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

从自然杂交区域的混合程度来评估世系之间的生殖隔离是一种经验和信息丰富的方法来划分物种，并允许对生殖隔离随着时间的推移或随着基因组差异的增加而积累的无与伦比的见解。我们建议研究古北陆两栖动物对谱系的杂交带；现有数据集将通过使用RADseq衍生的核位点对东古北的15个谱系对进行分析加以补充。作为第二个工作包，我们将估算300个两栖动物物种水平谱系的时间校准系统发育树，其中包括大约50个谱系对，其中杂交带信息是可用的。我们预测混合带斜的陡度和过渡的宽度将随时间和屏障位点的积累而成比例。根据种群基因组理论的预测，在一定数量的屏障位点出现后，生殖隔离以快速、非线性的方式增加，我们将进行实证检验。我们的数据集将进一步允许我们测试哪个因素更好地预测生殖隔离：分化时间或两个谱系之间的基因组分化。最后，我们将使用降采样程序来了解大致但可靠地估计杂交带宽度需要多少RAD位点，并根据这些结果，将探索一种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，用于下一代两栖动物的DNA条形码；通过将这种新方法应用于RADseq研究的相同样本，我们将评估其在杂交带模式初步评估和物种划分方面的潜力。

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.