Molecular Evolution of Dim-Light Visual Pigments in Neotropical Geophagine Cichlids by Shannon Refvik a Thesis Submitted in Conf
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Molecular Evolution of Dim-light Visual Pigments in Neotropical Geophagine Cichlids by Shannon Refvik A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Ecology and Evolutionary Biology University of Toronto c Copyright 2012 by Shannon Refvik Abstract Molecular Evolution of Dim-light Visual Pigments in Neotropical Geophagine Cichlids Shannon Refvik Master of Science Graduate Department of Ecology and Evolutionary Biology University of Toronto 2012 Neotropical cichlid fishes are highly diverse and occupy a wide range of environments. Evo- lution of visual pigments has been important in the diversification of the African rift lake cichlids, but relatively little is known of Neotropical cichlid visual systems. This thesis ad- dresses the molecular evolution of the dim-light visual pigment rhodopsin in the Geophagini tribe of Neotropical cichlids. We use various likelihood-based codon models of molecular evo- lution and newly isolated sequences for Neotropical cichlid rhodopsin to compare patterns of selective constraint among Neotropical, African rift lake, and African riverine cichlid rhodopsin; and provide evidence for differences in selective constraint among clades with positive selection occurring in both the Neotropical and African rift lake clades. We further investigate and find evidence for variation in selective constraint within the geophagine ci- chlids. Comparing the results obtained from different methods suggests that Clade model C is more appropriate than branch-site models for investigating variation in selective con- straint among clades. Neotropical cichlids, alone and in comparison with African cichlids, are emerging as an excellent system for investigating molecular evolution in visual pigments. ii Acknowledgements First and foremost, I would like to thank Hern´anL´opez-Fern´andezand Belinda Chang, my co-supervisors, for their support and advice throughout my degree. I came into this project with zero experience working either with fish or in molecular biology, but they were incredibly helpful in the ensuing learning process. I would also like to thank Hern´anfor providing me with opportunities to work in the field, and my committee members, Allan Baker and Nathan Lovejoy, for their helpful suggestions throughout. I would like to acknowledge members of the L´opez-Fern´andezand Chang labs for their support - particularly Jessica Arbour, who helped me wade through the finer points of graduate school adminstration processes; James Morrow, David Yu, and Ilke van Hazel, who patiently helped me learn lab procedures; and Cameron Weadick, who conducted some of the studies that motivated my research and helped me to implement the clade model he developed. My family, friends, and in particular my partner Jasper Palfree have been incredibly sup- portive - it was helpful and motivating to share my successes and discuss my challenges with such great people. On that note, I would like to thank the members of Toronto's Lindy Hop scene, who definitely kept me sane when the challenges seemed overwhelming. Lastly, I would like to acknowledge my funding sources for this project, NSERC and OGS. iii Contents 0.1 Statement of Contributions . .1 1 General Introduction 2 1.1 Biogeography of Cichlids . .2 1.2 Vertebrate Vision . .6 1.3 Visual systems of African and Neotropical cichlids . 10 1.4 Codon based models of molecular evolution . 13 1.5 Objectives . 19 1.5.1 Objective 1: Investigating differences in selective constraint between Neotropical and African dim light visual pigments . 19 1.5.2 Objective 2: Investigating differences in selective constraint within geophagine cichlid dim light visual pigments . 20 1.6 Figures . 20 2 Molecular Evolution of Dim-light Visual Pigments in Neotropical Geophagine Cichlids: Evidence for differences in selective constraint in comparison with African cichlids 22 2.1 Abstract . 22 2.2 Introduction . 23 iv 2.3 Methods . 26 2.3.1 Samples and Sequences . 26 2.3.2 Tree building . 27 2.3.3 Testing for selection . 28 2.4 Results . 32 2.4.1 Molecular dataset . 32 2.4.2 Site models . 33 2.4.3 Clade model C . 33 2.4.4 Branch-site Models . 34 2.4.5 Influence of positively selected sites on rhodopsin function . 35 2.5 Discussion . 37 2.5.1 Positive selection in Neotropical and African cichlid rhodopsins . 37 2.5.2 High average omega values . 38 2.5.3 Divergent selection between clades . 39 2.5.4 Non-overlapping BEB sites . 41 2.5.5 Clade model C vs. Branch-site Results . 43 2.5.6 Caveats . 45 2.5.7 Conclusions . 46 2.6 Tables . 46 2.7 Figures . 51 2.8 Supplementary information . 55 3 Patterns of Selective Constraint in Geophagine Cichlid Rhodopsin 60 3.1 Introduction . 60 3.2 Methods . 62 3.2.1 Species Included and Phylogenetic Relationships . 62 v 3.2.2 Clade Model C Analyses . 62 3.2.3 Branch-site Analyses . 63 3.3 Results . 63 3.3.1 Clade Model C . 