Structure and Evolution of Lizard Immunity Genes
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University of New Orleans ScholarWorks@UNO University of New Orleans Theses and Dissertations Dissertations and Theses Summer 8-7-2020 Structure and Evolution of Lizard Immunity Genes Trent Santonastaso University of New Orleans, New Orleans Follow this and additional works at: https://scholarworks.uno.edu/td Part of the Evolution Commons, Genetics Commons, Genomics Commons, Integrative Biology Commons, Molecular Biology Commons, Other Ecology and Evolutionary Biology Commons, Other Genetics and Genomics Commons, and the Population Biology Commons Recommended Citation Santonastaso, Trent, "Structure and Evolution of Lizard Immunity Genes" (2020). University of New Orleans Theses and Dissertations. 2819. https://scholarworks.uno.edu/td/2819 This Dissertation is protected by copyright and/or related rights. It has been brought to you by ScholarWorks@UNO with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in University of New Orleans Theses and Dissertations by an authorized administrator of ScholarWorks@UNO. For more information, please contact [email protected]. Structure and Evolution of Lizard Immunity Genes A Dissertation Submitted to the Graduate Faculty of the University of New Orleans in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Integrative Biology by Trenten T. Santonastaso B.S. Pennsylvania State University, 1994 M.S. Northeastern Illinois University, 2009 August, 2020 Dedicated to my mother and father, Linda Bogenschneider and Michael Santonastaso, who taught me to question, encouraged me to think, and introduced me to natural history. ii Acknowledgements I must thank everyone who instructed and guided me through this process. First, Dr. Nicola Anthony who set up so many opportunities in laboratories and field camps around the world. Her scientific mind was essential in focusing my efforts and pushing me to think through obstacles. Finally, her thorough editorial process maintained stringently professional product. My off- campus committee members who had the patience and will to train me and have me in their labs and field camps. Dr. Michael Jensen-Seaman who hosted me for a full summer to work in his lab at Duquesne University, and Dr. Susan Perkins who hosted me for weeks at a time, on Saba Island and in her labs in at American Museum of Natural History. Both of these professionals also were patient and invaluable in writing and processing the data. This couldn’t have been finished without them. Dr. Foufoupolous who hosted me for two weeks in his field camp in Greece as a simple field hand to enhance my understanding of that natural system. Dr. Barney Rees, Dr. Wendy Schluchter, and Dr. Steve Johnson who were instrumental in getting me through the bureaucratic steps towards graduation. I would also like to acknowledge my peer cohort for emotional support, practical advice, and camaraderie: Laura Alexander, Annie Cespedes, Jessica Edwards, Katy Morgan, Devin Prestin, Robinson Sudan, and Henry Wooley. iii Table of Contents List of Figures..…………………………………………………………………………………....v List of Tables..…………………………………………………………………………………….vi Abstract…………………………………………………………………………………………..vii Introduction………………………………………………………………………………………..1 Literature Cited……………………………………………………………………………6 Chapter 1..………………………………………………………………………………………..12 Abstract…………………………………………………………………………………..13 Introduction………………………………………………………………………………14 Materials and Methods…………………………………………………………………...17 Results……………………………………………………………………………………22 Discussion………………………………………………………………………………..32 Acknowledgements………………………………………………………………………36 Literature Cited…………………………………………………………………………..57 Chapter 2…………………………………………………………………………………………62 Abstract…………………………………………………………………………………..63 Introduction……………………………………………………………………………....64 Materials and Methods…………………………………………………………………...66 Results……………………………………………………………………………………72 Discussion………………………………………………………………………………..78 Acknowledgements………………………………………………………………………83 Supplementary Methods…………………………………………………………………83 Literature Cited…………………………………………………………………………..89 Chapter 3…………………………………………………………………………………………94 Abstract…………………………………………………………………………………..95 Introduction………………………………………………………………………………96 Materials and Methods………………………………………………………………….100 Results…………………………………………………………………………………..108 Discussion………………………………………………………………………………111 Acknowledgements……………………………………………………………………..118 Literature Cited…………………………………………………………………………118 Conclusion……………………………………………………………………………………...126 Vita……………………………………………………………………………………………...128 iv List of Figures Chapter 