Evidence for ancient interspecific gene flow through hybridization has been reported in many animal and plant taxa based on genetic markers. The study of genomic patterns of closely related species with allopatric distributions allows the assessment of the relative importance of vicariant isolating events and past gene flow. Here, we investigated the role of gene flow in the evolutionary history of four closely related freshwater fish species with currently allopatric distributions in western Iberian rivers—Squalius carolitertii, S. pyrenaicus, S. torgalensis and S. aradensis—using a population genomics dataset of 23,562 SNPs from 48 individuals, obtained through genotyping by sequencing (GBS). We uncovered a species tree with two well-differentiated clades: (i) S. carolitertii and S. pyrenaicus; and (ii) S. torgalensis and S. aradensis. By using D-statistics and demographic modelling based on the site frequency spectrum, comparing alternative demographic scenarios of hybrid origin, secondary contact and isolation, we found that the S. pyrenaicus North lineage is likely the result of an ancient hybridization event between S. carolitertii (contributing ~84%) and S. pyrenaicus South lineage (contributing ~16%), consistent with a hybrid speciation scenario. Furthermore, in the hybrid lineage, we identify outlier loci potentially affected by selection favouring genes from each parental lineage at different genomic regions. Our results suggest that ancient hybridization can affect speciation and that freshwater fish species currently in allopatry are useful to study these processes.

基于遗传标记的许多动植物类群中已经报道了古代种间基因通过杂交流动的证据。研究具有异域分布的密切相关物种的基因组模式可以评估变异分离事件和过去基因流的相对重要性。在这里，我们研究了基因流在伊比利亚西部河流中目前异域分布的四种密切相关的淡水鱼类进化史中的作用——Squalius carolitertii、S. pyrenaicus、S. torgalensis和S. aradensis— 使用来自 48 个个体的 23,562 个 SNP 的群体基因组学数据集，通过测序基因分型 (GBS) 获得。我们发现了具有两个分化良好的进化枝的物种树：（i）S. carolitertii和S. pyrenaicus；(ii) S. torgalensis和S. aradensis。通过使用基于站点频谱的 D 统计和人口统计模型，比较混合起源、二次接触和隔离的替代人口统计情景，我们发现S. pyrenaicus North 谱系可能是S. carolitertii之间古老杂交事件的结果（贡献约 84%）和S. pyrenaicus南方谱系（贡献约 16%），与混合物种形成情景一致。此外，在杂交谱系中，我们确定了可能受到不同基因组区域的每个亲本谱系的选择有利于基因影响的异常位点。我们的研究结果表明，古代杂交会影响物种形成，而目前异源中的淡水鱼类对研究这些过程很有用。