A DRAFT DE NOVO GENOME ASSEMBLY AND MITOCHONDRIAL POPULATION GENOMICS FOR THE NORTHERN BOBWHITE (COLINUS VIRGINIANUS) A Dissertation by YVETTE ASHTON HALLEY-SCHULTZ Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Christopher M. Seabury Committee Members, William J. Murphy Michael F. Criscitiello James E. Womack Intercollegiate Faculty Chair, Craig J. Coates December 2015 Major Subject: Genetics Copyright 2015 Yvette A. Halley-Schultz ABSTRACT Wild populations of northern bobwhites (Colinus virginianus; hereafter bobwhite) have declined across most of their historic U.S. range, and despite their importance as an experimental wildlife model for ecotoxicology studies, no bobwhite draft genome assembly has emerged. Herein, we present the first bobwhite draft de novo genome assembly, with more than 90% of the assembled bobwhite genome captured within < 40,000 final scaffolds (N50 = 45.4 Kb) despite evidence for approximately 3.22 heterozygous polymorphisms per Kb. Moreover, three annotation analyses produced evidence for > 14,000 unique genes and proteins. Bobwhite analyses of divergence with the chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) genomes revealed many extremely conserved gene sequences, and evidence for lineage-specific divergence of noncoding regions. Coalescent models for reconstructing the demographic history of the bobwhite and the scarlet macaw were concordant with how opposing natural selection strategies (i.e., skewness in the r-/K-selection continuum) would be expected to shape genome diversity and the effective population sizes in these species. Using genomic tools and resources developed via the draft genome assembly, we evaluated the concordance of population inferences and conclusions resulting from the analysis of short mitochondrial fragments (i.e., partial or complete D-Loop nucleotide sequences) versus complete mitogenome sequences for 53 bobwhites representing six ecoregions across TX and OK (USA). Median joining (MJ) haplotype networks demonstrated that analyses performed using small mitochondrial fragments were ii insufficient for estimating the true (i.e., complete) mitogenome haplotype structure, corresponding levels of divergence, and maternal population history of our samples. Notably, discordant demographic inferences were observed when mismatch distributions of partial (i.e., partial D-Loop) versus complete mitogenome sequences were compared, with the reduction in mitochondrial genomic information content observed to encourage spurious inferences in our samples. A probabilistic approach to variant prediction for the complete bobwhite mitogenomes revealed 344 segregating sites corresponding to 347 total mutations, including 49 putative nonsynonymous single nucleotide variants (SNVs) distributed across 12 protein coding genes. Evidence of gross heteroplasmy was observed for 13 bobwhites, with 10 of the 13 heteroplasmies involving one moderate to high frequency SNV. Haplotype network and phylogenetic analyses for the complete bobwhite mitogenome sequences revealed two divergent maternal lineages (dXY = 0.00731; FST = 0.849; P < 0.05), thereby supporting the potential for two putative subspecies. However, the diverged lineage (n = 103 variants) almost exclusively involved bobwhites geographically classified as Colinus virginianus texanus, which is discordant with the expectations of previous geographic subspecies designations. Tests of adaptive evolution for functional divergence (MKT), frequency distribution tests (D, FS) and phylogenetic analyses (RAxML) provide no evidence for positive selection or hybridization with the sympatric scaled quail (Callipepla squamata) as being explanatory factors for the two bobwhite maternal lineages observed. Instead, our analyses support the supposition that two diverged maternal lineages have survived from pre-expansion to post-expansion population(s), with the segregation of some slightly deleterious nonsynonymous mutations. iii DEDICATION I would like to dedicate this dissertation to my family. To my parents, Wendell and Monica Halley, and my little brother, William Halley, for all of their love, friendship, support and sacrifices; I am truly blessed to have belonged to such an incredible family. I would also like to dedicate this dissertation to my husband, Kyle Schultz. Thank you for being so understanding, loving and patient, I couldn’t have asked for a more supportive partner in life. Lastly, I would like to acknowledge all Texas A&M University former students who have donated to the Student Assistance fund, the assistance I received has left me eternally grateful. iv ACKNOWLEDGMENTS Foremost, I am very grateful to my committee chair, Dr. Christopher M. Seabury, for all of his support, guidance