63 3.3.2 Branch-site . 64 3.3.3 Divergently Selected Sites . 64 3.4 Discussion . 66 3.4.1 Divergent Selection Between Clades, with Positive Selection Throughout 66 3.4.2 Clade model C vs. Branch-site Results . 67 3.5 Tables . 69 3.6 Figures . 73 4 Conclusions and Future Directions 75 4.1 Conclusions . 75 4.2 Future Directions . 79 5 References 84 vi List of Tables 2.1 Parameter estimates, likelihood values, likelihood ratio tests, and significance values of PAML random site models using Neotropical or African RH1 sequences. 47 2.2 BEB sites in Neotropical and African cichlids . 48 2.3 Parameter estimates, likelihood values, test statistics, and p values for various data partitions in Clade Model C. 49 2.4 Likelihood values, test statistics, and p values for likelihood ratio tests for branch-site models. 50 2.1 Supplementary Table. Species list, museum catalogue numbers, and accession numbers for sequences used in this study. 55 2.2 Supplementary table: Parameter estimates, likelihood values, test statistics, and p values for various data partitions in Clade Model C with phylogenetically misplaced species removed. 57 2.3 Supplementary table: Likelihood values, test statistics, and p values for likeli- hood ratio tests for branch-site models with phylogenetically misplaced species removed. 58 2.4 Supplementary table: Detailed BEB output for Site Models, CmC, and Branch- site Models. 59 vii 3.1 Supplementary Table. Species list, museum catalogue numbers, and accession numbers for sequences used in this study. 69 3.2 Parameter estimates, likelihood values, test statistics, and p values for CmC analysis of a tree with three partitions: The \Satanoperca" clade, the \Geoph- agus" clade, and a clade of basal outgroups. 71 3.3 Likelihood values, test statistics, and p values for likelihood ratio tests for branch-site models. 72 viii List of Figures 1.1 3D images of dark-state and active state rhodopsin. 21 2.1 Maximum likelihood tree of RH1 sequences, constrained to be reciprocally monophyletic . 52 2.2 RH1 phylogeny and distribution of amino acid residues at positively selected sites in Neotropical and African cichlids. 53 2.3 Interface between rhodopsin molecules in a dimer . 54 2.4 Openings to retinal binding pocket in the active conformation of rhodopsin. 54 3.1 Amino acid residues at divergently selected sites in geophagine cichlids and some Neotropical basal outgroups . 74 ix 0.1 Statement of Contributions Chapters 1, 3, and 4 of this thesis were conceived of and written by Shannon Refvik. Chapter 2 of this thesis will be submitted as a paper co-authored by myself and my two co-supervisors, Belinda Chang and Hern´anL´opez-Fern´andez. The studies included in this chapter were designed collaboratively between myself and my supervisors. I conducted all of the data collection, performed the statistical analyses, and wrote the text of all work submitted in this thesis. 1 Chapter 1 General Introduction 1.1 Biogeography of Cichlids The rivers of South and Central America harbour the most diverse freshwater fish fauna on earth, with an estimated 7000 species that interact in a wide variety of structured commu- nities (Reis et al. 2003). Cichlids fishes are the third largest group of Neotropical fish with approximately 600 species, and are ubiquitous throughout the ecologically varied aquatic habitats of South and Central America from southern Patagonia to Texas (Reis et al. 2003). Cichlids exhibit diverse life histories, reproductive modes, and feeding strategies (Wimberger et al. 1998, Barlow 2000), with this diversity being well represented by the geophagine clade. Geophagines are a monophyletic group (L´opez-Fern´andez et al. 2010) restricted to South America and Southern Panama (Reis et al. 2003), and are one of the three most species rich tribes of Neotropical cichlids along with Cichlasomatini and Heroini (Kullander 1998, Smith et al. 2008, L´opez-Fern´andez et al. 2010). Within 17 genera, this clade includes species with a diversity of feeding modes including piscivorous species, substrate sifters, and water- 2 column feeders, as well as species that mouth brood their young (L´opez-Fern´andez et al. 2005a, 2012). Diet categories within the geophagines are highly correlated to morphological characteristics, indicating that ecomorphological specialization has occurred (Winemiller et al. 1995, L´opez-Fern´andez et al. 2012). Their ecological and morphological diversity, com- bined with the well-resolved genus-level phylogeny available for Neotropical cichlids (L´opez- Fern´andez et al. 2010), make them an ideal system for investigating the ecology and evolution of the freshwater fish fauna in the Neotropics. Neotropical cichlids make up a monophyletic clade that is sister to the African cichlids (Streelman et al. 1998, Farias et al. 1999, 2000, 2001; Sparks and Smith 2004, Smith et al. 2008, L´opez-Fern´andez et al. 2010), which includes the species-rich and well-studied African rift lake cichlids (reviewed in: Kocher 2004, Seehausen 2006).