ( R. pediculicola ; GenBank Accession Numbers AB263105, EF110570, and EF110571), human body lice ( R. pediculicola ; EF110569, EF110572, and AB236101), chimpanzee lice ( R pediculischaeffi ; EF110573), and human pubic lice ( R pthiripubis ; EF110574). Sequences were aligned using Clustal X (Thompson et al.

PAGE 39

39 1997) a nd manually adjusted using MacClade v. 4.06 (Maddison and Maddison 2000) These closely related sequences were easily aligned by eye with no ambiguity as to positional homology. Modeltest v. 3.7 (Posada and Crandall 1998) was used to determine a model of nucleotide evolution (GTR+G) for the Riesia 16S rDNA data as described above. A branch and bound ML analysis with a subsequent bootstrap analysis (200 replicates) was conducted using the best -fit model in PAUP* 4.0b10 (Swofford 2002) Reed et al. (2007) es timated divergence dates in the phylogenetic tree of primate lice, and estimated the split between the genera Pediculus and Pthirus to be 9.42 17.38 Ma ago. Because this node in the louse tree has a corresponding node of cospeciation in the endosymbiont t ree (Allen et al. 2007) we are able to calculate an absolute rate of nucleotide substitution within Riesia using the calibration range of 9.4217.38 Ma for the split between Pediculus and Pthirus endosymbionts. Divergence times were estimated using pena lized likelihood (TN algorithm) in the program r8s (Sanderson 2003) A smoothing parameter of 0.32 was determined using the cross -validation procedure. Substitution Rates Among Host/Endosymbiont Lineages To determine whether the age of the host/endosym biont association correlates with nucleotide substitution rate, we retrieved data from the literature of insect/pendosymbiont assemblages having both estimates of the age of the association (through fossil evidence) as well as either rates of pendosymbio nt nucleotide evolution for 16S rDNA or pairwise sequence divergences. In the absence of pairwise sequence divergences for a particular assemblage, we estimated these values by examining the two most divergent sequences as in Ochman et al. (Ochman et al. 1999) The systems

PAGE 40

40 examined included primate lice and Riesia (Allen et al. 2007) aphids and Buchnera (Moran et al. 1993) cockroach/termites and Blattabacterium (Bandi et al. 1995) whiteflies and Portiera (Thao and Baumann 2004) (date from Poinar (1992) ) tse tse flies and Wigglesworthia Askoy et al. 1995) Auchenorrhyncha (cicadas, hoppers and spittlebugs) and Sulcia (Moran et al. 2005) psyllids and Carsonella (Thao et al. 2000) weevils and Nardonella weevils and the S -clade of pendosymbionts (Lefevre 2006) and ants and Blochmannia (Degnan et al. 2004) Rates of nucleotide substitution were plotted against the age of host/endosymbiont association. Because there is error in both the estimate of the rate of nucleotide evolution and the age of association, a reduced major axis regression was performed on the log -transformed data to better estimate the relationship between the age of the association and rate of nucleotide evolution in these systems. R esults Age of Riesia/Louse Association The ag e of the association between the fast evolving pendosymbiont Riesia and the primate sucking lice in which it lives was previously unknown. In order to estimate this age, we examined the pendosymbiont from a closely related louse genus, Pedicinus The pendosymbiont from Pedicinus badii (a louse that parasitizes Old World monkeys) does not group with the anthropoid primate louse p endosymbionts (the Riesia lineage) in our Maximum Likelihood or Bayesian (not shown) phylogenetic analyses (Figure 31 ). The Maximum Likelihood analysis groups the pend o symbiont of Pedicinus badii at the base of a clade containing the pendosymbionts Wigglesworthia and Baumannia (p endosymbionts of tse -tse flies and leafhoppers, respectfully), some free-living bacteria, and the p endosymbionts of distantly related sucking lice of rodents

PAGE 41

41 (Figure 31 ). Bayesian phylogenetic trees were largely identical, and placed the pend o symbiont of Pedicinus badii at the base of the same clade. Analyses constraining the Pedicinus p endosymbi ont to group with the Riesia lineage produced trees that were significantly worse than the best Maximum Likelihood tree according to the Kishino Hasegawa ( p = 0.004) and Shimodaira-Hasegawa ( p = 0.004) tests. Furthermore, none of the suboptimal trees from the Bayesian analysis were consistent with this topological constraint ( p < 0.001). We can therefore formally reject the hypothesis that the p endosymbiont sequences from Pedicinus badii are sister to or embedded within the Riesia lineage. Because Pedici nus is the closest living relative of Pediculus and Pthirus this phylogenetic analysis demonstrates that the age of the association between Riesia and primate lice has an upper bound at 25 My for the split between Pedicinus and Pediculus and Pthirus Thus, the age of association between Riesia and their louse hosts is between 12.95 and 25 My, making this one of the youngest insect/p endosymbiont assemblages known. Absolute Rates Using the 9.42 17.38 My split between Pediculus and Pthirus as a cal ibration date (Reed et al. 2004, Reed et al. 2007) we estimated the divergence time between Riesia pediculicola (human head and body louse pendosymbionts) and Riesia pediculischaeffi (chimp louse p endosymbionts) at 5.42 My, which is very close to the ag es estimated for these lice and for their vertebrate hosts (Reed et al. 2004) We further estimate that the pendosymbionts of the human head lice originated 0.90 My (Figure 3 2), which is similar to the estimate of 1.2 My for the lice (Reed et al. 2004) The pairwise sequence divergence for Riesia p endosymbionts of Pediculus and Pthirus is 12.90% (GTR+I model), therefore the absolute rate of evolution of Riesia p -

PAGE 42

42 endosymbionts is 0.0037 -0.0684 substitutions per site per million years which translates to 18.56 -34.24% per 50 My (Table 3 -1). Substitution Rates Among Host/Endosymbiont Lineages When the rates of nucleotide substitution for Riesia are compared to other known insect/p endosymbiont systems, we find that the rate of substitutions in 16S rDNA decre ases with age of association and levels off after 100 My (Figure 3 -3). Although the majority of the systems are evolving at a rate similar to what was reported for Buchnera (1 -2% per 50 MY), the younger systems are evolving much faster (334% per 50 MY; F igure 33 ). Reduced major axis regression of the log -transformed data indicates that 78% of the variation in rates of nucleotide evolution can be explained by the age of the association (Figure 3 4) and that the decrease in rates is exponential. The pairw ise sequence divergence in Riesia calculated here (18.56 34.24% per 50 My) was corrected with a best -fit model of nucleotide substitution. Some previous studies did not use the best -fit evolutionary model to correct for multiple substitutions. Therefore, to test the impact of the substitution model, we also evaluated the same pairwise divergences using the Jukes -Cantor model. The Jukes -Cantor distances still provide a much faster rate of nucleotide substitution in Riesia (12.923.9% per 50 My), and it is important to note that this more simplistic model of molecular evolution underestimates the substitution rate by 30%. D iscussion Overview In this study, we find that there is considerable variation ( 15 to 30 -fold) in the rate of p endosymbiont nucleotide evolution for the 16S rDNA gene. The association between anthropoid primate lice and their pendosymbionts in the genus Riesia

PAGE 43

43 represents one of the youngest insect/pendosymbiont assemblages known to date (between 12.95 and 25 Ma), and compared to other insect/ p endosymbiont assemblages Riesia is experiencing the highest rate of nucleotide substitution yet measured (18.56 34.24% per 50 My; Figures 3 -3 and 3 -4). Among all insect/ p endosymbiont assemblages examined, we find that 78% of the variation in nuc leotide substitution rate can be explained by the age of the association (Figure 3 -4). Higher rates of nucleotide substitution are associated with the youngest host/ p endosymbiont assemblages despite correcting for multiple substitutions (Figures 3 3 and 3 4). Nucleotide substitution rates decrease to approximately 1-2% per 50 My when the insect/ p endosymbiont assemblages reach approximately 100 My of age. These findings are consistent with the hypothesis that selection reduces the effect of Mullers rat chet over time. An alternative explanation is that substitution rate variation is driven by variation in p endosymbiont population size (i.e., smaller populations evolv e faster than larger ones due to genetic drift ). Estimates of effective population siz e are not available for the taxa used in this study, therefore this cannot be tested directly. It is likely that p endosymbiont effective population size is governed largely by host effective population size, transmission dynamics, and other aspects of th e pendosymbiont/host relationship. Additional research, however is needed to directly test these hypotheses. As proposed in the mutational meltdown model, selection is likely the force reducing the number of deleterious mutations that become fixed in pe ndosymbiont populations. The slow but steady accumulation of deleterious mutations is predicted to impair the pendosymbionts ability to function if the process of Mullers ratchet goes

PAGE 44

44 unchecked. Our data suggest that selection may steadily grow strong er in older assemblages and thereby slow the rate of Mullers ratchet by removing individuals with the highest mutational load. An increase in selection over time explains why some p endosymbionts have ancient associations with their insect hosts and remain functional for hundreds of millions of years. However, younger assemblages do not always have a higher rate, especially among co endosymbionts. Weevils (Insecta: Coleoptera) have two lineages of p endosymbionts (termed the R and S -clades) that are ev olving at roughly the same rate even though R endosymbionts have been associated with their hosts for 75 My longer (Lefevre et al. 2004) The rate differences between weevil p endosymbionts, however, are minimal and fit well within the limits of oth er pe ndosymbionts (Figure 3-4). It has been predicted that selection plays a major role in slowing down or stopping Mullers ratchet. As slightly deleterious mutations go to fixation, they reduce the fitness of the host. If there were synergistic epistatic interactions between mutations we would expect an exponential increase in selection over time, which is consistent with our data. Endosymbiosis The importance of p endosymbionts to insects, concerning their radiation into nutrient poor niches, cannot be overstated (Douglas 1989, Moran 2007, Perotti et al. 2009) Yet, very little is known about how bacteria become endosymbionts, although it is thought that they might originate from attenuated pathogens (Corsaro et al. 1999, Moran and Wernegreen 2000, Dale et al. 2001, Braig et al. 2009) Regardless of the mechanism, the basic requirements for becoming an endosymbiont are substantial. The endosymbiont must overcome many host physical, cellular, and molecular barriers for internalization (Ochman and Moran 2001) and a mechanism must develop for

PAGE 45

45 transmission of the bacteria to the insects offspring (Gil et al. 2004) Within the Riesia lineage alone, these bacteria undergo two extra -cellular migrations and are housed in no fewer than four distinct mycetomes (P erotti et al. 2007) From an evolutionary perspective this complex host/ p endosymbiont interaction seems highly specialized and the likelihood of repeated endosymbiont replacement over time is unknown. If slightly deleterious mutations were to continue un abated in insect/p endosymbiont associations, then we would expect to see a steady increase in the number of nucleotide substitutions over time, maintaining a high rate of molecular evolution. Instead we see a decline in the substitution rate (Figure 3 3) Our interpretation is that as the host/ p endosymbiont association ages, and the mutational load of pendosymbionts increases, the role of selection increases and slows the rate of accumulation of slightly deleterious mutations. This is consistent with the studies of Tamas et al. (2002) van Ham et al. (2003) and Clark et al. (1999), who found that the rate of genome degradation and AT bias also decreases over time. Our findings are also consistent with that of Delmotte et al. (Delmotte et al. 2006) wh o found that the genes lost at the beginning of the association were those that were the least selectively constrained. We propose that there are selective constraints embodied in the process and maintenance of endosymbiosis that could mitigate the effect s of Mullers ratchet in late -stage or well established endosymbionts. Bergstrom and Pritchart (1998) suggested that long-term bottlenecks increase the selection pressure on deleterious mutations by increasing the variance in fitness among hosts. Therefore host -level selection may help to maintain the endosymbiosis over the long-term.

