SNP tools for studying the ecology and evolution of rainforest frogs

Our team presents new SNP tools for studying the ecology and evolution of rainforest stream frogs. We begin by outlining a relatively inexpensive and productive cDNA-based method of marker discovery, and then characterise levels of polymorphism for 10 new nuclear introns in multiple co-distributed frog species. We survey 3944bp of nuclear sequence, across 144 individuals representing 6 hylid frog species, identifying 30-199 informative SNPs per species. While the nuclear marker discovery routine we present draws heavily upon previous approaches, the technique remains under-utilised in molecular labs, and could rapidly expand access into the genomes of non-model organisms for ecological and historical research. Our strategy relies upon conserved exon priming to ensure broad utility across species for comparative studies. The discovery process combines extensive field, laboratory and bioinformatic components, including collection and preservation of organismal RNA samples, cDNA library construction, gene annotation, primer design and PCR optimisation. Most nuclear introns amplify across a broad range of co-distributed and speciose hylid frogs, often using the same standardised PCR protocol. We showcase the utility of these new SNPs tools in two ongoing research applications in comparative demography and the resolution of species boundaries. The first application compares gene estimates of population histories (divergence times and effective population sizes) to predictions from paleo-habitat distribution models, to assess how rainforest frog sensitivity to historical climate change varies with respect o current environmental niche breadth. Second, we re-assess the species boundaries of two recently described frog taxa, and test the hypothesis these forms segregate by forest habitat type, using comparative data from genes, morphology and environmental distributions. The marker discovery methods and data collection presented highlight research from several undergraduates sponsored by mentoring programs, including UC Berkeley’s Undergraduate Research Apprentice Program (KC) and NSF’s Research Experience for Undergrads (RB, JV).