Phylogeography, hybridization, and species discovery in the Etheostoma nigrum complex (Percidae: Etheostoma: Boleosoma)

The history of riverine fish diversification is largely a product of geographic isolation. Physical barriers that reduce or eliminate gene flow between populations facilitate divergence via genetic drift and natural selection, eventually leading to speciation. For freshwater organisms, diversification is often the product of drainage basin rearrangements. In young clades where the history of isolation is the most recent, evolutionary relationships can resemble a tangled web. One especially recalcitrant group of freshwater fishes is the Johnny Darter (Etheostoma nigrum) species complex, where traditional taxonomy and molecular phylogenetics indicate a history of gene flow and conflicting inferences of species diversity. Here we assemble a genomic dataset using double digest restriction site associated DNA (ddRAD) sequencing and use phylogenomic and population genetic approaches to investigate the evolutionary history of the complex of species that includes E. nigrum, E. olmstedi, E. perlongum, and E. susanae. We reveal and validate several evolutionary lineages that we delimit as species, highlighting the need for additional work to formally describe the diversity of the Etheostoma nigrum complex. Our analyses also identify gene flow among recently diverged lineages, including one instance involving E. susanae, a localized and endangered species. Phylogeographic structure within the Etheostoma nigrum species complex coincides with major geologic events, such as parallel divergence in river basins during Pliocene inundation of the Atlantic coastal plain and multiple northward post-glacial colonization routes tracking river basin rearrangements. Our study serves as a nuanced example of how low dispersal rates coupled with geographic isolation among disconnected river systems in eastern North America has produced one of the worlds freshwater biodiversity hotspots.

河川鱼类的多样性演化历史，在很大程度上是地理隔离的产物。能够降低乃至消除种群间基因流的物理屏障，可通过遗传漂变与自然选择推动种群分化，最终促成物种形成。对于淡水生物而言，物种分化往往是流域地貌重组的结果。在隔离历史较为新近的年轻演化支中，物种间的演化亲缘关系往往如同缠结的蛛网般难以厘清。

其中一类尤为棘手的淡水鱼类类群为约翰尼镖鲈（Johnny Darter, *Etheostoma nigrum*）物种复合群，传统分类学与分子系统发育学的研究均显示该类群存在基因流现象，且对物种多样性的推论存在冲突。

本研究通过双酶切限制性位点相关DNA测序（double digest restriction site associated DNA, ddRAD）构建基因组数据集，并运用系统基因组学与群体遗传学方法，对包含*E. nigrum*、*E. olmstedi*、*E. perlongum*以及*E. susanae*在内的该物种复合群的演化历史展开探究。

研究团队揭示并验证了多个可被界定为独立物种的演化谱系，凸显出对约翰尼镖鲈物种复合群的多样性开展正式分类描述的必要性。此外，本次分析还鉴定出近期分化的谱系间存在基因流，其中一例涉及*E. susanae*——一种局域分布的濒危物种。

约翰尼镖鲈物种复合群内的系统生物地理学结构，与重大地质事件高度契合，例如上新世时期大西洋沿岸平原被淹没期间各流域发生的平行分化，以及追踪流域地貌重组的多条冰期后向北殖民路线。

本研究为阐释北美东部水系间因扩散速率低下加之地理隔离，如何造就全球淡水生物多样性热点地区之一，提供了一个细致入微的范例。