Resolving higher-level phylogenetic networks with repeated hybridization in a complex of polytypic salamanders (Plethodontidae: Desmognathus)

Repeated hybridization between incipient lineages is a common feature of ecological speciation and ecomorphological diversification. However, computational constraints currently limit our ability to reconstruct network radiations from gene-tree data. Available methods are limited to level-1 networks wherein reticulations do not share edges, and higher-level networks may be non-identifiable in many cases. We present a heuristic method to recover information from higher-level networks across a range of potentially identifiable empirical scenarios, supported by a theorem and success in simulated data. When extrinsic information indicating the location and direction of recent or ancestral hybridization events is available, our method can yield successful estimates of non-level-1 networks, or at least a reduced possible set thereof. We apply this technique to the Pisgah clade of Desmognathus salamanders, which contains four to seven species exhibiting two discrete phenotypes, aquatic “shovel-nosed” and semi-aquatic “black-bellied” forms in the southern Appalachian Mountains of the eastern United States. Phylogenomic data strongly support a single backbone topology with up to five overlapping hybrid edges. These results suggest an unusual mechanism of ecomorphological hybrid speciation, wherein a binary threshold trait causes hybrids to shift between two microhabitat niches, promoting ecological divergence between sympatric hybrids and parentals. This contrasts with other well-known systems in which hybrids exhibit intermediate, novel, or transgressive phenotypes. Geographically proximate populations of both phenotypes exhibit admixture, and at least two black-bellied lineages have been produced via reticulations between shovel-nosed parentals, suggesting complex transmission dynamics. The genetic basis of these phenotypes is unclear and further data are needed to clarify the nature of selection and speciation in the group.

Methods
Morphological measurements from specimens, data for specimens collected in the wild, AHE data (NEWICK-format trees) from Pyron et al. (2022; Ecol. & Evol.), and GBS data assembled using ipyrad (Eaton & Overcast 2020).

初始谱系间的反复杂交是生态物种形成与生态形态分化的典型特征。但当前受限于计算能力，我们难以从基因树数据中重建网状辐射演化网络。现有方法仅适用于一级网状网络（level-1 networks），即其网状事件不共享演化分支；而高阶网状网络在多数场景下可能无法被识别。我们提出一种启发式方法，可在一系列潜在可识别的实证场景下从高阶网状网络中恢复演化信息，该方法得到了理论定理的支持，并在模拟数据集上验证有效。当存在可指示近期或祖先杂交事件发生位置与方向的外部信息时，本方法可成功推断非一级网状网络，至少可缩小其可能解集的范围。我们将该技术应用于美国东部阿巴拉契亚山脉南部的河溪蝾螈属（Desmognathus）蝾螈皮斯加演化支（Pisgah clade），该支系包含4至7个物种，存在两种离散表型：水生的“铲鼻型”与半水生的“黑腹型”。系统发育组学数据强烈支持一个包含至多5条重叠杂交边的核心拓扑结构。这些结果表明存在一种罕见的生态形态杂交物种形成机制：二元阈值性状使杂交后代在两种微生境生态位之间发生转换，促进了同域分布的杂交后代与亲本类群间的生态分化。这与其他知名研究系统中的情况形成对比，这些系统的杂交后代往往呈现中间型、新型或超亲表型。两种表型的地理邻近种群均存在遗传渐渗现象，且至少有两个黑腹型演化支由铲鼻型亲本间的网状演化事件产生，这暗示了复杂的传递动力学。这些表型的遗传基础仍不明确，尚需进一步的数据以阐明该类群中选择作用与物种形成的本质。

方法
本研究采用的数据包括：标本形态测量数据、野外采集标本的配套记录数据、Pyron等（2022；《Ecology & Evolution》）提供的AHE（锚定杂交富集）数据（NEWICK格式树文件），以及使用ipyrad（Eaton与Overcast，2020）组装的GBS（基因型分型测序）数据。