PAGE 46

46 Although our data show that Mullers ratchet slows through time, Mullers ratchet may not cease to act entirely in pendosymbionts, and three outcomes have been recorded. An e ndosymbiont may become so degraded that it effectively becomes an organelle such as Carsonella the p endosymbiont of psyllids (Nakabachi et al. 2006) Carsonella has been associated with its host for 100 to 250 My (Thao et al. 2000) and has a low rate of nucleotide evolution (Figure 3 3). It may be possible that Carsonella remains functional only because many of its genes have been transferred to the host genome and the products of these genes are shipped back to the symbiont (Moran 2 007) Alternatively, the biological functions of an endosymbiont may be so reduced that a second endosymbiont is required. This is the case with the co primary endosymbionts Baumannia and Sulcia which have lost so many metabolic genes that by themselves they would not be viable or functional as endosymbionts (McCutcheon and Moran 2007) Sulcia the more ancient pendosymbiont, has been associated with its host for 250 My and has a genome size of 245 kb (McCutcheon and Moran 2007) whereas Baumannia the younger p endosymbiont, has only been associated with sharpshooters for 25-40 My (Takiya et al. 2006) and has a larger genome of ~686 kb (Wu et al. 2006) These co -primary endosymbionts only survive by complementing each other. Finally, an endosymbiont ma y become so degraded it is eventually replaced, possibly out -competed, by another bacterial lineage. In fact, Anderson and Kurland (1998) suggested that obligate bacteria may replace each other at rates determined by Muller s ratchet. The gradual accumul ation of slightly deleterious mutations, slowly degrading the genome over time, may make the endosymbiont unable to compete with relatively benign pathogens that have the ability to participate in the mutualism. These

PAGE 47

47 less attenuated pathogens could then r eplace the older degraded p endosymbiont lineages, which may have been the case with some aphid lineages (Moran and Baumann 1994, Perez -Brocal 2006) weevils (Lefevre et al. 2004) and sucking lice (Hypsa and Krizek 2007) The relationship of the pathogen and host at this point would change to a mutualistic one thereby giving the new bacterial lineage the benefits of this relationship such as potentially escaping host immune defense through provision of various host transported mycetomes that protect the n ew p endosymbiont, which has been found in lice for many stages (Perotti et al., 2007) For the new mutualist, however, in some cases this new arrangement might hasten its extinction as Mullers ratchet engages. Table 3 1. Percent sequence divergence and rate of nucleotide substitution of the 16S rDNA gene of Riesia Riesia is the the p rimary endosymbio nt from anthropoid primate lice, these numbers are calibrated at 9.42 and 17.38 Million years 9.42 My 17.38 My Substitutions / My 0.0137 0.0074 Substitutions / site / My 0.0068 0.0037 Percent / 50 My 34.24% 18.56%

PAGE 48

48 Table 3 2. Bacteria taxa, their hosts, and GenBank accession numbers used in the phylogenetic analysis presented in Figure 3 1. Some bacteria lack a species name because they have not been characterized completely. Secondary rather than primary endosymbionts are indicated with an asterisk. Free-living bacteria have no associated host. Endosymbiont Host Species GenBank Riesia pediculicola Pediculus humanus capitis EF110571 Riesia pediculicola Pediculus humanus humanus EF110569 Riesia pediculischaeffi Pediculus schaeffi EF110573 Riesia pthiripubis Pthirus pubis EF110574 --Haematomyzus elephantis DQ076663 --Haematopinus apri DQ076665 --Haematopinus eurysternus DQ076661 --Haematopinus suis DQ076662 --Solenopotes capillatus DQ076664 --Polyplax spinulosa DQ076666 --Polyplax serrata DQ076667 Blochmannia ulcerosus Camponotus ants AY334375 Blochmannia sp. Camponotus ants AY334375 Sodalis glossinidius Glossina AJ245596 Baumannia cicadellinicola leafhoppers AY676882 Arsenophonus sp. Triatoma melanosoma DQ508172 Buchnera aphidicola Acyrthosiphon pisum BA000003 Wigglesworthia glossinidia Glossina brevipalpis BA000021 --* Melanococcus albizziae AF476106 ---* Planococcus citri AF476107 --* Erium globosum AF476105 --* Aphalaroida inermis AF263556 ---* Glycaspis brimblecombei AF263561 --* Glossina austeni GAU64869 --* Planococcus citri AF476107 Serratia symbiotica Aphis craccivora AY822594 Tatlockia micdadei -NA -AF227162 Legionella adelaidensis -NA -Z49716 Photorhabdus luminescens --NA -EF592562 Providencia alcalifaciens -NA -AY994312 Providencia vermicola Steinernema thermophilum AM040495 Arsenophonus arthropodicus Pseudolynchia canariensis DQ115535 Escherichia coli -NA -AP009048

PAGE 49

49 Figure 31. Maximum likelihood phylogram representing phylogenetic relationships of alll bacteria used in this study. C ommon insect p endosymbionts, and closely related taxa as determined from a BLAST search of each louse endosymbiont sequence. Numbers at nodes indicate maximum likelihood support values greater than 60. Gray lines indicate louse p endosymbionts. The p endo symbiont from Pedicinus badii (the louse that parasitizes Red Colobus monkeys) is shown in red demonstrating that it does not group with the Riesia p endosymbionts. There are now at least six distinct lineages of p endosymbionts sampled from sucking lice.

PAGE 50

50 Figure 32. Maximum likelihood phylogram representing phylogenetic relationships of Riesia p endosymbionts. Numbers above the node represent divergence dates (in millions of years) whereas numbers below the nodes are bootstrap support values (only num bers greater then 60 are shown). The divergence date calibration point of 9.42 17.38 My is indicated with an asterisk.

PAGE 51

51 Figure 33. Primary endosymbiont nucleotide substitution rates as a function of the age of insect/p endosymbiont association. Ages and rates were retrieved from the literature or were calculated from pairwise sequence divergences and are shown as percent per 50 My. Point estimates (diamonds) represent the median value between the upper and lower estimates (bars show the full range of dates). The dotted line indicates a rate of 2% per 50 My. It has been suggested that the tse tse fly and aphid pendosymbionts (Wigglesworthia and Buchnera, respectively) are closely related to each other and are the result of a more ancient endosymbiosi s event than represented here (Lerat et al. 2003, Canback et al. 2004). Using an older date to calculate rates, however, does not change the results (data available upon request).

PAGE 52

52 Figure 34. Reduced major axis regression of the log transformed data from Figure 33 The age of the association is on the x axis and the rate of nucleotide evolution is on the y axis. 78% of the variation in rate of nucleotide evolution can be explained by the age of the association suggesting that the rate of nucleotid e evolution does decrease over time in pendosymbionts of insects.

PAGE 53

53 CHAPTER 4 MULTIPLE LINEAGES OF BACTERIA IN ANOPLURA INDICATE A HIGH RATE OF BACTERIAL REPLACEMEN T ON A SHORT EVOLUTI ONARY TIME -SCALE Introduction Bacteria are one of the most common forms of life on the planet; they have evolved into almost every habitat and have every type of interaction with other organisms, from pathogenic to commensal to mutualistic. Humans harbor a community of bacteria that is thought to be critically important in human health and disease prevention (Eckberg et al. 2005) Cows and other foreand hindgut fermenters require bacteria to break down and provide nutrients lacking in their diet (Stewart el al. 1997). Many insects also have endosymbiotic bacteria that reside in specialized cells and provide nutrients to the insect (Buchner 1965). Primary endosymbionts have been implicated in helping insects become one of the most diverse animal groups by enabling them to exploit food sources that are lacking in important nutrients, such as plant sap, wood and blood (Buchner 1965), as is the case with blood sucking lice ( Pthiraptera: Anoplura), which parasitize mammals. It is thought that the radiation of many of these insect groups occurred a fter the insect ancestor acquired the bacteria and was able to exploit a new food source (Buchner 1965). In these insects, the bacteria are transmitted vertically from mother to offspring through the eggs to get incorporated into the next generation. As this process plays out over longer and longer time scales we see that as the insect diversifies, all species of the insect have the same lineage of bacteria. In fact, comparison of the phylogenetic tree of the insect host with that of the bacteria shows that the bacteria have radiated and cospeciated along with their hosts (Moran and Baumann 1994). Although most insect endosymbiont lineages have cospeciated with their hosts, a few

PAGE 54

54 cases have been found where the phylogenetic tree of the bacteria does not mat ch that of its host (Moran and Baumann 1994, Perez Brocal et al. 2006, Lefevre et al. 2004, Allen et al. 2009). In these cases, the endosymbiont has been replaced with a different endosymbiont lineage. Interestingly, for most of these cases, such replacem ents have been found only one or two times over hundreds of millions of years of shared evolutionary history between the bacteria and their hosts. These results suggest that bacterial replacement is a rare event. In Anoplura (sucking lice) however, a diffe rent pattern is found. Among the few louse species examined, six different bacterial lineages have been found to date (Hypsa and Kirizek 2007, Allen et al. 2009, Fukatsu et al. 2009). Interestingly, sucking lice are only 60 -80 million years old (Light et al. In Review ), which suggests that many bacterial replacements have happened in a short amount of time compared to other insect/endosymbiont systems (Moran et al. 1993, Bandi et al. 1995). If this trend continues as more lice are sampled, this will likely prove to be the group with the highest known rate of bacterial turnover. Wi th over 540 species of Anoplura represented across 15 families, a much wider sampling of this group is needed to be able to calculate the number of bacterial replacements that have occurred over the last 60 million years. To determine the rate of bacterial replacement in this group, I sequenced the 16S rDNA gene of primary endosymbiont from many species of Anoplura and analyzed them phylogenetically with other bacteria from Gammaproteobacteria. To understand the coevolutionary history of Anoplura and their primary endosymbionts, the results from the

PAGE 55

55 bacterial phylogeny were mapped onto and compared with the evolutionary history of their louse hosts. Methods Lice Sampling Overall, 27 specimens of lice were collected from museums and mammal collectors, representing 8 families and 21 species for molecular analysis. To remove external bacteria, lice were washed 3 times in 500ul of 5% bleach, and washed 2 times with sterile water (Meyer 2007). Lice were then completely crushed and extracted using a Qiagen micro kit (Cat No. 56304) according to the manufacturers protocol w ith the following modifications. L ice were placed in 80ul of Prot e inase K (Quiagen) and incubated overnight on a heat ing block at 55 C The DNA was eluted in 50ul of sterile water heated to 55 C Water was also extracted as a negative control to ensure no bacterial contamination was introduced during the extraction. 16S rDNA was amplified with general bacterial primers 27 f orward ( GAG TTT GAT CCT GGC TCA G ) and either 1525 ( AGA AAG GAG GTG ATC CAG CC ) or 1329 (GAC GGA TCCACG GGC GGT GTG TRC) r everse primers Primers were brought to a final concentration of 0.7uM using Stratagene Hi -Fidelity Mast er Mix (Cat No. 600650 51) Total PCR volume was 50ul. Cycling conditions included an initial denaturation step at 95C for 2 min, then 40 cycles of denaturation at 95 C for 40 sec, annealing at 50 C for 30 sec. and extension at 72 C for 2 min. A final ext ension step at 72C for 30 min was included to ensure that the majority of the products were polyadenylated for cloning. To separate the different bacterial sequences, p roducts were then cloned using the Invitrogen Cloning Kit ( Cat No. 450030), and 96 col onies were picked and

PAGE 56

56 sequenced in the forward direction at the ICBR sequencing facility at the University of Florida Complete sequences for the 16S rDNA region were generated for those products with unique matches when queried against the GenBank non-redundant nucleotide database using BLAST. Due to base pair composition, endosymbionts are likely to match against other endosymbionts in this database, so these matches provided a pool of putative primary endosymbiont sequences. Finally, the primer sequences were removed from all sequences. Bacterial Sampl ing Alignment and Tree Building We dow nloaded all Gammaproteobacteria 16S rDNA sequences from the Ribosomal Database Project (RDB, ~ 72, 000) and removed all identical sequences (leaving ~65,000 sequences ). N ext, we removed sequences that were shorter than 750 base pairs (roughly half the gene) to mitigate alignment issues and to retain individuals where the majority of information about the gene was available. The resulting 42,626 taxa were then split into 20 groups of approximately 2,000 taxa each, and one or two of the endosymbiont sequences from each louse were added to each group. Alignments were generated for each group using MUSCLE (Edgar 2004), and checked by eye. Profile alignments ( two alignments were maintained and aligned together by adding in gaps ) were then created, and again checked by eye. Overall, there were 20 individual MUSCLE alignments and 19 profile alignments. Finally, ambiguous sections of the alignment were removed. To investigate how ta xon sampling would affect the number of endosymbiont lineages identified in Anoplura, four datasets were created. First, all pairwise distances were calculated, and sequences that were at least 80% similar were combined into clusters. Datasets for phylogen etic analysis were generated by selecting one sequence

