Relaxed selection in erythropoietic gene hemogen among high-latitude Antarctic notothenioids by Carmen M. Elenberger B.A. in Anthropology, University of Florida A thesis submitted to The Faculty of the College of Science of Northeastern University in partial fulfillment of the requirements for the degree of Master of Science December 12, 2018 Thesis directed by H. William Detrich Professor of Biochemistry and Marine Biology 1 Copyright 2018 Carmen Elenberger 2 Acknowledgements First and foremost, I would like to thank my advisor, Dr. H. William Detrich, for his guidance and his support over the past four years. He challenged me to broaden my horizons and gave me the opportunity to travel to the ends of the earth in order to do so. I would also like to thank Dr. Thomas Desvignes, as well as Laura Goetz and Sierra Smith, for their assistance in conducting field work for this project. I would like to extend further thanks to Dr. Jacob Daane for permitting me to use his unpublished data to expand my analyses. Many thanks to Biology Open for allowing me to reproduce their figure with permission [1]. I would like to thank my committee members, Dr. A. Randall Hughes and Dr. Steve Vollmer, for their interest in my research and their advice in analyzing and framing the results of my research. I would also like to thank my labmate, Dr. Michael Peters, and our lab manager, Sandra Parker, for their advice, assistance, and encouragement over the years. Additionally, I would like to thank the faculty and staff of the Marine Science Center, as well as the funding sources for this research. Special thanks to the staff of Palmer Station and the crew of the Laurence M. Gould for a productive and memorable field season. Finally, I would like to thank my friends and family for their unwavering support and encouragement, now and always. 3 Abstract of Thesis Antarctic icefish (Channichthyidae) are the only vertebrate taxon with an erythrocyte-null phenotype, and present an interesting model for studying the evolution and regulation of erythropoiesis. The gene hemogen has been identified to encode a protein which plays a role in regulating erythropoietic processes in vertebrates. hemogen may have been potentially impacted by the loss of globin-expression. I investigated possible relaxed selection at the hemogen locus by looking for evolutionary change to the regulatory elements or segments encoding the Hemogen protein, and assessed the evolutionary processes that drove hemogen variation among Antarctic notothenioids. While regulatory mechanisms remain intact, icefish show a significant 90bp indel in exon 3 of hemogen that would disrupt conserved modules in the Hemogen protein that are critical for erythropoiesis. Despite this, hemogen still remains expressed at low levels in adult icefish and possesses a novel splice variant that encodes a truncated protein possibly serving as a dominant negative for wild-type Hemogen. I conclude that while hemogen has undergone relaxed selection and accumulated mutations that would impact erythropoietic function in non-Antarctic fish, the observed mutations may be tolerated due to erythrocyte and hematocrit modifications in notothenioid blood phenotypes. hemogen may have a decreased— but still important—role to play in icefish, possibly functioning as a dominant negative for hemogen’s role in erythropoiesis. 4 Table of Contents Acknowledgements 3 Abstract of Thesis 4 Table of Contents 5 List of Tables 6 List of Figures 7 List of Abbreviations 9 Introduction 11 Methods 15 Results 21 Discussion 29 Tables and Figures 40 References 67 5 List of Tables 1 Primers used in PCR and qRT-PCR reactions to amplify hemogen gDNA and cDNA in Antarctic notothenioids (pg 40) 2 Species sequenced and included in study of Antarctic notothenioid hemogen (pg 41) 3 Codon usage bias for hemogen (total coding sequence) among Antarctic notothenioids (pg 42) 4 Mean pairwise dN/dS for within-family comparisons of Antarctic notothenioid families (pg 43) 5 Mean pairwise dN/dS for between-family comparisons of Antarctic notothenioid families (pg 44) 6 Results of codon-based site tests conducted in CodeML on the Antarctic radiation (pg 45) 6 List of Figures 1 Zebrafish Si:dkey-25o16.2 and human Hemogen are orthologous and encode related proteins that differ in size (pg 46) 2 Icefish transcript variants for hemogen and their putative effects on translation illustrated in representative species Champsocephalus gunnari (pg 48) 3 Maximum likelihood tree used to test for positive selection on the branch leading to the Antarctic notothenioid clade (pg 50) 4 Maximum likelihood tree used in site-tests for positive/pervasive selection among Antarctic notothenioids (pg 51) 5 RELAX tree shows relaxed selection on the branches contained