and encouragement throughout my graduate education. I would also like to thank Dr. William J. Murphy, Dr. James E. Womack, and Dr. Michael F. Criscitiello for serving on my committee and providing me with valuable advice. I also acknowledge the help and support provided by Eric K. Bhattarai and David L. Oldeschulte; thank you for your comradery and friendship. Additionally, I would also like to extend my thanks to all my collaborators whose cooperative efforts helped make the work within this dissertation possible: Dr. Jared Decker, Dr. Scot Dowd, Dr. Joshua Hill, Dr. Charles Johnson, Dr. Richard Metz, Dr. Markus J. Peterson, Dr. Steven Presley, Dr. Dale Rollins, Rebekah Ruzicka, and Dr. Jeremy F. Taylor. This project wouldn’t have been possible without the financial support of Joe Crafton and Park Cities Quail, the Rolling Plains Quail Research Foundation, and the Reversing the Quail Decline in Texas Initiative with Upland Game Bird Stamp Fund, based on a collaborative effort between Texas Parks and Wildlife Department and Texas A&M AgriLife Extension Service. Thank you to all the hunters who donated samples, and to Texas Parks and Wildlife Department, I am also grateful to Doug Schoeling, Jena Donnell, and the Oklahoma Department of Wildlife. Lastly, I would like to extend my thanks to the Texas AgriLife Genomics and Bioinformatics Core, Texas A&M University, and the Missouri Sequencing Core (Nathan Bivens; Sean Blake) at the University of Missouri for high quality sequencing services. v NOMENCLATURE Abbreviation Description cDNA Complementary DNA BAC Bacterial Artificial Chromosome PE Paired-end MP Mate pair Gbp Gigabase Pairs Kbp Kilobase Pairs SNP Single Nucleotide Polymorphism MYA Million Years Ago ORF Open Reading Frame MHC Major Histocompatibility Complex GWAS Genome-wide Association Studies SAM Sequence Alignment/Map Format SRA Sequence Read Archive WGS Whole Genome Shotgun LINE Long Interspersed Elements SINE Short Interspersed Elements LTR Long Terminal Repeats KYA Thousand Years Ago PSMC Pairwise Sequentially Markovian Coalescent vi TABLE OF CONTENTS Page ABSTRACT ..................................................................................................................... ii DEDICATION ................................................................................................................ iv ACKNOWLEDGMENTS ................................................................................................ v NOMENCLATURE ........................................................................................................ vi TABLE OF CONTENTS ............................................................................................... vii LIST OF FIGURES .......................................................................................................... x LIST OF TABLES .......................................................................................................... xi CHAPTER I INTRODUCTION ...................................................................................... 1 Bobwhite Biology and Ecology ............................................................................ 1 Bobwhite Population Decline in the U.S. ............................................................. 5 Bobwhite Harvest in the U.S. ................................................................................ 6 Historic Bobwhite Research .................................................................................. 8 Bobwhite Research Needs ................................................................................... 11 CHAPTER II GENOME SEQUENCING AND DE NOVO ASSEMBLY ................... 13 Introduction ......................................................................................................... 13 Results and Discussion ........................................................................................ 14 Methods ............................................................................................................... 18 Source of Bobwhite (Colinus virginianus) Genomic DNA ............................ 18 Genome Sequencing Strategy .......................................................................... 18 Genome Assembly........................................................................................... 19 CHAPTER III ANNOTATION AND COMPARATIVE ANALYSIS OF THE BOBWHITE GENOME .............................................................................................. 22 vii Introduction ......................................................................................................... 22 Results and Discussion .......................................................................................