View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Carolina Digital Repository THE EVOLUTION OF MORPH-BIASED GENES IN SPADEFOOT TOADS Aaron Reid Leichty A thesis submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Master of Science in the Department Biology Chapel Hill 2011 Approved by: Advisor: David W. Pfennig Reader: Corbin Jones Reader: Karin Pfennig ABSTRACT AARON REID LEICHTY: The evolution of morph-biased genes in spadefoot toads (Under the direction of David W. Pfennig) Polyphenisms allow for the expression of two or more discrete phenotypes from a single genotype in an environment dependent manner. These phenotypes are associated with extensive differences in levels of gene expression and recent work suggests that genes with differential expression between phenotypes (biased genes) evolve at elevated rates relative to genes showing no difference between phenotypes (unbiased genes). The causes of this difference in evolutionary rate remain relatively unexplored. Using a combination of molecular and genomic approaches I identified genes with biased expression between the environmentally induced alternative morphs of spadefoot toad tadpoles (genus Spea). I further characterized these morph-biased genes in closely related spadefoot toad species that do not express alternative tadpole morphologies (genus Scaphiopus). Phylogenetic estimates of protein evolutionary rates in Spea reveal that morph-biased genes are evolving more quickly than unbiased genes, but this was dependent on rate estimation method. Surprisingly, in Scaphiopus morph-biased orthologs are evolving more quickly than unbiased orthologs, regardless of estimation method. Finally, morph-biased genes in Spea are evolving no more quickly than their orthologs in Scaphiopus. In total, our work suggests that genes with higher rates of evolution are more likely to evolve differential expression, but differential expression has no direct influence on a gene’s evolutionary rate. ii ACKNOWLEDGEMENTS I would like to thank my advisor, David Pfennig. I appreciate many things about David, but it is his openness that I value most. It is because of this openness that I found myself at UNC and now I find myself transitioning to Pennsylvania. I cannot imagine a better advisor. I also thank Karin Pfennig. Without her support my projects would have never happened. I truly hope I was a worthy investment. I thank Corbin Jones for intellectual reassurance at important stages in my work and for a lab away from lab. Finally, I would like to thank all of the family and friends that have been supportive of me throughout my time at UNC. iii TABLE OF CONTENTS LIST OF TABLES..………………………………...…………………….....................….v LIST OF FIGURES..………………………………...………………………...................vi Chapter I. GENERAL INTRODUCTION…………………………...….....….……………...1 II. EVOLUTION OF GENES WITH MORPH-BIASED GENE EXPRESSION……..………………………………...………………………...7 Summary………………………………...………………………………………...7 Introduction………………………………...……………………………………...8 Methods……………………………...…………………………………………….8 Results……………………………...…………………………………………….15 Discussion……………………………...………………………………………...17 III. CONCLUSIONS……………………………...…………………………………31 APPENDIX……………………………...…………………………………….................36 REFERENCES……………………………...……………………………………...........60 iv LIST OF TABLES Table 2.1. Results from quantitative PCR in S. bombifrons………………………………..22 A.1. Primers used for quantitative PCR in S. bombifrons…………………………...37 A.2. Candidate genes for differential expression between tadpole morphs from microarray experiment…………………………………..........47 A.3. Genbank accessions for all Xenopus sequences used in the rate analyses……..………………………………………………………….....58 v LIST OF FIGURES Figure 1.1. Environmentally induced tadpole morphs from the genus Spea…………………………………………………………………...6 2.1. Volcano plot of genes tested with qRT-PCR in S. bombifrons.……………………………………………………………………..27 2.2. Comparisons of gene expression measured in lab reared and wild caught tadpoles……………………………….........................