Bathydraconidae and Channichthyidae, demonstrating a trend of relaxed selection in hemogen on the way to the erythrocyte-null phenotype (pg 53) 6 Gene structure and size remains conserved among red-blooded and white-blooded notothenioids, including regulatory regions conserved among teleost fish (pg 54) 7 Conservation of conserved non-coding elements CNE1 and CNE2 in Antarctic notothenioids relative to Gasterosteus aculeatus and Danio rerio (pg 56) 8 hemogen exon 3 deletions in representative species from Channichthyidae relative to a red-blooded notothenioid, and their predicted effects on transcription and translation (pg 57) 9 Variant forms of hemogen “exon 3” deletion mapped onto the Channichthyidae species tree (pg 59) 10 hemogen indels in Antarctic notothenioids mapped onto a maximum parsimony tree (pg 60) 11 Pairwise dN/dS comparisons plotting total dN/dS of whole Hemogen-encoding sequence with the dN/dS values for the N-terminus and C-terminus of notothenioid Hemogen, within families Nototheniidae (A & B) and Channichthyidae (C & D). (pg 62) 12 Pairwise dN/dS trends between families Nototheniidae and Channichthyidae, plotting whole-Hemogen dN/dS vs the N-terminus (A) or C-terminus (B). (pg 63) 13 qPCR quantification of hemogen transcript variants in representative icefish species C. aceratus and C. gunnari, comparing adult head kidney hemogen expression with N. coriiceps adult head kidney for both hemgn-L and hemgn-s splice variants (pg 64) 7 14 Changes to the bipartite nuclear localization signal in icefish (Champsocephalus gunnari) relative to red-blooded notothens (Notothenia coriiceps). (pg 66) 8 List of Abbreviations aa amino acid bp base pair CAI Codon Adaptation Index cDNA complementary DNA CNE conserved non-coding element dN nonsynonymous mutation rate DNA deoxyribonucleic acid dN/dS ratio of nonsynonymous to synonymous mutation rates dS synonymous mutation rate EDAG erythroid differentiation-associated gene GATA1 GATA-binding protein 1 gDNA genomic deoxyribonucleic acid HoxB4 homeobox B4 KLF4 Krueppel-like Factor 4 -lnL negative log likelihood MMCT Middle Miocene Climate Transition MRCA most recent common ancestor Mya million years Myb MYB Proto-Oncogene, Transcription Factor NLS nuclear localization signal p300 histone acetyltransferase p300 PCR polymerase chain reaction 9 qPCR quantitative polymerase chain reaction RNA ribonucleic acid Sox9 transcription factor SOX-9 UTR untranslated region 10 INTRODUCTION Cold-driven evolution of the Antarctic notothenioid lineage began roughly 46 Mya [2] concurrent with the emergence of the Drake Passage (55-41 Ma) [3] and the initial formation of the Antarctic Circumpolar Current [4]. The development of antifreeze glycoproteins [5, 6] permitted colonization and persistence in the Southern Ocean [7] and set the stage for further diversification during successive cooling periods and accompanying geological events. The radiation of the high latitude Antarctic notothenioids (Cryonotothenioidea) occurred during a period of diversification driven by intensified cooling of the Southern Ocean during the Middle Miocene Climate Transition (MMCT) [7, 8], with species diversification beginning ~14 Mya and accelerating ~11 Mya during the Late Miocene [7, 9-11]. Cooling during the MMCT led to contemporary Antarctic conditions (-2℃ to + 2℃) and resulted in the scouring of continental shelves by ice [12, 13]. This opened ecological niches for potential colonization by removing more temperate adapted competitors [14] and leading to rapid morphological and ecological diversification [15]. Current day Antarctic notothenioids comprise 77% of Antarctic teleost diversity and constitute a marine species flock [16] derived via adaptive radiation [17-19]. High levels of morphological diversity and intense speciation make Antarctic notothenioids a useful evolutionary model for studying cold adaptation. Antarctic notothenioids possess a number of remarkable changes to erythropoiesis and the oxygen-transport system at large that resulted in the evolution of the only known vertebrate clade devoid of erythrocytes—the family Channichthyidae, characterized by a “white-blooded” phenotype [20]. It has been hypothesized that the high oxygen concentration in polar seawater could lead to potential relaxed selection on erythrocytes and other oxygen-binding pigments, as hypoxic stress becomes less of a relevant factor with oxygen in such high abundance [21]. 11 Evidence for such relaxed selection can be seen in changes to blood content: a study of “red- blooded” Antarctic species from McMurdo Sound showed decreased numbers of erythrocytes, lowered hematocrit, and lowered hemoglobin concentrations when compared with temperate fish [22]. General trends throughout the radiation show that the more derived