Species are the fundamental unit of biodiversity studies. However, many species complexes are difficult to delimit, especially those characterized by complicated patterns of population structure. Salamanders in the family Plethodontidae often form species by slowly fragmenting across a landscape over space and time. They thus provide many examples of species complexes in which gradual Darwinian evolution has resulted in multiple units of varying degrees of differentiation, including incompletely separated lineages. Herein, we report on a molecular systematic investigation of woodland salamanders in the Plethodon wehrlei group, a group that has recently been split from two species into five species. To quantify patterns of genetic variation, we collected genetic samples from 24 individuals from 20 populations, including all species and representing a carefully selected subset of previous work. From these samples, we obtained genomic data by using anchored hybrid enrichment, resulting in 319 loci averaging 1300 bp in length. Biallelic single-nucleotide polymorphisms were randomly selected from 316 of these loci for some analyses. We examined patterns of genetic structure by using a combination of multivariate statistics and methods based on evolutionary models (such as the Bayesian program STRUCTURE) and found that all of the recognized species formed genetic clusters; however, P. wehrlei and P. punctatus were relatively weakly differentiated and STRUCTURE identified three separate clusters within P. jacksoni. Species trees inferred using the weighted accurate species tree algorithm (wASTRAL), Bayesian phylogenetics and phylogeography (BPP), and TreeMix all recovered the same topology, with P. dixi sister to the other taxa, which included a northern clade (P. wehrlei, P. punctatus, P. pauleyi) and a southern clade (P. jacksoni, with three separate groups). TreeMix only inferred one gene flow event. We evaluated the candidate species by using BPP and the genealogical divergence index (gdi). Although BPP delimited all candidate species with strong support (all posterior probabilities = 1.0), the gdi only strongly supported P. dixi and P. pauleyi, both of which have only been recently described. We discuss the difficult problem of species delimitation in groups that form species via range fragmentation. We also provide a vision for future research, with the aim of better testing and diagnosing the species diversity within the P. wehrlei group.

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