Hybrid zones among Palearctic amphibian lineages as a model to understand temporal patterns of species formation and refine methods of species delimitation

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Assessing reproductive isolation between lineages from the extent of admixture across their natural hybrid zones is an empirical and informative approach to delimit species, and allows unparalleled insights into the buildup of reproductive isolation over time, or with increasing genomic divergence. We propose to study hybrid zones of pairs of lineages of Palearctic amphibians; an existing data set will be complemented by analysis of 15 lineage pairs of the Eastern Palearctic using nuclear loci derived from RADseq. As a second work package, we will estimate a time-calibrated phylogenetic tree of 300 amphibian species-level lineages, including about 50 lineage pairs for which hybrid zone information is available. We predict the steepness of the hybrid zone cline and the width of the transition will scale with time, and with the accumulation of barrier loci. We will empirically test predictions from population genomic theory according to which reproductive isolation increases in a fast, non-linear fashion after the origin of a certain number of barrier loci. Our data set will furthermore allow us to test which factor better predicts reproductive isolation: divergence time or genomic divergence between two lineages. Finally, we will use downsampling procedures to understand how many RAD loci are needed for roughly but reliably estimating hybrid zone width, and informed by these results, will explore the prospects of an cost-effective multiplex PCR approach to amplify and sequence a set of 20-50 universal nuclear markers for next-generation DNA barcoding of amphibians; by applying this novel approach to the same samples studied by RADseq, we will assess its potential for initial assessments of hybrid zones patterns, and for species delimitation in general.