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

评估生殖隔离血统之间的混合物的程度跨越其自然杂交区是一个经验和翔实的方法来界定物种，并允许无与伦比的见解随着时间的推移，生殖隔离的积累，或随着基因组的分歧。我们建议研究混合区的古北两栖动物的血统对现有的数据集将补充分析的15个血统对东部古北使用来自RADseq的核基因座。作为第二个工作包，我们将估计一个时间校准的系统发育树的300种两栖动物物种水平的血统，其中包括约50血统对混合区的信息。我们预测的陡度的混合带渐变和过渡的宽度将规模与时间，并与障碍位点的积累。我们将实证检验预测从人口基因组理论根据生殖隔离增加在一个快速的，非线性的方式后，一定数量的障碍位点的起源。我们的数据集将进一步使我们能够测试哪个因素更好地预测生殖隔离：两个谱系之间的分歧时间或基因组分歧。最后，我们将使用降采样程序来了解需要多少RAD基因座来粗略但可靠地估计杂交区宽度，并通过这些结果，将探索具有成本效益的多重PCR方法的前景，以扩增和测序一组20-50个通用核标记，用于两栖动物的下一代DNA条形码;通过将这种新方法应用于RADseq研究的相同样本，我们将评估其在混合区模式的初步评估和物种划界方面的潜力。

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.