PAGE 57

57 from each cluster, and adding all endosymbiont sequences to yield an alignment of 76 taxa. This process was then repeated with progressively more stringent sequence similarity thresholds to yield datas ets of 215 taxa (85% similarity), 865 taxa (90% similarity) and 4,275 taxa (95% similarity). Each dataset was nested within subsequent datasets, i.e., all 76 taxa from the 80% dataset were included in the 85% dataset, and all taxa in the 85% dataset were i ncluded in the 90% dataset etc. Phylogenetic trees were created for each of these datasets using a maximum likelihood framework with a GTR + G model of molecular evolution in the program RAxML (Stamatakis 2006), and bootstrap values were calculated from 20 0 replicates. The number of endosymbiont lineages found on each tree was then calculated by counting all of the nodes that grouped sucking lice endosymbionts together to the exclusion of all other bacteria with > 50% bootstrap support. Finally because endo symbionts are thought to have greater than 50% AT bias (Lambert and Moran 1998) in their 16S rDNA, AT% was calculated for all of the putative endosymbionts, this is another indicator that the bacterial sequences came from an endosymbiont. Freeliving bacte ria have < 50% AT composition at the 16S rDNA. Results Endosymbionts Overall, 27 louse species were examined and 21 putative endosymbiont sequences found (Table 41); in one instance two distinct endosymbiont sequences were found within a single louse ( An cistroplax crocidur ae) For six specimens, I could not recover any sequence that met either the sequence similarity or the base composition percentage criterion for endosymbionts. Most of these came from one louse family, Hoplopleruidae, and five of the si x lice species harbored another known

PAGE 58

58 louse pathogen, Bartonella (Proteobacteria: alphaproteobacteria). Interestingly, in one specimen, I found both a putative endosymbiont sequence and a Bartonella sequence (Table 41). A total of 30 sequences of putative endosymbionts were recovered: 22 generated from this study and 8 downloaded from GenBank. Together, these 30 sequences cover 8 families of Anoplura (Table 4 1). All 30 sequences showed sequence similarity to known endosymbionts in a BLAST search, and 27 o f those sequences had AT% of 49 or above. Three sequences from distantly related lice, including the second endosymbiont sequence from Ancistroplax crocidurae had AT% of 45, suggesting these may not be a primary endosymbiont based on the 50% rule. The endo symbionts I sequenced from the genus Pedicinus had an average pairwise sequence divergence of 3.7% from another known Pedicinus endosymbiont, P. obtusus (Fukatsu et al. 2009), where the fluorescent in -situ hybridization (FISH) has established that the bact erial sequence is indeed found within the mycetome. Phylogenetic Analysis Interestingly, most of the endosymbionts grouped fairly close together within the large bacterial trees (Figure 41) and were nested within the genus Arsenophonus Other insect endosymbionts are also in this part of the tree, like Buchnera (Aphids) and Wigglesworthia (Tsetse flies). Only one group of endosymbionts had a distinctly different placement in the tree, Polyplax sp These endosymbionts were classified to the genus Legionella in the Ribosomal Database Project As the number of taxa in the phylogenetic analysis increased, the number of endosymbiont lineages increased as well, the total number of endosymbiont lineages therefore was 13 with a higher number of taxa represented (Table 42) A few

PAGE 59

59 endosymbiont groups had phylogenetic relationships that mirrored those of their hosts. These included Candidatus Riesia found in Great Ape lice, Pedicinus (Old World Monkey Lice), Haematopinus (Pig Lice), Polyplax (Rodent lice), and Ancistroplax (Rodent Lice) (Figure 4-2). Only one group was found that did not match their hosts evolutionary history, and it was found consistently in all of the trees with high bootstrap support. This group consists of the endosymbiont from a seal louse ( Proechinophthirus fluctus ) in the family Echinopthiridae, the second putative endosymbiont sequence from Ancistroplax crocidurae family Hoplopleuridae, and the endosymbiont sequence from Sathrax durus family Polyplacidae ( Figure 4-2 ) These endosymbiont sequences had only 45% AT bias, which suggests they are either not a primary endosymbiont or a very young endosymbiont and have not had time to accumulate AT bias. Discussion Endosymbionts Examination of the results of the base pair composition analysis reveals some interesting findings. Although most endosymbi ont sequences had greater than 50% AT bias, a few had less Two end osymbionts from lice in the genus Ancistroplax had 49% and 50% res pectively, and they grouped together. Three endosy m bi on t s had 45% AT bias much less than the 50% cut off for primary endosymbionts and these three end o symb ionts grouped together with high bootstrap support in all of the analyses They are also the only group that does not mirror h ost evolutionary history. These endosymbionts are from three species of Anoplura that are very distantly related on the louse tree. One possible explanation is that they are grouping together due to base pair composition and not phylogenetic history. However, when we calculate average pairwise distances in this group we get 0.0074, which is much smaller than the

PAGE 60

60 endosymbionts from Pedicinus (0.0373), Haematopinus (0.0329) and Candidatus Riesia (0.0579), which all group together wi th high bootstrap support with the same branching pattern as their hosts. One explanation for this surprising finding is that this bacterium may actually be a secondary endosymbiont, or a pathogen that is found in many lice, but only sampled in this study a few times. One louse ( Ancistroplax sp .) had both this sequence and another endosymbiont sequence with 49% AT bias, which is more similar to other primary endosymbionts, so there is some evidence of this lineage cooccurring with other lineages that more closely fit the criteria of a primary endosymbiont. FISH analysis to determine where in the louse this sequence is found would illuminate where it is located. Interestingly, it is thought that the transition to the mutualistic lifestyle speeds up the rat e of molecular evolution, if this lineage of bacteria is not in the mycetome, and not the primary endosymbiont, its rate of molecular evolution may not have increased, which may explain why such a small amount of divergence is found between these bacteria from distantly related hosts. Taxonomy In 2007 we proposed to name the endosymbiont of the gorilla louse ( Pthirius gorillae ), Riesia pthirigorillae (Allen et al. 2007); however, recently it has been suggested that Riesia along with other insect endosymbionts, should be placed in the genus Arsenophonus (Moran et al. 2009). Here, w e find similar results with most of our Anopl ura endosymbionts grouping within the genus Arsenophonus In light of this, it is likely that the gen us Riesia will be subsumed with in Arsenophonus However, for louse endosymbionts, w e propose that the naming system conti n ues by describing the host name as the species name. Ultimately, however, before names can be formally

PAGE 61

61 described the FISH work needs t o be completed to demonstrate that the sequences do come from the mycetome. Bartonella as an Endosymbiont Bartonella is a known louse pathogen from a different group of bacteria (Alphaproteobacteria). For many louse taxa, we found this pathogen, and for a few lice I did not sequence a primary endosymbiont. One explanation would be that Bartonella has become the primary endosymbiont in this group. To determine if this is the case, I downloaded all Bartonella sequences from RDB, removed the sequences having l ess than half of the gene, aligned them using MUSCLE, and checked them by eye. Using a GTR + G model of molecular evolution, I created a best Maximum Likelihood tree in RAxML with 100 bootstrap replicates. Here, I found that only the Bartonella sequences f rom this group of Hoplopleuran lice group together with high bootstrap support (Figure 4 -3). The other Bartonella sequences were found outside of this clade and throughout the Bartonella tree (data not shown). This pattern suggests that in this louse group, this Bartonella lineage is different. One possible explanation is that it has become the primary endosymbiont in this group. Number of lineages We expect that as we add taxa to a phyl ogenetic analysis, some clades will break up, and therefore the number of independent endosymbiont lineages (those that group together to the exclusion of all other taxa) would increase. Also, because there are a finite number of endosymbiont lineages sampled, we expect that the number of endosymbiont lineages will remain the same once a sufficient number of taxa have been included in the analysis. Here, we find that the number of distinct lineages of endosym b i onts increased from 10 to 13 (Table 2) at 865 t axa, and remained at 13 at

PAGE 62

62 with 4,275 taxa represented. This suggests that sampling 865 taxa from clusters of sequences that are 90% similar from Gammaproteobacteria, is adequate to calculate the number of independent lineages of endosymbionts in this grou p, and may provide clues as to the appropriate number of taxa needed to describe phylogenetic relationships among Gammaproteobacteria with 16S rDNA. We found 13 distinct lineages of bacteria in Anoplura; however, i t is likely that there are more because only 8 of the 15 families have been examined. If we suggest that Bartonella is the endosymbiont in that clade of Hoplopleuran lice, then we have 14 replacements over 80 million years, which gives us a rate of 0.175 changes/million years, or one turnover every 6 million years. We know that the age of the Riesia /Louse association is between 8 and 25 million years old, which supports the finding that these bacteria get replaced roughly every six million years. Evolution of Sucking Lice and their Endosymbionts There are five groups of endosymbionts that grouped together with high bootstrap support those of the louse genera Pediculus and Pthirus Pedicinus Haematopinus Ancistroplax and Farenholzia (Figure 4-2 ). Within these groups, they have the same branching pattern as their hosts, which suggests that, after a bacterial replacement, the new endosymbiont then coevolved and cospeciated with its louse host for some time. It has been suggested that endosymbionts are replaced at a rate corresponding to their rate of genome degradation (Andersson and Kurland 1998). As endosymbionts accumulate slightly deleterious mutations over time, they are unable to compete with invading bacteria and may be replaced. However, there have been a number of papers suggesting that selection may act to reduce the rate of genome degradation in this group ( Clark et al. 1999, Tamas et al. 2002 and van Ham et al. 2003, Allen et al. 2009),

PAGE 63

63 and may prevent the endosymbionts from being replaced. Anoplura as a whole is only around 80 million years old, and many of these endosymbiont groups are likely much younger than 50 My.(Light et al In Review) In this group though, they are being replaced at a much higher rate. Blood Feeding as a Source of Endosymbiont s Many insects whose endosymbionts grouped within the Arsenophonus genus also feed on mammalian blood. For example, the endosymbiont from Great Ape lice, Candidatus Riesia (Great Ape Lice endosymbionts), grouped together with endosymbion ts from Hippoposcid flies (many genera), deer keds (Liptoptena) and a dog tick (Dermacentor) (Data not shown). One way to interpret this is that there may be some common Arsenophonus lineages in mammalian blood that have the tendency to become primary endo symbionts in insects. Therefore, when lice switch to a new mammalian host, they may come into contact with a different lineage of Arsenophonus that is then able to outcompete the myectomic bacteria and become the primary endosymbiont. In lice however, thi s bacterium is likely to have a short evolutionary history with its host as it will likely get replaced sometime later.

PAGE 64

64 Table 4 1. Louse taxa used in this study (arranged by family), host associations, and GenBank accession numbers. Museum acronyms for host taxa installed in Natural History Museums are as follows: Louisiana State University Museum of Natural Science (LSUMZ), Moore Laboratory of Zoology, Occidental College (MLZ), New Mexico Museum of Natural History (NMMNH), and University of Alaska Museum of the North (UAM). indicates endosymbionts sequenced, X indicates no endosymbiont sequenced, other bacteria sequenced as indicated. Louse Family and Species (Locality) Taxon Label Host (Order: Family; Museum Voucher) Endosymbiont %AT Echinophthiriidae Proechinophthirus fluctus (USA: AK) Echin3.17.09.2 Callorhinus ursinus (Carnivora: Otariidae) Yes 45% Haematopinidae Haematopinus eurysterunus -Bos taurus (Artiodactyla: Bovidae) DQ076661 52% Haematopinus suis (Florida, USA) Hpsu7.14.09.4 Sus scrofa (Artiodactyla: Suidae) Yes 52% Haematopinus suis -Sus scrofa (Artiodactyla: Suidae) DQ076662 52% Haematopinus apri -Sus scrofa (Artiodactyla: Suidae) DQ076665 52% Hoplopleuridae Ancistroplax crocidurae 1 (Vietnam) Axcro4.26.09.1 Crocidura sp. (Soricomorpha: Soricidae) Yes 50% Ancistroplax crocidurae 2 (China) Axsp7.14.09.5 Crocidura attenuata (Soricomorpha: Soricidae) Yes(2) 49%, 45% Hoplopleura ferrisi 2 (MX: Puebla) Hofer7.14.09.8 Peromyscus difficilis (Rodentia: Cricetidae; LSUMZ 36247) No Bartonella Hoplopleura hirsuta (USA: TX) Hosp4.17.09.7 Sigmodon hispidus (Rodentia: Cricetidae; LSUMZ 36377) No Bartonella Hoplopleura onychomydis (USA: AZ) Hoony8.27.08.6 Onychomys torridus (Rodentia: Cricetidae; NMMNH 4394) No Bartonella Hoplopleura reithrodontomydis 2 (USA: AZ) Hosp7.14.09.6 Reithrodontomys sp. (Rodentia: Cricetidae; NMMNH 4411) No Hoplopleura sicata (China) Hosic7.14.09.9 Niviventer fulvescens (Rodentia: Muridae) No Bartonella Linognathidae Linognathus spicatus (Zimbabwe) Linog6.22.09.1 Connochaetes taurinus (Artiodactyla: Bovidae) Yes 52% Solenopotes capillatus -Bos tarus (Artiodactyla:Bovidae) DQ076664 50% Pedicinidae Pedicinus badii (Uganda) Qnbad7.24.06.8 Piliocolobus tephrosceles (Primates: Cercopithecidae) Yes EU827263 53% Pedicinus pictus 1 (Ivory Coast) Qnpic3.31.08.1 Piliocolobus badius (Primates: Cercopithecidae) Yes 54% Pedicinus pictus 2 (Ivory Coast) Qnpic6.30.09.2 Colobus polykomos (Primates: Cercopithecidae) Yes 53% Pedicinus pictus 3 (Ivory Coast) Qnsp3.31.08.3 Colobus polykomos (Primates: Cercopithecidae) Yes 54% Pedicinus obtusus (Nagano, Japan) -Macaca fuscata (Primates: Cercopithecidae) AB478979 54% Pediculidae Pediculus humanus capitis Pdcap1.19.05.1 Homo sapiens (Primates: Hominidae) Yes EF110571 51%

