University of Mississippi eGrove Electronic Theses and Dissertations Graduate School 2017 The Reptile Gut Microbiome: Its Role In Host Evolution And Community Assembly Timothy Colston Colston University of Mississippi Follow this and additional works at: https://egrove.olemiss.edu/etd Part of the Biology Commons Recommended Citation Colston, Timothy Colston, "The Reptile Gut Microbiome: Its Role In Host Evolution And Community Assembly" (2017). Electronic Theses and Dissertations. 387. https://egrove.olemiss.edu/etd/387 This Dissertation is brought to you for free and open access by the Graduate School at eGrove. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of eGrove. For more information, please contact [email protected]. THE REPTILE GUT MICROBIOME: ITS ROLE IN HOST EVOLUTION AND COMMUNITY ASSEMBLY A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MISSISSIPPI BY TIMOTHY JOHN COLSTON, MSC. IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY CONFERRED BY THE DEPARTMENT OF BIOLOGY THE UNIVERSITY OF MISSISSIPPI MAY 2017 © Timothy John Colston 2017 ALL RIGHTS RESERVED ABSTRACT I characterize the endogenous (gut) microbiome of Squamate reptiles, with a particular focus on the suborder Serpentes, and investigate the influence of the microbiome on host evolution and community assembly using samples I collected across three continents in the New and Old World. I developed novel methods for sampling the microbiomes of reptiles and summarized the current literature on non-mammalian gut microbiomes. In addition to establishing a standardized method of collecting and characterizing reptile microbiomes I made novel contributions to the future direction of the burgeoning field of host-associated microbiome research. Through persistent and rigorous fieldwork I amassed the largest dataset of non- mammalian vertebrate microbiomes in existence. By incorporating emergent next generation sequencing technologies and combining cross-disciplinary methods from the fields of phylogenetics and community ecology, I show that the core reptile gut microbiome is comprised of members of the Proteobacteria, Firmicutes and Bacteroidetes; and that reptile gut bacterial communities are more similar to those of birds than mammals—a previously untested hypothesis. I show that the reptile gut microbiome is strongly influenced by host phylogeny and several ecological traits including parity and foraging mode. My analyses reveal that the composition of the reptile gut microbiome is influenced by who (phylogeny), where (geographic locality) a host organism is and how she lives. Broadly, this work reflects that the microbiome of reptiles is both a phylogenetic and ecological trait that is influenced by selection and that host- associated microbiomes harbor a wealth of natural history information waiting to be explored. ii DEDICATION I dedicate this work to my wife, Dr. Katya Colston, and eldest daughter, Ms. Maddison Colston, for their unceasing love, dedicated and unwavering support and encouragement during the pursuit of my academic goals and lengthy (often years) of absence abroad to collect data. iii ACKNOWLEDGEMENTS I would like to thank all those who facilitated my fieldwork, in no particular order, for without them this work would not have been possible. Guy and Yvonne Levene, The Frankfurt Zoological Society, Matt Thornton, Ramona Arechega, James N’Dungu, Sissay Taye, Teddy Tedila, Zenebe Beyene, Will Norton, Abebe Getahun, Thachuor Biel, the Ethiopian Wildlife Conservation Authority, Oromia Forestry Service, faculty and staff in the Zoology Department at Addis Ababa University and the public affairs office of the U.S. Embassy in Addis Ababa were all instrumental in some way to my research in Ethiopia. Kathy Slater and the teams of volunteers and students of Operation Wallacea Mexico as well as Dr. Lenin Rodriguez made my work in Mexico possible. World Wildlife Fund Guyanas, Global Wildlife Conservation and the Coypu Foundation supported my fieldwork in Guyana. Frederico G. R. França, Gabriel Costa, Daniel Mesquita, Guarino Colli, Gustavo Vieira, Hussam Zaher, Felipe Grazziotin and the herpetology labs at the federal universities of Brasilia and João Pessoa provided me with assistance, samples and support while in Brazil. Kenny Wray and members of the Rokyta lab at Florida State University assisted with sample collection in Florida. Lastly I would like to thank (again no particular order) Nathan Lujan, Stuart Nielsen, Megan Smith, Andrew Snyder, my committee, and all of the members of the Noonan Lab at Ole Miss during my tenure for their assistance and friendship. Support for this dissertation came from a variety of sources including a J. William Fulbright Fellowship, US National Science Foundation, American Museum of Natural History, Society for Integrative and Comparative Biology, Percy Sladen Memorial Fund, Linnean Society, and UM Graduate School. I am deeply indebted to all of these organizations. iv TABLE OF CONTENTS ABSTRACT……………………………………………………………………………………....ii DEDICATION…………………………………………………………………………………....iii ACKNOWLEDGEMENTS……………………………………………………………………....iv LIST OF TABLES……………………………………………………………………………….xi LIST OF FIGURES……………………………...