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

近年来，更强大的高通量DNA测序方法迅速崛起。这些可以产生数据，在更精细的尺度上定义生物体的进化谱系，从而界定物种，但比较测试不同类型标记的性能的实证研究很少。在拟议的项目中，我们计划以两栖动物为模型，在定量物种划分方法中评估基因组标记的性能，目标是开发一个负担得起的、有效的基因组工具箱来划分这一动物群体中的物种。我们将首先用不同的方法为一组17个物种和物种复合体生成欧洲两栖动物的高通量测序数据，即转录RNA测序(RNAseq/转录组学)、混合测序、限制位点相关DNA测序(DdRADseq)、使用RAD探针的杂交捕获(HyRADseq)和分子反转探针(MIP测序)。欧洲的目标物种将代表从非常年轻和几乎没有分化的种内谱系到高度分化、形态分化但有时仍在杂交的谱系的整个连续体。我们将使用这些物种的现有分类，作为基准，比较不同的标记集在基于多物种合并方法的物种划分中的性能。我们将测试不同方法学来源、长度、变异性和生物功能的标记在正确划分这些欧洲两栖动物物种方面的表现。为了避免分类学上的膨胀，表现良好的标记不能错误地将细小的种内种群群界定为物种，但它们也应该足够敏感，以识别密切相关的隐蔽物种。基于欧洲两栖动物的经验数据，我们将选择一套性能良好的标记和生物信息学程序来进行可靠的两栖动物统计物种划定，旨在制定简单和负担得起的实验室方案。然后，我们将在快速分类修订的背景下，将选定的工作方案应用于分类上错综复杂的热带两栖动物群体(来自马达加斯加的Brygoomantis亚属青蛙)，在此期间，我们设想描述20多个新物种。

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.