PAGE 65

65 Table 4 1. Continued. Louse Family and Species (Locality) Taxon Label Host (Order: Family; Museum Voucher) Endosymbiont %AT Pediculus humanus capitis (USA: FL) Pdcap9.20.05.2 NW Homo sapiens (Primates: Hominidae) Yes 51% Pediculus humanus humanus (USA:MD) Pdhum5.19.05.2 Homo sapiens (Primates: Hominidae) Yes 51% Pediculus schaeffi (Uganda) Pdsch4.30.03.8 Pan troglodytes (Primates: Hominidae) Yes EF110573 51% Polyplacidae Fahrenholzia ehrlichi 1 (USA: TX) Fzehr8.20.08.1 Liomys irroratus (Rodentia: Heteromyidae; LSUMZ 36395) Yes Bartonella 52% Fahrenholzia ehrlichi 2 (MX: Puebla) Fzehr6.30.09.4 Liomys irroratus (Rodentia: Heteromyidae; LSUMZ 36299) Yes 51% Lemurpediculus verruculosus 1 (Madagascar) Lesp4.26.09.2 Microcebus rufus (Primates: Cheirogaleidae) Yes 53% Linognathoides marmotae 1 (USA: CO) Lnlae6.30.09.3 Marmota flaviventris (Rodentia: Sciuridae) Yes 54% Neohaematopinus neotomae (USA: CA) Neneo8.20.08.2 Neotoma lepida (Rodentia: Cricetidae; MLZ 1921) No Bartonella Neohaematopinus sciuropteri (USA: OR) Nescp6.30.09.5 Glaucomys sabrinus (Rodentia: Sciuridae) Yes Bartonella 53% Sathrax durus (Vietnam) Sathrax4.26.09. 3 Tupaia belangeri (Scandetia: Tupaiidae) Yes 45% Polyplax serrata -Apodemus sylvaitcus (Rodentia:Muroidea) DQ076667 52% Polyplax spinulosa -Rattus norvegicus (Rodentia:Muridae) DQ076666 53% Pthiridae Pthirus gorillae (Uganda) Ptgor9.14.08.1 Gorilla gorilla (Primates: Hominidae) Yes 53% Pthirus pubis (Scotland) Ptpub8.14.06.2 Homo sapiens (Primates: Hominidae) Yes EF110571 53% Rhynchophthirina Haematomyzus elephantis 059_ Haem_elep hantis Elephas maximus (Proboscidea: Elephantidae) DQ076663 55%

PAGE 66

66 Table 4 2. Taxon sampling, and endosymbiont determination. Here we built phylogenetic trees with different numbers of taxa, found by clustering all of the gammaproteobacterial sequences according to % similarity and selecting a representative sequence from each cluster. As the number of taxa examined increased, there was an increase in the number of distinctly different endosymbiont li neages found, ranging from 10 to 13. However, going from 865 to 4275 taxa did not change the endosymbiont number, suggesting that 865 taxa is adequate sampling to identify the number of endosymbiont lineages. C luster 80% 85% 90% 95% No. Taxa 76 217 865 4275 No. Endosymbionts 11 10 13 13

PAGE 67

67 Figure 41. Bacterial tree showing placement of Anopluran endosymbionts. Maximum likelihood tree of Gammaproteobacteria from the 90% cluster (865 taxa). Red lines indicate the placement of the endosymbionts from sucking lice (Anoplura). These lice are mos tly nested within the Genus Arsenophonus with the exception of the primary endosymbionts from Polyplax sp Identified to be in the genus Legionella from the Ribosomal Database Project.

PAGE 68

68 Figure 42. Phylogenetic tree of Louse taxa from Light et al. (In R eview). Names on the right are the Host Family names. Thicker branches indicate ones with a putative endosymbiont sequence. Colors represent endosymbionts that group together phylogenetically and also show relationships the same as their hosts, suggestin g cospeciation of the bacteria and their hosts in these groups. Asterisks represent independent lineages of bacteria, Solenopotes capilatus is not represented on the tree because we did not have the molecular data, but an endosymbiont sequence was on GenBank. Subscript A represents the three putative endosymbionts that always group together. Zero indicates a potential loss of the endosymbiont or a switch to a different type of endosymbiont like Bartonella.

PAGE 69

69 Figure 43. Maximum Likelihood tree of 279 Bar tonella sequences Bartonella from Sucking lice are shown in red, and t hey group together with 74% bootstrap support suggesting that in this group of Hoplopleuran lice Bartonella may in fact be the endosymbiont. The other Bartonella endos y mbion t s sequenced from Anoplura are nested within different groups on this tree (not shown).

PAGE 70

70 CHAPTER 5 POPULATION GENETICS OF HABITAT SENSITIVE RED COLOBUS SUGGEST LONG -TERM STABILITY OF KI BALE NATIONAL PARK Introduction Red Colobus monkeys (Cercopithecidae: Colobus: Piliocolobus ) are considered to be one of the most endangered primates in the world (IUCN 2010). These monkeys are patchily distributed across equatorial, all in the genus Piliocolobus The r adiation of Colobines began in the Plio cene around 7.5 ( 1.2) Ma and the Red Colobus (Procolobus (Piliocolobus )) diverged from the Olive Colobus ( Procolobus (Procolobus )) around 6.4 ( 1.1) Ma (Ting, 2008) Recent molecular work suggests that the extant forms of Pilocolobus radiated only around 3 Ma (Ting 2008). The taxonomy of this group has been debated for some time, with estimates of the number of species ranging from one to 16 (Ting, 2009). Mittermeier et al. (2009) recognized nine members of this genus, and ranked three within the top 25 most endangered primates. The IUCN (2010) lists six members of the genus Pilio colobus, and five of those are considered endangered or critically endangered. Red Colobus are critically endangered in part because they are very sensitive to habitat change. They are large, folivorous monkeys (Struhsaker, 1975, Oates 1996b) that live in social groups ranging from 25 to 1 50 individuals (Struhsaker 1975, Chapman pers. com.), necessitating large tracts of land for foraging (Oates, 1987), Due to this most populations of Red Colobus today are threatened by habitat destruction (Struhsaker and Leland 2008). Another issue facing Red Colobus is hunting pressure. Because of their large size, they are often hunted by humans and other predators (Oates and Davies 1994); it has been documented that one population of Red Colobus

PAGE 71

71 in west Africa was recently eradicated due to human hunting within the last 30 years (Oates et al., 2000). Currently, it is thought that th ere is only one area where Red Colobus are not threatened, and that is Kibale National Park, Uganda ( Struhsaker 1997). Kibale National Park (KNP) (795 km2) is located in western Uganda near the foothills of the Ruwenzori Mountains (Struhsaker, 1975; 1997). Currently, KNP harbors 13 different species of primates including the highest numbers of Red Colobus in the world; census estimates range from 30,000 80,000 monkeys (Chapman pers. com.). These red colobus are thought to have diverged from a sister p opulation less than 600,000 years ago (Ting 2008). They are also the most extensively studied population where data have been collected on behavior (Struhsaker 1975), dietary preferences (Chapman et al. 2005), and even viral load (Goldberg et al. in press ). However, the reasons these Red Colobus are so abundant in KNP while other populations are on the verge of local extinction remain unclear. Because they are so sensitive to habitat change, the protection the monkeys have received inside the park likely plays a major role. There are good records on human impacts (i.e. logging) in the park that go back at least 30 years, and evidence of disturbances (clearings) within the last 2,000 years (Chapman et al. 2000). I nterpreting how anthropogenic disturbances hav e affected KNP monkey populations in the recent past can provide insight into the stability of this species and inform conservation measures in the future. A critical first step is to develop a basic understanding of the population genetic structure of the se monkeys. To understand the population history of Red Colobus in KNP, we used genetic data to first determine the number of populations. Based on those results, we calculated

PAGE 72

72 the effective population size, coalescent point, and looked for evidence of a population bottleneck. Together with the information about disturbances in the park, these analyses illuminate how the population size of the Red Colobus has been affected by habitat change. Methods DNA Collection and Storage Blood and fecal samples were collected from 6 groups throughout KNP from 2007 2009: Small Camp (SC), Large Mikana (LM) K 30, Dura, Sebatolli, Mainaro (Figure 5 1). The first two groups have been part of an ongoing behavior study, and blood samples were obtained when individuals we re fitted with collars. Later, to increase the number of individuals sampled for this study, non-collared monkeys were followed to obtain fecal material. For the remainder of the groups, at least five individuals were sampled to look for evidence of gene f low throughout the park (Figure 5 -1). Blood samples were stored on a Classic FTA Card (Whatman WB -12 -0205). Fecal samples were treated with the two -step procedure described by Nsubuga et al. (2004): samples were pl aced in 95% ethanol for 2436 hours, then moved to silica beads (Sigma S7625) for longer -term storage. DNA Extraction and Quantification DNA was extracted from b lo od samples using the FTA protocol described by the manufacturer Fecal samples were extracted using a QIAmpStool kit (Qiagen) acc ording to the manufacturers protocol. Feces only contain a small amount of DNA from the cells lining the rectum, which can make genotyping unreliable (Goosens et al 2000). In order to obtain adequate DNA for genotyping analyses, DNA was extracted

PAGE 73

73 from 2 -4 fecal samples from each indivi dual (Taberlet et al. 1996 Goosens et al. 2000). Overall, this resulted in 230 extracts. We quantified the DNA in a sub -sample of these extracts (N=143), in order to determine the number of PCR amplifications necessary to o btain an accurate genotype (after Harris et al. 2009; Arandjelovic et al. 2009). Extracts had DNA concentrations of 0.48 to 50pg per reaction, consistent with other fecal studies (Harris et al., 2009, Arandjelovic et al 2009) A majority (>70%) of samples had greater than 5 pg of DNA per extract, which previous studies have determined requires 23 amplifications from independent extracts per individual to determine accurate genotypes (Harris et al., 2009, Arandjelovic et al 2009) Genotype analysis Eleve n loci, originally identified from the human genome, (Table 5 1) and found to be variable in Red Colobus (Difore pers com.), were amplified using a three-primer nested PCR as in Schuelke ( 2000) with one primer a universal M13 fluorescently labeled forward primer. PCR reactions were conducted in a total volume of 25 l: 12.5 l of the Type -it Microsatellite PCR Kit (Qiagen), final concentrations of 0.04 each of the primers (nonlabeled forward primer, labeled universal M13 primer and reverse primer), water, and .48 50pg of DNA per reaction. Cycling conditions included an initial activation step of 95 for 5 min, a first round of cycling consisting of 15 cycles of 95 for 30 seconds, 52 for 1 min 30 seconds and 72 for 30 seconds, followed by a second round of cycling consisting of 28 cycles of 95 for 30 seconds, 48 for 1 min 30 seconds and 72 for 30 seconds, and a final extension step of 72 for 30 min. Final products had a fluorescently -labeled universal M13 primer ( 21) at the 5 end and were run on a AB3730xl 96 capillary automated sequencer and scored

PAGE 74

74 using the program GeneMarker (Softgenetics). Only one person (JMA) did all of the scoring to reduce error. Each PCR included a positive control (Monkey 13) to ensure proper scoring and a negative control (water) to check for contamination. Each locus was amplified from at least two different extracts. If the two reactions produced different genotypes (i.e. missing one allele), another reaction was conducted until two reactions produced consistent genotypes, with up to four reactions conducted for each locus. To decrease the probability of allelic dropout, homozygotes were verified by at least 3 successful reactions. If four PCR reactions failed to yield two consistent genotype s the sample was scored as missing data. Finally, individuals that had fewer than 9 loci typed were excluded. The final dataset included 85 individuals (SC=38, LM=26, K30=5, Dura=5, Sebatoli=6, Mainaro=5). Number of Populations The number of populations of Red Col obus was calculated three ways in order to verify our results, because all subsequent analyses relied on these results. First, using RSTCALC (Goodman 1997), which estimates RST (an estimation of FST from microsatellite data) and Nm (number of migrants per generation) and secondly, using STRUCTURE 2.3.3 (Pritchard et al., 2000), which assigns individuals to populations where the number of populations is defined a priori by the user. STRUCTURE analysis was performed using an admixture model and correlated allele frequencies among populations. Because six troops were sampled throughout the park, the a priori number of populations (k) was tested from 1 to 6. Each test was allowed to run for 1 X 106 generations, after excluding a burn in of 1 X 105 generations Each k was tested ten times to examine the variation between runs The likelihood scores for each run were examined visually for stationarity, and the maximum likelihood from each run plotted

