Evaluating gene flow and habitat connectivity between Salamandra salamandra lineages across heterogeneous landscapes

Southern Iberia is a geologically, climatically and topographically heterogeneous region associated with speciation events and phylogeographic breaks across a variety of taxa. The Fire salamander (Salamandra salamandra) shows high diversity in this region, with five subspecies co-occurring in a relatively small area. Salamandra s. crespoi and S. s. morenica show a relatively continuous distribution (with punctually occurrences of S. s. gallaica and S. s. bejarae, as this is the southern limit of their ranges) along a west-east axis from the Alentejo in Portugal to Murcia in Spain, with a potential contact zone near the Portuguese-Spanish border, whereas S. s. longirostris is geographically isolated from the other taxa, south of the Guadalquivir river basin. Previous studies based on phylogenetic analyses of mitochondrial DNA sequences have identified S. s. longirostris as the sister group to all remaining S. salamandra lineages, which, in addition to its allopatric distribution, and specific morphology, has led some authors to propose full species status for S. s. longirostris. However, no previous studies have assessed patterns of gene flow or environmental differences among neighboring Salamandra subspecies. Here we combined genetic (mtDNA, nuclear genealogies and microsatellites) and spatial data (climate and landcover) in an integrative approach to: (1) delineate the distribution of the studied subspecies; (2) quantify gene flow between them; (3) assess potential environmental differences between the two main southern Iberian lineages, (4) characterize patterns of genetic diversity and structure for each southern Iberian subspecies, and (5) evaluate connectivity among S. s. longirostris populations. While mtDNA analyses recovered five clades associated with the five subspecies, nuclear genealogies show allele sharing among all taxa that could be the result of incomplete lineage sorting, or introgression events, under the influence of specific evolutionary mechanisms (e.g. sex-biased dispersal or mitochondrial selection). Microsatellite analyses found no evidence of gene flow between the allopatric isolated S. s. longirostris and other subspecies. Additionally, nuclear admixture was detected in two different contact zones, one involving S. s. crespoi and S. s. gallaica, and another among these two subspecies together with S. s. morenica, presenting again a possible scenario for nuclear introgression under the hypothesis of male-biased dispersal. Genetic structure analysis among S. s. longirostris populations, together with habitat connectivity analysis, detected a potential isolated population in the eastern extent of S. s. longirostris distribution range, while for other population no lack of connectivity was identified. Geographic distance was identified as the main driver of genetic differentiation Evaluating gene flow and habitat connectivity between S. salamandra lineages across heterogeneous landscapes FCUP v among S. s. longirostris populations, however it could be a byproduct of the extremely isolated eastern population. Ecological niche models (ENMs) for the present identified the Guadalquivir river basin as a highly unsuitable area, separating S. s. longirostris from neighboring S. salamandra lineages. Past projections of ENMs (LIG, LGM, Mid-Holocene), together with current projections, identified two isolated refugia in southern Iberia, associated with this two different S. salamandra lineages. Regarding niche comparisons, S. s. longirostris niche was identified as non-equivalent to those of other subspecies. Our results provide a comprehensive view of the historical and contemporary processes affecting biodiversity across southern Iberian Peninsula. This study supports the existence of multiple isolated refugia in southern Iberia, allowing S. salamandra to persist in this area during past climatic fluctuations, fitting in a scenario of range contractions leading to allopatric isolation (associated with speciation processes), and posteriorly expansions resulting in secondary-contact zones (allowing gene flow). Currently, contact zones involving the parapatrically distributed subspecies (S. s. crespoi, S. s. morenica and S. s. gallaica), showing patterns of nuclear introgression possibly caused by male-biased dispersal. Salamandra s. longirostris was allopatrically isolated from other subspecies, with the Guadaquivir river basin identified as a barrier to gene flow, which seems to be playing an important role in species formation in southern