Molecular phylogeny and species delimitation unravel climate-driven diversification and cryptic radiation in Iranian steppe Vipers (Vipera ursinii-renardi complex)

Accurate delineation of biodiversity units is essential for effective conservation, particularly in species complexes with cryptic diversity and fragmented distributions. In this study, we investigated the genetic structure, phylogenetic relationships, and phylogeographic history of Iranian populations within the Vipera ursinii-renardi complex by analysing two mitochondrial genes (cyt b, ND4) and three nuclear loci (PRLR, BDNF, NT3) from 12 individuals sampled across the species' known range. Multilocus phylogenetic analyses revealed two well-supported evolutionary lineages, including a previously unreported northwestern lineage (Lineage NW), which is sister to the Central Alborz lineage (V. ebneri sensu stricto; Lineage CA). Genetic clustering and haplotype network analyses further supported the distinctiveness of these lineages, indicating limited or no gene flow between them. Molecular-clock dating suggests that the divergence between Lineage CA and Lineage NW occurred during the Late Pleistocene, likely driven by climatic oscillations that promoted ecological isolation. Based on reciprocal mitochondrial monophyly, nuclear differentiation, and geographic separation, we recognize Lineage CA and Lineage NW as two evolutionarily significant units in Iran. Recognizing and conserving this cryptic diversity is vital for preserving the evolutionary legacy of Iranian steppe vipers and for guiding effective conservation strategies under increasing environmental and anthropogenic pressures.