PAGE 75

75 against k (Figure 52). Finally, MIGRAT E 3.14 (Beerli 2006, 2008), which uses Bayesian inference and coalescent theory to calculate the likelihood of a one population model and a two population model clustering the data into the two populations based on the Rst results above (Beerli 2010). For the one population model, MIGRATE was run for 100 X 106, and a 10% burn-in period was excluded. Multiple tests of the twopopulation model were run, using different ranges for the prior distribution of M ( number of migrants per allele per generation per mutation rate) from 0 -1, 05, 0-10, 015, 025, and 0 -500. Each model was run for 100 X 106 generations, with 10% burn-in, and repeated twice to examine variability between the runs. The results were uploaded into TRACER, and likelihood scores examined visually for stationarity. Marginal likelihoods of the models were then compared using bayes factors (Beerli 2010). Analysis To look for evidence of genotyping errors (scoring errors, null alleles and allelic dropout), the loci were examined in Micro-Checker 2. 2.3 (van Oosterhout et al., 2004). Evidence of non-random associations between alleles was examined in FSTAT 2.9.3.2 using 550,000 randomizations among the 11 loci (Gouded, 2002), and deviations from Hardy Weinberg equilibrium were calculated in POPGENE 1. 31 using a likelihood ratio (G2) test (Yeh et al, 1997) Estimates of Theta, Population Size and Bottlenecks The results from the analyses above strongly suggested that KNP Red Colobus make up a single population. Therefore, for the remainder of the analys is, all samples were included as a single population. We calculated effective population size ( Ne ) by calculating from MIGRATE 3.14 with a one-population model. For diploid biparental loci, is equal to 4Ne, scaled by mutation rate () ( Watterson 1975; Tajima 1983;

PAGE 76

76 Hudson 1990; Hein et al. 2004). The human mutation rate of 5 X 104 was used as a proxy for Red Colobus microsatellite mutation rates (Whittaker et al. 2003). Finally, to look for evidence of a bottleneck, the data were examined in M_p_val.exe (Garza and Williamson 2001) and BOTTLENECK 1.2.02 (Cornuet and Luikart 1996). Results Genotyping Errors, Linkage and Hardy Weinberg Equilibrium Evidence of null alleles was found in only two loci, D1S207 and D17S1290, and there was also e vidence of scoring error s in D17S1290 due to an excess of homozygote s. No evidence for allelic dropout was found. Out of 55 tests, only one locus pair (D2S1399 and C2A) was found to be significantly non-randomly associated at the B onferroni corrected alpha of <0.001 (Rice 1989). Estimates of homozygosity ranged from 0.3 to 0.9 and heterozygosity from 0.5 to 0.85 (Table 51), and only one locus (D20S206) significantly deviated from Hardy -Weinberg equilibrium (HWeq) using the Bonferroni corrected pvalue in P OPGENE. To determine if this was consistent across different analyses, FSTAT was used to calculate Weir and Cockerhams (1984) Fis value with 11,000 randomizations and permutation tests Here, D20S206 was not found to deviate from HWeq; interestingly, howe ver, D17S1290 was found to deviate from Hardy Weinberg in the FSTAT analysis. Taken together, we found minimal evidence for null alleles and genetic disequilibrium (only one locus pair out of 55 tests) The most problematic locus, D17S1290, had high homoz ygosity levels and deviated from Hardy Weinberg in only one analysis Micro -Checker suggested that the heterozygote deficiency at this locus is due either to null alleles or to possible scoring errors, and is therefore due to

PAGE 77

77 methodological, not biological reasons. Because it was not consistently found to be out of HWeq, and to increase power of the analysis, this locus was retained in the analysis. Number of Populations R STCALC gave an overall mean RHO of 0.036, which was not found to be significantly different from zero (p=0.142; Table 5 -2). A mean Nm of 11.48 was calculated with confidence intervals that include negative numbers, which indicate that the number of migrants is so high that it cannot be quantified (Goodman 1997). Not all pairwise Rst (RHO) values overlapped zero (7 of 15), although none were found to be significantly different from zero (p>0.05 for all tests; Table 52) The results from STRUCTURE indicate strong support for one population (Figure 5-2). The onepopulation model had the hi ghest likelihood for all 10 runs and the least amount of variation between them Because the 95% confidence intervals of all RHO values did not overlap zero between all comparisons, the genetic distances ( 2) from RSTCALC for each population were analyzed with a Neighbor Joining tree (data not shown). Here two clusters were apparent, one cluster included populations SC, Sebatoli and Mainaro (Cluster1), and a second cluster included LM, K 30, and Dura (Cluster2), showing some support for these two cluster s as populations. Furthermore, the average 2 is less for each group (Cluster1 = 0.62 and Cluster2 =1.3) relative to average 2 across the groups ( 1.46). Therefore, these two populations were then used in a two-population model in MIGRATE. Interestingly, for low values of M, the likelihood score for the one-population model is better than that of a two-population model (Bayes Factor >5) (Figure 53). As the prior distribution of M was increased, our likelihood score increased above that of

PAGE 78

78 the one population model, suggesting that a two-population model is more appropriate for the data. Because M is the number of migrants per generation scaled by mutation rate, if we assume the mutation rate is constant, then, as the prior distribution of M is increased, the number of migrants per generation ( Nm ) is increasing. Our likelihood scores increase above the onepopulation model when the values of Nm increase from 23.8 to 43.5 (Figure 5-3). The highest likelihood score was for the prior distribution of M going from 0 100, with an Nm of 171.47 migrants per generation. Because this level of migration suggests a single population even when the two population model produces a higher likelihood score, all calculations of theta ( ), coalescence time and bottleneck es timates were done considering all of the dataset as one population. Effective population size, Coalescent Point and Bottleneck Our overall theta ( ) value estimated from MIGRATE, integrating across all loci, was 7.03. We used a microsatellite mutation rate of 5 X 104 (Whittaker et al. 2003) to calculate an effective population size ( Ne ) of 3,905 individuals. A ratio of 1:5 effective population size to census size has been suggested (Tempelton, 1998) and if we take that into account, our estimate of th e census size is 19,527 Red Colobus in Kibale National Park. Using the one population mode in MIGRATE we get a coalescence time of 21,708 generations translating to 108,542 years using a generation time of 5 years (Thomas Struhsaker pers com.). Finally no evidence of a bottleneck was found in the program M_P_Val (Garza and Williamson 2001) or BOTTLENECK ( Cornuet and Luikart 1996).

PAGE 79

79 Discussion Since Red Colobus are one of the most endangered primates in the world (IUCN 2010) and the only area where they are not considered to be threatened is Kibale National Park, Uganda where census estimates range from 30,000 80,000 monkeys in the park (Chapman pers com.). Here we examine the number of populations in the park, and based on those results calculate the coalescent point of the population and look for evidence of population bottlenecks, to determine if the Red Colobus in Kibale have gone through a population reduction. Number and size of populations When we used MIGRATE to compare a one population model agains t a twopopulation model we found that, at low migration rates, a one-population model best describes the data, and, at higher migration rates, a two-population model is superior. Our likelihood scores for the twopopulation model increase above those for the one population model when the values of Nm (number of migrants between the populations) are high enough to keep two populations genetically similar (Mills and Alendorf 1996). If this trend were to continue, the highest likelihood score would be that of a two population model with infinite numbers of migrants between the two populations essentially one panmictic population. Therefore, based on the results from STRUCTURE, RST and MIGRATE, we propose that all of the Red Colobus in Kibale constitute one lar ge population. Here, we find the effective population size ( Ne ) of the Red Colobus is around 4,000 monkeys translating to a census estimate of 20,000 monkeys and correlates to a density of 23 to 100 monkeys per kilometer. Interestingly, two previous census estimates of Red Colobus in Kibale range from 30,312 to 80,675; however, it is thought

PAGE 80

80 that the true number lies on the lower end of those two estimates (Chapman pers. com.). Our 95% confidence intervals put our estimate of Red Colobus between 6,204 and 39,400, within and below range of the census. We know that this population of Red Colobus is femalebiased and male bonded, so the contribution to the next generation is not equal between the sexes which would make our estimates of Ne smaller compared to the census population sizes, and may explain why our numbers are on the lower end of the census estimates. With this high density of primates, it is therefore not surprising that there is no genetic structure across the park. History of Red Colobus in Kib ale National Park Although Red Colobus are sensitive to habitat change (Struhsaker 1997), they have an extremely large population in Kibale, which may suggest that there has not been a lot of disturbance over the long term Here we did not find any evidence of a population bottleneck and preliminary runs in BEAST also suggest that this population has been stable over its history (data not shown). Based on our MIGRATE runs, we get a coalescence point of 108,542 years, and this lineage of Red Colobus is thought to have diverged from its sister taxon around 600,000 years ago (Ting 2008). Taken together, these results suggest that there has been a stable population of Red Colobus in Kibale for over 100 thousand years, with no evidence of a population decline. Interestingly there is evidence that KNP has a history of some disturbance. For example, logging has recently been an issue (Chapman et al. 2000), and evidence from the recruitment rate of trees in Kibale suggests that there was a large -scale disturbance sometime in the more distant past (100 3,000 years ago, Taylor et al. 1999, Chapman et al In Prep). S ome possible explanations are drought (Bessems et al 2008), elephant activity, o r human clearing. However, based on our results, these disturbances have not

PAGE 81

81 been at the scale where they affected the Red Colobus, likely due to a large trac t of land being available to them. Because they are so sensitive to habitat change these results suggest that Kibale National Park may have been a refugium for the Red Col obus over the last 100,000 years and therefore has likely been a refugium for other species of animals during that time as well. Finally with a small sampling of COI from lice from the two large troops I find a high level of population structure, even t hough all of these lice have been identified to the same species. Overall average pdistances range from 00.15 (ave = 0.049, N=6) on a single host, in the SC 0 -0.16 (ave= 0.81, N=6) and LM 0 -0.004 (ave = 0.0013). Up to 15% difference on a single host suggests quite a bit of structure exists in the parasite populations, which have all been identified as the same species. Taken together we find that there is no population structure in the Red Colobus in KNP and find some preliminary evidence of population structure in their lice. Because the lice only move between host individuals during direct host -to host contact these results suggest that perhaps the lice are not moving around as much as their hosts. Alternatively, the lice may maintain population str ucture in the absence of host population structure. The next step of this work will be to examine the microsatellite dataset of the lice to look for gene flow between lice to determine the level of population structure in the parasites

PAGE 82

82 Table 5 1. Micr osatellite loci genotyped for Red Colobus ( Piliocolobus tephrosceles ) from Kibale National Park. N number of individuals, Na Number of alleles, Ne effective number of alleles, Ho o bserved heterozygosity, He expected heterozygosity calculated in POPGENE 1.3.1. Fis values were calculated in FSTAT 2.9.3.2 indicate s significance at the bonferron i corrected pvalues (0.0045). ** indicates overall FIS value for all loci calculated in GDA with 95% confidence intervals which overlap zero suggesting overall no Homozygote or Heterzygote excess when all loci are accounted for. No locus was found to deviate from Hardy -Weinberg equilibrium using both the the G -test and the Fis permutation test su ggesting these loci are suitable for other population genet ic analysis. Locus N Na Ne Ho He Fis D14S306 84 6 4.25 0.76 0.77 0.01 D3S1766 82 9 6.31 0.76 0.85 0.11 D2S1399 84 10 6.57 0.91 0.85 0.06 D7S1817 78 9 4.88 0.69 0.80 0.14 D20S206 79 7 5.08 0.79 0.81 0.03 D8S260 82 4 1.91 0.54 0.48 0.12 D8S165 85 3 2.07 0.55 0.52 0.63 D1S207 85 14 6.12 0.74 0.84 0.12 D17S1290 80 4 1.66 0.30 0.40 0.25* C2A 85 7 2.39 0.55 0.59 0.06 D5S1457 82 8 3.62 0.67 0.73 0.08 Average 82 7.36 4.08 0.66 0.69 0.047** ( 0.0024 0.0987)

PAGE 83

83 Table 5 -2. Results from RSTCALC. Each of the 6 populations were compared and RHO estimates calculated. Nm number of migrants per generation included many negative numbers which suggest that the number of migrants is so high that it cannot be quantified (Goodman 1997). indicate where the 95% confidence intervals did not overlap zero for RHO and include negative numbers for Nm. Group 1 Group 2 RHO Nm P value SM LM 0.000 516.56 0.47 SM K 30 0.014 17.89 0.55 SM DURA 0.045* 5.28* 0.07 SM SEB 0.013 19.01 0.23 SM MAIN 0.036* 6.70* 0.13 LM K 30 0.040 5.94 0.97 *LM DURA 0.067* 3.48* 0.06 LM SEB 0.013 19.12 0.33 LM MAIN 0.063* 3.74* 0.10 K 30 DURA 0.052* 4.55* 0.09 *K 30 SEB 0.005 48.09 0.31 K 30 MAIN 0.062 3.76 0.23 *DURA SEB 0.082* 2.81* 0.10 DURA MAIN 0.082* 2.83* 0.21 SEB MAIN 0.049 4.90 0.32 TOTAL PT 0.015 17.42 0.42 TOTAL 95% BS ( 0.008 0.1117) ( 80.48 94.77)

PAGE 84

84 Figure 51. Map of Kibale National Park (KNP). Map shows orientation in Uganda and black points indicate populations of Red Colobus ( Piliocolobus ) that were sampled in this study.

