Doctoral Dissertation Research: Paul Hime: Assessing Gene- and Site-Specific Support for Deep Amphibian Relationships Across Nuclear Loci that Interact with Mitochondria and Ribosomes

OVERVIEW: The relationships among the three extant amphibian orders have been notoriously difficult to resolve, and despite hundreds of studies using different suites of morphological and molecular characters and differing degrees of taxon sampling, the topology of deep branches in the amphibian phylogeny remains recalcitrant. Different data sets drawn from different sets of taxa have generally recovered two competing hypotheses about inter-ordinal amphibian relationships, namely that salamanders and caecilians are sister taxa (the Procera hypothesis), or that salamanders and frogs form a monophyletic clade (the Batrachia hypothesis). Yet, which of these mutually exclusive topologies reflects higher order relationships has significant implications for understanding the timescale of amphibian diversification and the relationships among extant tetrapods. This apparent conundrum is exacerbated by the fact that different studies have found relatively strong support for these competing hypotheses. Batrachia and Procera are both supported by non-overlapping suites of morphological characters. Similarly, a large body of phylogenetic research using nuclear, mitochondrial, and ribosomal DNA sequences in amphibians have failed to resolve the Batrachia/Procera issue. In general, mtDNA and rRNA sequences have tended to support Procera, while combined nuclear and mtDNA data sets have lent support for Batrachia. Few studies have used large numbers of nuclear loci to investigate this question, yet those that have provide tantalizing support for Procera. As my main dissertation project, I have generated a massive phylogenomic data set comprising 300 nuclear loci, complete mitochondrial genomes, and complete rRNA sequences for 300 species of amphibians with the aim of resolving the topology of the amphibian phylogeny. In the course of analyzing these data, we have found a signal of strong support for Procera from the vast majority of nuclear loci, whereas analysis of the rRNA and mtDNA sequences provide strong support for Batrachia. INTELLECTUAL MERIT: Here we propose an integrative suite of objectives aimed at reconciling these seemingly conflicting phylogenetic signals arising from analysis of different functional and organellar components of the amphibian genome. Building on amphibian genomic and transcriptomic resources which we have developed for 15 diverse taxa spanning all three orders, we will design a novel gene capture system to enrich for and sequence a diverse suite of nuclear encoded ribosomal protein-coding loci from 300 amphibian taxa. Because these target loci are intimately associated with these conflicting genetic regions, this study will allow us to test hypotheses about molecular evolutionary convergence among different regions of the amphibian genome and the ways in which this can inform our ability to accurately reconstruct phylogeny and to reconcile conflicting topologies from different types of loci in the context of functional molecular genetic associations. BROADER IMPACTS: The proposed project represents a substantial extension beyond co-PI Hime’s thesis research and will contribute directly to the improvement of his dissertation as well as providing opportunities for professional and academic development. This work will provide important avenues for training in bioinformatics and high-throughput sequencing data analysis, key skills in the genomic era. Beyond providing graduate training opportunities, this research will be an excellent vehicle for engaging undergraduate researchers in the process of conducting scientific investigation, and at the University of Kentucky, we are well positioned to engage undergraduates from traditionally under-represented groups here in rural Appalachia. Additionally, this research is of broad interest to evolutionary biologists working across the amphibian Tree of Life and the genomic resources generated here will advance the field of amphibian phylogenetics and contribute to broader-scale initiatives to understand the functional genomic bases of molecular evolutionary convergence and its implications for reconstructing deep branches in the tree of life.