…28 2.3. Comparison of protein evolutionary rates (dN/dS)…………………………………………………………………….................29 2.4. Proposed scenario for the evolution of polyphenic gene expression……………………………………………………….....30 vi CHAPTER I GENERAL INTRODUCTION Polyphenisms allow for the expression of two or more discrete alternative phenotypes from a single genotype in an environment dependent manner (sensu Mayr 1963). This extreme form of phenotypic plasticity allows for individuals of a population to adaptively respond to environmental variation, facilitating the utilization of multiple niches by a single species. Most commonly the alternative phenotypes are characterized by morphological, physiological and behavioral differences and it is generally thought that this variation is important in processes such as speciation and adaptive radiation (West-Eberhard 2003; Pfennig et al. 2010). These evolutionary and ecological consequences of polyphenic development have received a great deal of attention, but we still remain ignorant of the molecular mechanisms and evolutionary origins of polyphenic development (Aubin-Horth & Renn 2009). Recent advances in genomic technologies have greatly increased understanding of the molecular mechanism of polyphenism relative to knowledge of its evolutionary origins. Numerous studies have now documented large suites of genes showing differential expression between the alternative phenotypes of polyphenic species (partially reviewed in Snell-Rood et al. 2010). A common theme emerging from these studies is that differences between environmentally mediated alternatives often rival the differences between genetically mediated alternatives in both number of genes and magnitude of expression (e.g. aphids, Brisson et al. 2007; beetles, Snell-Rood et al. 2011). This finding is important because it suggests that the unique selection pressures operating on alternative phenotypes under genetic control may also be acting in systems with environmentally mediated alternatives. The differential expression associated with sexual dimorphism is a well-studied example of genetically controlled alternative phenotypes. This differential expression between males and females, sex-biased expression, is often mediated by a few genetic differences. Despite these minor genetic differences, at the level of gene expression a large portion of the genes in the genome differ between sexes. It is now well documented that genes with sex-biased expression are evolving more quickly than genes expressed equally between sexes (reviewed in Ellegren & Parsch 2007), and a great deal of work has demonstrated this is primarily due to adaptive evolution in genes with expression limited to reproductive tissues (Pröschel et al. 2006; Meisel 2011). Recent work examining genes with differential expression between alternative phenotypes of polyphenic species, morph-biased genes, have revealed that they too evolve at elevated rates compared to genes shared between phenotypes (unbiased genes) (Snell-Rood et al. 2010; Hunt et al. 2010; Van Dyken & Wade 2009). The causes and generality of this rate difference remain unknown. There are at least four different possible causes for morph-biased genes evolving more rapidly than their unbiased counterparts. Genes differentially expressed between morphs may be experiencing high levels of adaptive evolution driven by the unique requirements of each alternative phenotype’s ecological niche. No study has yet presented evidence of pervasive positive selection in morph-biased genes, but Hunt et al. 2 (2010) interpreted an increase in evolutionary rate of queen-biased genes in bees (Apis mellifera) as a result of adaptive evolution. Morph-biased genes may experience high levels of neutral and/or deleterious evolution due to a relaxation of evolutionary constraint. Evidence exists that genes expressed in only one alternative phenotype (sex or morph-specific genes) evolve more quickly than genes expressed in both due selection’s inability to remove deleterious alleles in unexpressing individuals (sex-specific genes: Brisson & Nuzhdin 2008; morph- specific genes: Van Dyken & Wade 2009). If selection is proportional to degree of morphs-biased expression then morph-biased genes may behave as morph-specific genes (Snell-Rood et al. 2010). To date, there have been no tests of this idea, but some authors have interpreted higher rates of evolution in morph-biased genes as being consistent with this hypothesis (Snell-Rood et al. 2011). It is also possible, from a phylogenetic perspective, that genes with high rates of evolution are more likely to evolve morph-biased expression. This would give the impression that morph-biased expression has a direct effect on evolutionary rate, but in fact is a consequence of a preexisting evolutionary rate. Hunt et al. (2010) presented evidence that queen-biased genes are historically more dispensable (a gene’s degree of importance to organismal fitness) and can be interpreted as support for this hypothesis. The findings of Snell-Rood et al. (2011) could also be explained by this hypothesis. Finally, morph-biased genes may actually experience a reduction in evolutionary rate due to morph-biased expression. If genes with higher rates of evolution are more likely to evolve morph-biased expression (third scenario) then a shift from unbiased to biased expression may be accompanied by a decrease in a gene’s level of dispensability. 3 This