PAGE 85

85 Figure 52. Log likelihood scores for output from Structure. It was run using an admixture model and correlated allele frequencies among populati ons. The number of populations (k) was tested from 1 to 6 for 1 X 106 generations after a burnin of 1 X 105 generations Each k was tested ten times to examine the variation between runs The likelihood scores for each run were examined visually for stat ionarity

PAGE 86

86 Figure 53. Log likelihood scores for the program Migrate. Dotted line indicates likelihood score for one population run for 10 X 106 generations with a burnin of 10%. Two populations were tested using different prior distributions of M (m/ migrants by mutation rate) between the two populations from 0 1, 0-5, 0 -10, 0-15, 025, and 0100 each was run for 10 X 106 generations with 10% burnin and repeated twice to examine variability between runs. Numbers by nodes are number of migrants per generation ( Nm ). This graph indicates that for low values of M the one population model has a higher likelihood score, but for higher values of M the two -population model has a better likelihood score. As M increases the number of migrants between the two p opulations is increasing effectively making the two populations panmictic, and therefore we conclude that here is one population.

PAGE 87

87 CHAPTER 6 CONCLUSIONS As stated at the beginning of this discussion, the evolutionary history of an organism is strongly affected by its interactions with other species in a shared environment (e.g. predator -prey interactions), and these interactions may affect the entire evolu tionary trajectory of a lineage. I have examined two associates of sucking lice, their primary endosymbionts and their mammalian hosts to determine the phylogenetic as well as population genetic patterns of these two associates. Part I: Lice and their Pri mary E ndosymbionts Primary endosymbiotic bacteria (pendosymbionts) are thought to be partially responsible for the incredible diversification of insects. P endosymbionts are found in insects that specialize on nutrient poor diets, where they supplement th eir insect hosts diet with nutrients (Buchner 1965). It has been suggested that the acquisition of a bacterial lineage in insect ancestors has played a key role in the species diversification of some insect groups. Endosymbiotic bacteria cannot live outs ide of the insect hosts and are transmitted from mother to offspring through the eggs. Some insect groups have had the same lineage of endosymbiont for millions of years, such as aphids and their endosymbionts in the genus Buchnera. These long -term associ ations suggest a long coevolutionary history between the insect -bacteria partners. The primary endosymbiont in the human head louse was first discovered over three hundred years ago (Hooke 1664), but was only recently described and formally named (Allen et al., 2007, Fukatsu et al. 2007). This was the first pendosymbiont to be identified from sucking lice. It is thought that all sucking lice have a pendosymbiont that synthesizes vitamins lacking in the louses specialized diet of mammalian blood

PAGE 88

88 (Buchne r, 1965). I hypothesized that like aphids, all sucking lice would have the same genus of endosymbiont. I found, however, that there have been as many as 14 different genera of endosymbionts in sucking lice (Allen et. al, 2009, Allen et al. In Prep. ). Whi le multiple independent origins of endosymbiotic bacteria is one possible explanation for this pattern, because lice cannot survive without endosymbionts (Buchner 1965) it is more likely that the ancestor of all lice acquired an endosymbiont, and among some louse groups, the endosymbiont has been out -competed and replaced by new bacterial lineages over time. Bacterial endosymbionts are weakened over time by a high rate of irreversible and harmful mutations due to their small population sizes and lack of re combination (Moran, 1996). It is thought that frequent mutations will eventually degrade the pendosymbiont genome and diminish the reproductive health of the bacteria (Moran, 1996) enabling other bacteria to outcompete the pendosymbionts and replace them Interestingly only a few groups of insects have shown pendosymbiont replacement similar to that found in sucking lice (Allen et. al, 2009). Further work has verified that the rate of molecular evolution for the endosymbiont in human sucking lice is an order of magnitude faster than most primary endosymbionts suggesting that they are accumulating harmful mutations at a faster rate than other pendosymbionts and likely being outcompeted and replaced faster as well ( Allen et al., 2009). The cause of this unusual mutation rate is unknown, although it is interesting that the mutation rate of the louse itself is high relative to other insects (Yoshizawa and Johnson 2003). Part II: Sucking Lice and their Mammalian H osts Organisms involved in obligate relationships are affected on both a long-term species level time scale and short term population-level time scale (Clayton and

PAGE 89

89 Johnson 2003). For example, the size of a parasite population is strictly tied to the social str ucture, dispersal ability and population size of its host In the case of lice and mammals, lice primarily move between hosts during direct host -to host contact. Therefore, louse population dynamics are particularly constrained by the social structure of t heir hosts (e.g. which hosts come into contact and how often they come into contact). The effect of mammalian host population dynamics on louse population dynamics remains poorly understood. A louse host, the Red Colobus monkey, breeds and forages in stab le social groups called troops. I have determined that there is a high level of migration and gene flow across Kibale National Park, the number of host individuals is large in particular for primates and is around 20,000 4 0,000 individuals, providing a large host population for their sucking ice, as well as opportunities for movement across the park. Interestingly, with a small sampling of COI from lice from the two large troops I find a high level of population structure, even though all of these lic e have been identified to the same species. Overall average pdistances range from 00.15 (ave = 0.049, N=6) on a single host, in the SC 0 0.16 (ave= 0.81, N=6) and LM 0 0.004 (ave = 0.0013). Up to 15% difference on a single host suggests quite a bit of structure exists in the parasite populations, which have all been identified as the same species. The next step of this work will be to examine the microsatellite dataset of the lice to look for gene flow between lice on different hosts and determine how m any populations of parasites exist on one large population of hosts. This work will provide insight into long-term changes in louse population structure, and we may in fact find that there is genetic structure in the parasite populations in the

PAGE 90

90 absence o f host population structure. This would then suggest that the lice are not able to move as easily between host populations, and may be constrained by the amount of physical interactions between host troops. My future goal is to examine obligate relationshi ps on both evolutionary and population level time scales and examine how population level processes affect long -term evolutionary patterns.

PAGE 91

91 LIST OF REFERENCES Adams, T.S. 1999. Hematophagy and hormore release. Annals of the Entomological Socitey of Americ a 92:1 13. Allen, J M D.L. Reed, M.A. Perott i, and H.R. Braig. 2007. Evolutionary relationships of Candidatus Riesia spp.," endosymbiotic E nterobacteriaceae living within hematophagous primate lice. Applied and Environmental Microbiology 73 : 1659 1664. Andersson S. G E. and C.G. Kurland. 1998. Reductive evolution of resident genomes. Trends in Microbiology 6 : 263268. Arandjelovic M ., K. Guschanski and G. Schubert 2009 Two -step multiplex polymerase chain reaction improves the speed and accuracy of g enotyping using DNA from noninvasive and museum samples. Molecular Ecology Resources 9 : 28 36. Atwood. K. C L.K. Schneider, and F.J. Ryan. 1951. Selective m echanisms i n b acteria. Cold Spring Harbor Sympo sia on Quantitative Biology 16:345355. Aschner, M. 1 934. Studies on the symbiosis of the body louse. I. Elimination of the symbionts by centrifugation of the eggs. Parasitology 26 :309314. Aschner, M., and E. Ries. 1933. Das verhalten der kleiderlaus beim ausschaltender Zoomorphology 26 :529. Askoy S. A.A. Pourhosseini and A. Chow. 1995. Mycetome endosymbionts of tsetse flies constitute a distinct lineage related to Enterobacteriaceae. Insect Mol ecular Biol ogy 4 : 15 -22. Bandi C M. Sironi G. Damiani L. Magrassi, and C.A. Nalepa. 1995. The establishment of i ntracellular symbiosis i n an ancestor of cockroaches and t ermites. Proceedings of the Royal Society of London Series B 259: 293299. Beerli, P. 2006. Comparison of Bayesian and maximu m likelihood inference on population genetic parameters. Bioinforma t ics 22 :341345. Beerli, P. 2008. MIGRATE version 3.0 a maximum likelihood and Bayesian estimator of gene flow using the coalescent. Beerli, P., M. Palczewski 2010. Unified framework to evaluate panmixia and migration direction amond multiple sampling locations. Genetics10.1534/genetics.109.112532.

PAGE 92

92 Bergstrom C T and J. Pritchard. 1998. Germline bottlenecks and the evolutionary maintenance of mitoc hondrial genomes. Genetics 149:2135 2146. Bessems, I. D. Verschuren, J.M. Russell, J. Has, F. Mees, B.F. Cumming. 2008. Paleolimnological evidence for widespread late 18th century drought across equatorial East Africa. Palaeogeography, Palaeoclimatology, Palaeoecology 259:107 -120. Beuning, K.R., K. Kelts, J. Russell, and B.B. Wolfe. 2002. Reassessment of Lake Victoria-Upper Nile River paleohydrology from oxygen isotope records of lake sediment cellulose. Geology. 30 :559562. Boutin -Ganache I., M. Rapo so M. Raymond C.F. Deschepper 2001. M13 -tailed primers improve the readability and usability of micros atellite analyses performed with two different allele -siz ing methods. Biotechniques 31 :2428. Braig H R B.D. Turner and M.A. Perotti. 2009. Symbio tic Rickettsia. In: Bourtzis K, Miller TA, editors. Insect Symbiosis 3. Boca Rat on: Taylor and Francis. pp. 221252. Brynnel, E. U., C. G. Kurland, N. A. Moran, and S. G. Andersson. 1998. Evolutionary rates for tuf genes in endosymbionts of aphids. Molecular Biology and Evolution 15:574582. Buchner, P. 1965. Endosymbiosis of animals with plant micro organisms. Interscience, New York. Busse, 1977. Chimpanzee predation as a possible factor in the evolution of red colobus monkey social organization. Evolution 31 :907-911. Butcher D 1995. Mullers Ratchet, Epistasis and Muta tion Effects. Genetics 141 :431437. Buxton, P. A. 1946. The Louse, 1st ed. Williams & Wilkins Co., Baltimore, MD. Chapman, C.A., L.J. Chapman, R.W. Wrangham, G. IsabiryeBasuta, K. Ben -David. 1997. Spatial and temporal variability in the structure of a tropical forest. African Journ al of Ecology 35 : 287302. Chapman, C.A., S.R. Balcomb, T. Gillespie, J. Skorupa, T.T. Struhsaker T.T. 2000. Longterm effects of logging on African primate communities: A 28 year comparison from Kibale National Park, Uganda. Conservation Biology 14 : 207-217. Canbac k B I. Tamas and S.G.E. Andersson. 2004. A phylogenomic study of endosymbiotic bacteria. Molecular Biology and Evolution 21 : 11101122.

