Candidate-species delimitation in Desmognathus salamanders reveals gene flow across lineage boundaries, confounding phylogenetic estimation and clarifying hybrid zones.

Dusky Salamanders (genus Desmognathus ) currently comprise only 22 described, extant species. However, recent mitochondrial and nuclear estimates indicate the presence of up to 49 candidate species based on ecogeographic sampling. Previous studies also suggest a complex history of hybridization between these lineages. Studies in other groups suggest that disregarding admixture may affect both phylogenetic inference and clustering-based species delimitation. With a dataset comprising 233 Anchored Hybrid Enrichment (AHE) loci sequenced for 896 Desmognathus specimens from all 49 candidate species, we test three hypotheses regarding (i) species-level diversity, (ii) hybridization and admixture, and (iii) misleading phylogenetic inference. Using phylogenetic and population-clustering analyses considering gene flow, we find support for at least 47 candidate species in the phylogenomic dataset, some of which are newly characterized here while others represent combinations of previously named lineages that are collapsed in the current dataset. Within these, we observe significant phylogeographic structure, with up to 64 total geographic genetic lineages, many of which hybridize either narrowly at contact zones or extensively across ecological gradients. We find strong support for both recent admixture between terminal lineages and ancient hybridization across internal branches. This signal appears to distort concatenated phylogenetic inference, wherein more heavily admixed terminal specimens occupy apparently artifactual early-diverging topological positions, occasionally to the extent of forming false clades of intermediate hybrids. Additional geographic and genetic sampling and more robust computational approaches will be needed to clarify taxonomy, and to reconstruct a network topology to display evolutionary relationships in a manner that is consistent with their complex history of reticulation.