……………………………………………...xiii CHAPTER I—MICROBIOME EVOLUTION ALONG DIVERGENT BRANCHES OF THE VERTEBRATE TREE OF LIFE: WHAT’S KNOWN AND UNKNOWN……………………..1 CHAPTER II—PYLOGENETIC ANALYSIS OF BACTERIAL COMMUNITIES IN DIFFERENT REGIONS OF THE GASTROINTESTINAL TRACT OF AGKISTRODON PISCIVOROUS, THE COTTONMOUTH SNAKE…………………………………………….55 CHAPTER III—EVOLUTION OF THE SQUAMATE REPTILE GUT MICROBIOME……..76 CHAPTER IV—INVESTIGATING THE INFLUENCE OF THE MICROBIOME ON HOST COMMUNITY ASSEMBLY USING SNAKES AND THEIR GUT BACTERIA………………………………………………………………………………95 BIBLIOGRAPHY………………………………………………………………………………118 APPENDIX……………………………………………………………………………………..142 VITAE…………………………………………………………………………………………..171 v LIST OF TABLES Chapter I. Table 1. Summary of host organism, technology used.………………………………………...…4 Chapter II. Table 1. Voucher numbers, locality information and region sampled for Agkistrodon piscivorus used in this study…………………………………………...…...59 Table 2. Statistical analyses of bacterial 16S rRNA gene pyrosequence data obtained from the gastrointestinal tract (GIT) of individual (ID) Agkistrodon piscivorus (numbered 103-130).………………………………………...…64 Table 3. Results of ANOSIM analyses comparing the bacterial communities in different regions of the GIT of individuals of Agkistrodon piscivorus……………...…...68 Table 4. Distribution and classification of 16S rRNA-defined bacterial OTUs representing >1% of the total proportion of reads obtained from the GIT of individuals of Agkistrodon piscivorus……………………………………………….…..70 Chapter III. Table 1. Bacterial OTUs that show significant differences in their distribution across the lizard and snake microbiome……………………………………………...…85 Table 2. Gut bacterial community is structured significantly by geographic location of the host………………………………………………………………….…..86 Table 3. Summary of unweighted UniFrac tests for significant phylogenetic clustering of gut bacterial community membership in snakes…………………………...89 vi Chapter IV. Table 1. Summary of snake host species used in this study………………………………........100 Table 2. Results of one-way ANOVA of measures of bacterial diversity in snake gut microbiomes compared across the three snake host communities……………………..105 Table 3. Gut bacterial community structure is significantly influenced by various ecological traits of hosts…………………………………………………………….….110 vii LIST OF FIGURES Chapter I. Figure 1. Distribution of microbiome studies across vertebrates…………………………………6 Figure 2. Increase in the number of microbiome studies for different classes of vertebrates over a 25 year period from 1990 to 2015…………………………………12 Chapter II. Figure 1. Nonmetric multidimensional plots of bacterial communities………………...……….65 Figure 2. Agkistrodon piscivorus GIT bacterial abundance……………………………………..66 Figure 3. Bacterial abundance present in the cloaca of Agkistrodon piscivorus……………...…69 Figure 4. Bacterial community similarity by region and phylogenetic reconstruction of bacterial communities found in Agkistrodon piscivorus………...……71 Chapter III. Figure 1. Family level phylogeny of Squamata based on Pyron et al. 2013…………...………..79 Figure 2. Percentage of bacterial phyla in pooled dataset of 390 reptile gut microbial samples……………………………………………………………...…….83 Figure 3. Relative abundance of bacterial classes present in Squamate reptile gut microbial samples………………………………………...…………………………86 Figure 4. Nonmetric multidimensional scaling plots of bacterial community structure based on Yue & Clayton’s theta (thetayc) similarities of bacterial communities………………………………………..………………………….87 viii Chapter IV. Figure 1. Diversity measures from the three snake host communities; gut bacterial samples from 36 snake species in Brazil, 24 snake species in Mexico and 23 snake species in the USA. …………………………………….…....106 Figure 2. Nonmetric multidimensional scaling plots of snake gut bacterial community structure based on Yue & Clayton’s theta (thetayc) similarities of bacterial communities………………………………………………………………..…107 Figure 3. Relative abundance of gut bacterial subphyla in a tropical snake community…….…108 Figure 4. Relative abundance of gut bacterial subphyla in a subtropical snake community…...111 Figure 5. Relative abundance of gut bacterial subphyla in a temperate snake community…….113 ix CHAPTER I MICROBIOME