PAGE 93

93 Chapman, C.A. T. Webb, R. Fronstin, M.D. Wasserman, and A.M. Santamaria. 2005. Assessing dietary protein of colobus monkeys through feacal sample analysis: A tool to evaluate habitat quality. African Journal of Ecology 43:276278. Charlesworth D M.T. Morgan, and B. Charlesworth. 1993. Mutation accumulationin finite outbreeding and inbreeding populations. Genetic Res earch 61 : 3956. Clark M A. N.A. Moran, and P. Baumann. 1999. Sequence evolution in bacterial endosymbionts having extreme base compositions. Molecular Biology and Evolution 16 :1586 -1598. Cornuet, J.M., and G. Luikart 1996. Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144:20012014. Corsaro D D. Venditti M. Padula and M. Valassina 1999. Intracellular life. Critical Rev iews in Microbiology 25 : 3979. Cunningham, C. W., H. Z hu, and D. M. Hillis. 1998. Best -fit maximum likelihoodmodels for phylogenetic inference: empirical tests with known phylogenies. Evolution 52:978987. Dale C S.A. Young D.T. Haydon S.C. Welburn. 2001. The insect endosymbiont Sodalis glossinidius util izes a type III secretion system for cell invasion. Proc eedings of the Nat ional Acad emy of Sci ences USA 98 : 1883 -1888. Dale, C., M. Beeton, C. Harbison, T. Jones, and M. Pontes. 2006. Isolation, pureculture, and characterization of Candidatus Arsenophonus arthropodicus," anintracellular secondary endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis. Applied and Environmental Microbiology 72:2997 -3004. Dedeine, F. C. M. Bandi, M. Boultreau, and L.H. Kramer 2003. Insights into Wolbachia obligatory symbiosis. In: Bourtzis K, Mille r T, editors. Insect Symbiosis CRC Press: LLC. 267 -282. Delmotte F C. Rispe J. Schaber F.J. Silva and A. Moya. 2006. Tempo and mode of early gene loss in endosymbiotic bacteria from insects. B MC Biology. 6 :5 6. Degnan P H A.B. Lazarus C.D. Brock and J.J. Wernegreen. 2004. Host -symbiont stability and fast evolutionary rates in an ant -bacteri um association: Cospeciation of Camponotus species and their endosymbionts, Candidatus Blochmannia. Sys tematic Biol ogy 53 : 95110. Douglas, A. E. 1989. Mycetocyte symbiosis in insects. Biological Reviews, Cambrige Philosophical Society 64 :40934.

PAGE 94

94 Durden, L. A., and G. G. Musser. 1994. The sucking lice (Insecta: Anoplura) of theworld: A taxonomic checklist with records of mammalian hosts and geographical distributions. Bulletin of the American Museum of Natural History 218:90. Eberle, M. W., and D. L. McLean. 1983. Observation of symbiote migration in Human body lice with scanning and transmission electron microscopy. C anadian Journal of Microbiol. 29 :755-762. Eckburg, P.B., E.M. Bik, C.N. Bernstein, E. Purdom, L. Dethlefsen, M. Sargent, S.R. Gil, K.E. Nelson and D.A. Relman. 2005. Diversity of the human intestinal microbial flora. Science 308:16351638. Felsenstein J 1974. Evolutionary Advantag e of Recombination. Genetics 78 :737756. Fuchs, B. M., G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann.1998. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16SrRNA for fluorescently label ed oligonucleotide probes. Applied and Environmental Microbiology 64 :4973 -4982. Fukatsu T., T. Hosokawa, R. Koga, N. Nikoh, T. Kato, S. Hayama, H. Takefushi and I. Tanaka. 2009. Intestinal endocelluar symbiotic bacterium of the macaque louse Pedicinus obtusus: Distinct endosymbiont origins in anthropoid primate lice and the old world mokey louse. Applied and Environmental Microbiology 75:3796 3799. Garza, J.C. and E.G. Williamson 2001. Detection of reduction in population size using data from microsatellite loci. Molecular Ecology 10:305-318. Geiger, A., S. Ravel, R. Frutos, and G. Cuny. 2005. Sodalis glossinidius (Enterobacteriaceae) and vectorial competence of Glossina palpalis gambiensis and Glossina morsitans morsitans for Trypanosoma congolense savannah type. Current Microbiology 51 :35. Gil, R A. Latorre, and A. Moya. 2004. Bacterial endosymbionts of insects: insights from comparative genomics. Environmental Microbiology 6 : 1109 -1122. Goldberg,T.L., D.M. Sintasath, C.A. Chapman, K.M. Cameron, W.B. Karesh, S. Tang, N.D. Wolfe, I.B. Rwego, N. Ting, and W.M. Switzer. Co infection of Ugandan red colobus ( Procolobus [Piliocolobus] rufomitratus tephrosceles ) with novel, divergent delta ,lenti and spumaretroviruses. Journal of Virology (In Press). Goldb erger, J., and J. F. Anderson. 1912. The transmissions of typhus fever, with special reference to transmission by the head louse ( Pediculus capitis ). Public Health Reports 27 :297 -308. Goldman, N J.P. Anderson A.G. Rodrigo. 2000. Likel ihoodbased tests oftopologies i n phylogenetics. Systematic Biology 49:652670.

PAGE 95

95 Goodman, S.J. 1997. RST CALC2.2: A collection of computer programs for calculating unbiased estimates of genetic differentiation and determining their significance for microsatellite data. Molecular Ecology 6 :881885. Goossens, B., L. Chikhi, S.S. Utami, J. Ruiter, and M.W. Bruford. 2000. A multi samples, multi extracts approach for microsatellite analysis of faecal samples in an arboreal ape. Conservation Genetics 1:157-162. Goudet, J. 2002. FSTA T Software, v. 2.9.3.2. http://www2.unil.ch/popgen/softwares/fstat.htm Haldane J B.S. 1937. The effect of variation on fitness. Am erican Nat uralist 71 :337349. Harris, T.R., D. Caillaud, C.A. Chapman, and L. Vigiland, 2009. Neither genetic nor observational data alone are sufficient for understanding sex biased dispersal in a social group-living species. Molecular Ecology 18:1777 -1790. Hein, J., M. Schierup, C. Wiuf. 2004. Sequence Variation, Genealogies and Evolution Oxford University Press, New York, New York. Hooke, R. 1664. Micrographia or some physiological description of minute bodies made by magnifying glasses with observations and iquiries thereupon. Council of the Royal Society of London for Improving Natural Knowledge, London, United Kingdom. Hypsa V., and J. Krizek. 2007. Molecular evidence for polyphyletic origin of the primary symbionts of sucking lice (Phthiraptera, Anoplura). Micro bial Ecol ology 54 : 242251. Huelse nbeck J P and B. Rannala. 1997. Phylogenet ic methods come of age:Testing hypotheses in an evolutionary context. Science 276:227 -232. Huelsenbeck, J. P., and F. Ronquist. 2001. MR -BAYES: Bayesian inference of Phylogeny. Bioinformatics 17 :754-755. Hudson, R.R. 1990. Gene genealogies and the coalescent process. In: Oxford Surveys in Evolutionary Biology Oxford University Press United Kingdom Hurst, L., and G. Mcvean. 1996. Evolutionary genetics. and scandalous symbionts.Nature 381:742742. IUCN 2010. IUCN Red List of Threatened Species. Version 2010.1. Ihlen, P.G., and S. Ekman. 2002. Outline of phylogeny and character evolution inRhizocarpon (Rhizocarpaceae, lichenized Ascomycota) based on nuclear ITS and mitochondrial SSU ribosomal DNA sequences. Biologic al Journal of the Linnean Society 77 :535546.

PAGE 96

96 Johnson, K. P., R. H. Cruickshank, R. J. Adams, V. S. Smith, R. D. M. Page, and D. H. Clayton. 2003. Dramatically elevated rate of mitochondrial substitution in lice (Insecta: Phthiraptera). Molecular Phylogenetics and Evolution 26:231-242. Khuner, M. 2008. Coalescent Geneology samplers: windows into population history. Trends in Ecology and Evolution 24 :86 -93. Kishino H and M. Hasegawa. 1989. Evalu ation of the maximum -likelihood estimate of the evolutionary t re e t opologies from DNA -sequence data, and the branchi ng rrder In Hominoidea. Molecular Ecology 29: 170179. Kittler, R., M. Kayser, and M. Stoneking. 2003. Molecular evolution of Pediculus humanus and the origin of clothing. Current Biology 13:1414 -1417. Klasson L S.G.E. Andersson. 2004. Evoluti on of minimal -gene -sets in host dependent bacteria. Trends in Microbiol ogy 12 : 37 43. Kondrashov, A.S. 1994. Mullers ratchet under epistatic s election. Genetics 136: 14691473. Kyei -Poku, G. K., D. D. Colwell, P. Coghlin, B. Benkel, and K. D. Floate. 2005. On the ubiquity and phylogeny of Wolbachia in lice. Molecular Ecology 14 :285 -294. Lefvre, C H. Charles A. Vallier B. Delobel and B. Farrell B. 2004. Endosymbiont phylogene sis in the Dryophthoridae weevils: Evidence for bacterial replacement. Molecular Biology and Evolution 21 : 965 -973. Lehane, M.J. 2005. The biology of blood feeding insects, 2nd ed. Cambridge University Press, Cambridge, United Kingdom. Leo, N. P., and S. C. Barker. 2005. Unravelling the evolution of the head lice andbody lice of humans. Parasitological Research 98 :44 47. Lerat E, V. Daubin and N.A. Moran. 2003. From gene trees to organismal phylogenyin prokaryotes: The case of the gamma-proteobacteria. P L o S Biology 1 : 101109. Light, J.E., V.S. Smith, J.M Allen, L.A. Durden, and D.L. Reed. 2010. Evolutionary history of mammalian sucking lice (Pthiraptera: Anoplura). In Review : BMC Biology. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, Yadhukumar, A. Buchner, T. Lai, S. Steppi, G. Jobb, W. Forster, I. Brettske, S. Gerber, A. W.Ginhart, O. Gross, S.Grumann, S. Hermann, R. Jost, A. Konig, T. Liss, R.Lussmann, M. May, B. Nonhoff, B.Re ichel, R. Strehlow, A. Stamatakis, N.Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode, and K. H. Schleifer. 2004. ARB: a software environment for sequence data. Nucleic Acids Research 32 :13631371.

PAGE 97

97 Lutzoni F and M., Pagel. 1997. Accelerated evolution as a consequence of transitions to mutualism. Proceedings of the National Academy of Sciences, USA 94 : 11422 11427. Lynch M ., and W. Gabriel. 1990. Mutation load and the survival of s mall populations. Evolution 44 :1725 -1737. Maddison, W P. and D.R. Maddi son. 2000. MacC lade: Analysis of phylogeny and character e volution. Sunderland, Massachusetts: Sinauer Associates. Mittermeier, R. A., J. Ratsimbazafy, A.B. Rylands, L. Williamson, J.F. Oates, D. Mbora, J.U. Ganzhorn, E. Rodrguez Luna, E. Palacios, E.W. H eymann, M.C.M. Kierulff, L. Yongcheng, J. Supriatna, C. Roos, S. Walker, and J.M. Aguiar. 2007. Primates in Peril: The Worlds 25 Most Endangered Primates, 2006 2008. Primate Conservation 22 :1 -40. Mills, S.,L., and F.W. Allendorf. 1996. The onemigrant per generation rule in conservation and management. Conservation Biology 10 :1509 -1518. McCutcheon, J P. and N.A. Moran. 2007. Parallel genomic evolution and metabolic interdependence in an ancient s ymbiosis. Proceedings of the National Academy of Sciences, USA 104:1939219397. Moran N A. M.A. Munson P. Baumann, and H. Ishikawa. 1993. A molecular clock in endosymbiotic bacteria is calibrated using the insect hosts. Proceedings of the Royal Society of London Series B 253 : 167171. Moran N and P. Baumann. 1994. Phylogenetics of cytoplasmically i nherited microorganisms of ar thropods. Trends in Ecology and Evolution 9 : 15 20. Moran, N. A., C. D. von Dohlen, and P. Baumann. 1995. Faster evolutionary rates in endosymbiotic bacteria than in cospeciating insect hosts. Journal of Molecular Ecology 41 :727-731. Moran N A. 1996. Accelerated evolution and Muller's rachet in endosymbiotic bacteria. Proceedings of the National Academy of Sciences, USA 93 : 2873 2878 Moran N A. and P. Baumann. 2000. Bacterial en dosymbionts in animals. Current Op inions in Microbiology 3 : 270-275. Moran N A. and J.J. Wernegreen. 2000. Lifestyle evolution in symbiotic bacteria: Insights from genomics. Trends Ecol ology and Ev ol ution 15 : 321326. Moran N A. P. Tran, and N.M. Gerardo. 2005. Symbio sis and insect diversification: An ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes. Applied and Environmental Microbiology 71 : 8802 -8810.