1…………………………………………………………………………………………12 Figure 1…………………………………………………………………………………..12 Figure 2…………………………………………………………………………………..21 Figure 3…………………………………………………………………………………..23 Figure 4…………………………………………………………………………………..27 Chapter 2…………………………………………………………………………………………62 Figure 1…………………………………………………………………………………..62 Figure 2…………………………………………………………………………………..67 Figure 3…………………………………………………………………………………..74 Figure 4…………………………………………………………………………………..76 Figure 5…………………………………………………………………………………..77 Figure 6…………………………………………………………………………………..78 Supplementary figure S-01………………………………………………………………86 Supplementary figure S-02………………………………………………………………87 Supplementary figure S-03………………………………………………………………88 Supplementary figure S-04………………………………………………………………88 Supplementary figure S-05………………………………………………………………89 Chapter 3…………………………………………………………………………………………94 Figure 1.………………………………………………………………………………….94 Figure 2…………………………………………………………………………………101 Figure 3…………………………………………………………………………………107 Figure 4…………………………………………………………………………………108 Figure 5…………………………………………………………………………………111 Figure 6…………………………………………………………………………………113 v List of Tables Chapter 1…………………………………………………………………………………………12 Table 1……………………………………………………………………………………25 Table 2……………………………………………………………………………………31 Table 3……………………………………………………………………………………34 Table 4……………………………………………………………………………………36 Supplementary Table 1...…………………………………………………………………37 Supplementary Table 2…………………………………………………………………...44 Supplementary Table 3…………………………………………………………………...47 Supplementary Table 4…………………………………………………………………...51 Chapter 2…………………………………………………………………………………………62 Table 1……………………………………………………………………………………68 Table 2……………………………………………………………………………………72 Table 3……………………………………………………………………………………75 Table 4……………………………………………………………………………………79 Chapter 3…………………………………………………………………………………………94 Table 1…………………………………………………………………………………..105 Table 2…………………………………………………………………………………..106 Table 3…………………………………………………………………………………..106 Table 4…………………………………………………………………………………..107 Table 5…………………………………………………………………………………..110 Table 6…………………………………………………………………………………..112 Table 7…………………………………………………………………………………..112 Table 8…………………………………………………………………………………..115 Table 9…………………………………………………………………………………..117 vi Abstract One of the most important gene families to play a role in adaptive immunity is the major histocompatibility complex (MHC). MHC class II loci are considered to be the most variable loci in the vertebrate genome, and studies have shown that this variability can be maintained through complex co-evolutionary dynamics between host and parasite. Despite the rich body of research into the MHC, there is comparatively little understanding of its genomic architecture in reptiles. Similarly, loci associated with innate immunity have received little attention in reptiles compared to other vertebrates. In the first chapter, we investigated the structure and organization of the MHC in the Anolis carolinensis genome by sequencing and annotating five bacterial artificial chromosomes (BAC) from the green anole genome library. We were able to identify three mhc2a, four mhc2b, and up to 15 mhc1 loci in A. carolinensis. Furthermore, we were able to link 17 scaffolds and provide sequence data to fill two significant gaps in the genome assembly. In the second chapter, we investigated the relative importance of drift and selection in shaping mhc2 variability in the reptile Podarcis erhardii. We sequenced the mhc2 gene from lizard populations from 14 islands in the Aegean that have experienced bottlenecks of differing duration and intensity. Despite signals of balancing selection, patterns of mhc2 variation were similar to microsatellites, providing evidence that the dominant evolutionary force in this system is drift. In the third chapter, we investigated how parasite infection rates impact innate immune variability in A. sabanus, a lizard indigenous to Saba Island where natural fluctuations in Plasmodium infection rates have been documented. We developed primers and sequenced part of the peptide binding region of three Toll-like receptors (TLRs) - tlr4, tlr6, and tlr13 and several beta-defensin (BD) loci. Although we were unable to characterize BD variability, we found three different haplotypes in tlr4, and five in tlr6. However, nucleotide variability was low (π < 0.005) and was not associated with infection status. We nevertheless present primers for multiple TLR genes and two BDs that could be of use in future studies of reptile innate immunity. Keywords – MHC, TLR, BD, BAC sequencing, bottleneck, host/parasite coevolution, Anolis, Podarcis vii Introduction Ecological immunology is an emerging field of study investigating how biotic and abiotic