The effect of hybrids on phylogenomics and subspecies delimitation in Salamandra, a highly diversified amphibian genus.

Traditional methods of phylogenetic reconstruction and species delimitation may be impeded by frequent hybridization among lineages. In this study, we conducted phylogenetic and clustering analyses of ddRAD genomic data on the entire genus Salamandra, which includes six species and over 25 subspecies of terrestrial salamanders. We expanded previous datasets to include missing subspecies and incorporated new samples, with an emphasis on secondary contact zones. Results obtained from a full dataset of 392 individuals (356,874 bp; 24,192 SNPs) were compared with those obtained after excluding substantially admixed individuals (n = 95; 835,467 bp; 51,557 SNPs) to explore the consequences of introgression on phylogenetic inference and taxonomic arrangement of subspecies. We found conflicting phylogenetic placements for taxa represented by many admixed individuals (identified by clustering ancestries). In contrast, a time-calibrated tree constructed without hybrids largely agrees with previous phylogenetic hypotheses. Within S. atra, we found paraphyly of S. atra atra, suggesting an additional candidate subspecies. Within S. infraimmaculata, two lineages are assignable to known subspecies and we additionally identified a third, deeply diverged lineage sampled near the Turkish/Syrian border. In S. algira, we found limited admixture between the subspecies S. a. tingitana and S. a. splendens despite their geographic proximity. Finally, within S. salamandra, we detected significant levels of hybridization between subspecies, which blurred their phylogenetic relationships, although the removal of admixed samples in subset analyses clarified the situation in most cases. Monophyly was recovered for subspecies that were previously found paraphyletic, including S. s. salamandra, S. s. gallaica, and S. s. fastuosa. Salamandra s. "alfredschmidti" was confirmed to be a junior synonym of S. s. bernardezi. Previously disputed subspecies, like S. s. "molleri" and S. s. "hispanica", correspond to separated lineages but are affected by admixture with other lineages. Further newly identified candidate subspecies in S. salamandra included a southern lineage within S. s. wer-neri and a western lineage within S. s. bernardezi. Finally, we re-evaluate the status of recognized subspecies in Salamandra, based on evidence from multiple delimitation criteria. Given that the evolutionary history could not be resolved for all subspecies, we highlight taxa within Salamandra that warrant further molecular examination and taxonomic revision, notably within the S. s. gallaica/"molleri"/bejarae complex. This study illustrates the impact of hybridization in phylogenetic analyses and its downstream effects in the identification of conservation units and their naming in the Linnean classification. [ABSTRACT FROM AUTHOR]