Developing, calibrating and applying a genomic toolbox for species delimitation in Palearctic and tropical amphibians

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Recent years have seen a meteoric rise of ever more powerful high-througput DNA sequencing methods. These can yield data to define evolutionary lineages of organisms at ever finer scales and thus delimite species, but empirical studies comparatively testing the performance of different kinds of markers for this purpose are scarce. In the proposed project we plan assesing the performance of genomic markers in quantitative species delimitation approaches, using amphibians as a model, and with the goal to develop an affordable and efficient genomic toolbox to delimit species in this animal group. We will first generate for a set of 17 species and species complexes of European amphibians high-throughput sequencing data with various methods, i.e., sequencing of transcribed RNA (RNAseq / transcriptomics), hybrid enrichment sequencing, restriction site associated DNA sequencing (ddRADseq), hybridization capture using RAD probes (hyRADseq), and molecular inversion probes (MIP sequencing). The European target species will represent the whole continuum from very young and barely differentiated intraspecific lineages to deeply divergent, morphologically differentiated but sometimes still hybridizing lineages. We will use the current taxonomy of these species, well established by a plethora of previous thorough integrative studies, as benchmark to compare the performance of different sets of markers in species delimitation, based on multispecies coalescent methods. We will test the performance of markers of different methological origin, length, variability, and biological function in correctly delimiting these species of European amphibians. To avoid taxonomic inflation, well-performing markers must not falsely delimit fine-scale intraspecific population clusters as species, but they also should be sensitive enough to identify closely related cryptic species. Based on the empirical data from European amphibians, we will select a set of well-performing markers and bioinformatic procedures for reliable statistical species delimitation in amphibians, aiming for simple and affordable laboratory protocols. We will then apply the selected work protocol to a taxonomically convoluted group of tropical amphibians (frogs of the subgenus Brygoomantis from Madagascar) in the context of a fast-track taxonomic revision during which we envisage describing over 20 new species.