Extreme mitochondrial population subdivision in southern Appalachian paleoendemic spiders (Araneae: Hypochilidae: Hypochilus), with implications for species delimitation

A prior study of molecular phylogenetic relationships in southern Appalachian Hypochilus taxa revealed unusually high intraspecific mitochondrial sequence divergences, but was limited by small intraspecific sample sizes. A subsequent in-depth population genetic study focused on a single species (H. thorelli Marx 1888), revealing genetic patterns consistent with extremely limited female-based gene flow among rock-outcrop limited populations. Here we extend the study of mitochondrial population genetic structuring to four remaining Appalachian Hypochilus species. Genetic inferences are based on a sample of COI mitochondrial sequences generated for over 250 specimens from 85 sampled locations. This geographic sample comprehensively covers the geographic distributions of all described taxa. Phylogenetic, network-based, and genealogical sorting index analyses reveal ubiquitous genetic structuring in all Hypochilus taxa. A majority of sampled locations possess limited genetic variation, with site-specific haplotypes forming genealogically exclusive microclades, consistent with limited female-based gene flow at the spatial scales sampled. At deeper phylogenetic levels, four of five described species are recovered as monophyletic on mitochondrial gene trees. Hypochilus pococki Platnick 1987 is recovered as paraphyletic, and is fragmented into five genetically divergent, allopatric phylogroups. These phylogroups, and multiple clades within one of the H. pococki phylogroups, are also recovered as distinct clusters in a generalized mixed Yule-coalescent (GMYC) analysis, suggesting the possibility of multiple cryptic species in the Appalachian fauna. However, a qualitative survey of male palpal variation fails to reveal morphological differences that distinguish these highly divergent genetic lineages. We suggest that a nuclear gene tree perspective is ultimately needed to resolve this contrast.