PAGE 98

98 Moran, N. A., and H. E. Dunbar. 2006. Sexual acquisition of beneficial symbionts in aphids. Proceedings of the National Academy of Sciences, USA 103:1280312806. Moran N A. 2007. Symbiosis as an adaptive process and a source of phenotypic complexity. Proceedings of the National Academy of Sciences, USA 104: 8627 8633. Muller H J 1964. The relation of recombination to mutational advance. Mut ation Research 1 :2 9. Murray, R. G. E., and K. H. Schleifer. 1994. Taxonomic Notes a proposal for recording th e properties of Pputative taxa of prokaryotes. International Journal of Systematic Bacteriology 44 :174-176. Murray, R. G. E., and E. Stackebrandt. 1995. Taxonomic Note Implementation of the provisional status Candidatus for incompletely described prokary otes. International Journal of Systematic Bacteriology 45 :186 -187. Nakabachi A. A. Yamashita, H. Toh, H. Ishikawa, and H.E. Dunbar. 2 006. The 160 kilobase genome of the bacterial endosymbiont Carsonella Science 314:267 -267. Nsubuga, A.M., M.M. Robbins, A.D. Roeder. 2004. Factors affecting the amount of genomic DNA extracted from ape faeces and the identification of an improved sample storage method. Molecular Ecology 13: 2089 2094 Oates, J.F. 1987. Food distribution and foraging behavior. In Primate societies, ed. B.B. Smuts, D.L. Cheney, R.M. Seyfarth, R.W. Wrangam and T.T. Struhsaker, 169209. Chicago: University of Chicago Press. Oates, J.F. and A.G. Davies. 1994. Conclusions: Past, present and future of the colobines. In Colobine monkeys: Their ec ology, behavior and evolution, ed. A.G. Davies and Oates, J.F. 4573. Cambridge: Cambridge University Press. Oates, J.F. 1996b. Habitat alteration, hunting and the conservation of folivorous primates in African forests. Australian Journal of Ecology. 21 :1 -9. Oates, J.F., M. Abedi Larty, S.W. McGraw, T.T. Struhsaker, and G.H. Whitesides. 2000. Conservation Biology 14 :1526 -1532. Ochman, H S. Elwyn and N.A. Moran. 1999. Calibrating bacterial evolution. Proc eedings of the Nat ional Acad ademy of Sci ences. USA 96 : 12638 12643. Ochman, H and N.A. Moran. 2001. Genes lost and gene s found: Evolution of bacterial pathogene sis and symbiosis. Science 292: 1096 1098.

PAGE 99

99 O'Fallon, B. 2008. Population structure, levels of selection, and the evolution of intrace llular symbionts. Evolution 62 :361 -373. Perez -Brocal V R., Gil S. Ramos A. Lamelas and M. Postigo. 2006. A smallmicrobial genome: The end of a long symbi otic relationship? Science 314 : 312313. Perotti, M. A., S. S. Catala, A. D. Ormeno, M. Zelazowska, S. M. Bilinski, and H.R. Braig. 2004. The sex ratio distortion in the human head louse is conserved overtime. BMC Genetics 5 :10. Perotti, M.A., H.K. Clarke, B.D. Turner and H.R. Braig. 2006. Rickettsia as obligate and mycetomic bacteria Federation of American Societies for Experimental Biology 20:2372 -2374. Perotti, M. A., J. M. Allen, D. L. Reed, and H. R. Braig. 2007. Host-symbiont interactions of the primary endosymbiont of human head and body li ce. Federation of American Societies for Experimental Biology 21:1058 -1066. Perotti M A ., D.L. Reed and H.R. Braig. 2 009. Endosymbionts of l ice. In: Bourtzis K, Miller TA, editors. Insect Endosymbionts 3. Boca Raton: Taylor andFrancis. pp. 205-220. Poin ar G O 1992. Life in Amber: Stan. Univ. Press Polak, M. 1996. Ectoparasitic effects on host survival and reproduction: The Drosophila Macrocheles Association. Ecology 77: 13791389. Posada, D., and K. A. Crandall. 1998. MODELTEST: Testing the model of D NA substitution. Bioinformatics 14 :817-818. Posada, D and T.R. Buckley. 2004. Model s election and model averaging in phylogenetics: Advantages of akaike i nformation cr iterion and bayesian approaches over likelihood ratio tests. Systematic Biology 53 : 793 -808. Pritchard, J.K. M. Stephens, and P. Donnelly. 2000. Inference of Population Structure Using Multilocus Genotype Data. Genetics 155:945-959. Puchta, O. 1955. Experimentelle untersuchungen uber die symbiose der kleiderlaus Pediculus vestimenti Burm eister. Z Parasitenkd 17 :1. Reed, D. L., and M. S. Hafner. 2002. Phylogenetic analysis of bacterial communities associated with ectoparasitic chewing lice of pocket gophers: A culture independent approach. Microbial Ecology 44 :7893.

PAGE 100

100 Reed, D. L., V. S. Sm ith, S. L. Hammond, A. R. Rogers, and D. H. Clayton. 2004. Genetic analysis of lice supports direct contact between modern and archaichumans. PLoS Biology 2 : 1972 1983 Reed DL, J.E. Light J.M. Allen and J.J. Kirchman. 2007. P air of lice lost or Parasite s regained: the evolutionary history of anthropoid primate lice. B MC Biology 5 : 1 11 Ries, E. 1931. Die symbiose der lause und federlinge. Zeitschrift fuer Morphologie und Oekologie der Tiere 20 :233367. Rice, W.R. 1989. Analyzing tables of statistical tes ts. Evolution 43 :223-225. Robinson, D., N. Leo, P. Prociv, and S. C. Barker. 2003. Potential role of headlice, Pediculus humanus capitis as vectors of Rickettsia prowazekii Parasitological Research 90:209. Saito, T., and S. Bjornson. 2006. Horizontal transmission of a microsporidium fromThe convergent lady beetle, Hippodamia convergens Guerin-Meneville (Coleoptera: Coccinellidae), to three coccinellid species of Nova Scotia. Biological Control 39:427. Sander son, M J 2003. r8s: inferring absolute r ates of molecular evolution and divergence times in the absence of a mole cular clock. Bioinformatics 19: 301302. Sasaki -Fukatsu, K., R. Koga, N. Nikoh, K. Yoshizawa, S. Kasai, M. Mihara, M.Kobayashi, T. Tomita, and T. Fukatsu. 2006. Symbiotic bacteria associated with Stomach Discs of Human Lice. Applied and Environmental Microbiology 72:7349 7352. Schuelke, M. 2000. An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology 18 :233-234. Shimodaira. H and M. Hasegawa. 1999. Multiple comp arisons of log -likelihoods with applications to phylogenetic inference. Molecular Biology and Evolution 16 :1114 1116. Southworth, J., H. Hartter, M.W. Binford, A. Goldman, C.A. Chapman, L.J. Chapman, P. O meja, and E. Binford 2010. Parks, people and pixels: evaluating landscape effects of and East Afrian national park on its surroundings. Tropical Conservation Science 3 :122142. Stewart, C.S., H.J. Fligt, M.P. Bryant. 1997. The rumen bacteria In: The rumen microbial ecosystem. P.N Hobson and C.S. Stewart (eds.) Blackie Academic and Professional. Struhsaker, T.T. 1975. The red colobus monkey. University of Chicago Press.

PAGE 101

101 Struhsaker, T.T. and M. Leakey. 1990. Prey selectivity by crowned hawk eagles on monkeys in Kibale Forest, Uganda. Behavioral Ecology and Sociobiology 26:435443. Struhsaer, T.T. 1997. Ecology of an African rain forest: logging in Kibale and the conflict between conservation and exploitation. University of Florida Press, Gainesville. Swofford D. L. 2002. PAUP*: Phylogenetic analysis using parsimony (*and othermethods). 4.0 b10 ed. Sinauer, Sunderland, MA. Tajima, F. 1983. Evolutionary relationship of DNA sequences in finite populations. Genetics 105 : 437460. Tamas I L. Klasson, B. Canback A.K. Naslund A.S. Eriksson. 2002. 50 millionYears of genomic stasis in endos ymbiotic bacteria. Science 296: 23762379. Takiya D M P.L. Tran, C.H. Dietrich and N.A. Moran. 2006. Co-cladogenesis spanning three phyla: leafhoppers (Insecta: Hemipter a: Ci cadellidae) and their dual bacterial symbionts. Mol ecular Ecol ogy 15 : 4175 4191. Taylor, D., R.A. Marchant, P. Robertshaw. 1999. A sediment based history of medium altitude forest in central Africa: A record from Kabata Swamp, Ndale volcanic field, Uganda. Journal of Ecology 87 :303-315. Tempelton, A. 1998. Human Races: A genetic and evolutionary perspective. American Anthropologist 100:632-650. Ting, N. 2008. Mitochondrial relationships and divergence dates of the African colobines: evidence of Miocene origins for the living colobus monkeys. Journal of Human Evolution 55:312325. Thompson J D T.J. Gibson, F. Plewniak F. Jeanmougin, and D.G. Higgins.1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by qua lity analysis tools. Nuc leic Acids Research 25: 4876 -4882. Thao, M L N.A. Moran P. Abbot E.B. Brennan and D.H. Burckhardt. 2000. Cospeciation of psyllids and their primary prokary otic endosymbionts. Applied and Environmental Microbiology 66 : 28982905. Thao, M L and P. Baumann. 2004. Evolutionary relat ionships of primary prokaryotic endosymbionts of whiteflies and their hosts. Applied and Environmental Microbiology 70 :3401 -3406. Hypsa, V, and J. Kirizek. 2007. Molecular evidence for the polyphyletic origin of the primary symbionts of sucking Lice (Pthiraptera: Anoplura). Microbial Ecology 54:242251.

PAGE 102

102 van Ham R J. Kamerbeek C. Palacios C. Rausell and F. Abascal. 2003. Reducti ve genome evolution in Buchnera aphidicola. Proceedings of the National Academy of Sciences, USA 100:581586. van Oosterhout, C., W.F. Hutchinson, D.P. Wills, and P. Shipley. 2004. MICRO CHECKER: Software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes 4 :535 -538. Wallace B. 1987. 50 Years Of Genetic L oad. Jounal of Heredety 78 : 134-142. Watterson, G.A. 1975. On the number of segregating sites in genetical models without recombination. Theoretical Population Biology 7 :256-276. Weir, B.S. and C.C. Cockerham. 1984. Estimating F -statistics for the analysis of poulation structure. Evolution 38:1358 -1370. Wernegreen, J. J., and N. A. Moran. 1999. Evidence for genetic drift in endosymbionts (Buchnera): analyses of protein-coding genes. Molecular Biology and Evolution 16:83 -97. Whittaker, J.C., R.M. Harbord, N. Boxall, I. Mackay, G. Dawson, and R.M. Sibly. 2003. Likelihoodbased estimation of microsatellite mutation rates. Genetics 164:781787. Wing, L.D., I.O. Buss. 1970. Elephants and forests. Wildlife monographs 19. Woolfit, M., and L. Bromham. 2003. Increased rates of sequence evolution in endosymbiotic bacteria and fungi with small effective population sizes. Molecular Biology and Evolution 20 :15451555. Wu D S.V. Daugherty S.E. Van Aken, G.H. Pai and K.L. Watkins. 2006. M etabolic complementarity and genomics of the dual b acter ial symbiosis of sharpshooters. PLoS Biology 4 :e188. Yeh, F.C., and T.J.B. Boyle. 1997. Population genetic analysis of co dominat markers and quantitative traits. Belgian Journal of Botany 129:157. Yong, Z., P. E. Fournier, E. Rydkina, and D. Raoult. 2003. The grographical segregation of human lice preceded that of Pediculus humanus capitis and Pediculus humanus humanus Canadian Research on Biology 326:565574. Yoshizawa, K. and K.P. Johnson. 2003. Phylogenetic position of Pthiraptera (Insecta: Paraneoptera) and elevated rate of evolution in mitochondrial 12S and 16S rDNA. Molecular Phylogenetics and Evolution 29 :102-114.

PAGE 103

1 03 BIOGRAPHICAL SKETCH Julia M. Allen was born in Provo, Utah in 1978. She grew up in Sandy, Utah and attended Skyline High School where she graduated in 1997. She then attended Southern Utah University for a f ew years before moving to the University of Utah. Here, she worked with Dr. Jim Ehlringer and Dr. Joan Coltrain studying the stale isotope chemistry of the large mammals of the LaBrea Tar Pits. She graduated from the University of Utah in 2003 with a B.S c. in Biology and a chemistry minor. She worked for a year with Dr. Denise Dearing studying hantavirus transmission of desert rodents in central Utah. She then started graduate school at the University of Florida in the Zoology Department in 2004. Here she studied evoluionary biology of sucking lice and their obligate partners.