An integrative insight into the diversity, distribution, and biogeography of the freshwater endemic clade of the (Teleostei: Gobiidae) in the Caucasus biodiversity hotspot.

Freshwater habitats of the Caucasus biodiversity hotspot represent a center of endemism for the gobiid genus Iljin, 1927. Hitherto, large-scale molecular studies, owing to restricted taxon and geographical sampling, have failed to give an elaborate picture of diversity and evolutionary history of these species. Here, to contribute to filling this gap, we assessed taxonomic diversity, phylogeography and evolutionary history for the south Caspian populations of presently classified as and , using an integrative taxonomic approach comprising an entire geographic range sampling, and analyses of mitochondrial DNA haplotypes, the head lateral line system, otolith shape, and meristic and morphometric variation. All freshwater samples of the belong to a monophyletic clade with two main subclades: a small subclade confined to the upper Sefidroud sub-basin including the type locality of and a large subclade with three geographically constrained haplogroups (Hg1, Hg2, and Hg3), comprising the rest of the distribution. Hg1 showed an eastern distribution including the type locality of , while Hg2 and Hg3 are sister groups with central and western-central distributions, respectively. The freshwater clade diverged from during the Tyurkyanian low stand (~150 m b.s.l. lasting ~0.1 Myr), while the divergence of and and radiations within took place during the Bakunian high stand (up to 50 m a.s.l. lasting ~378-480 kya). Species delimitation analyses indicated two distinct species, corresponding to each main subclade. Although the otolith shape and lateral line analyses did not reflect with phylogeographic pattern, PCA and DFA plots of meristic and morphometric data showed a clear separation of the two major subclades corresponding to and , suggesting the presence of significant morphological variation meriting formal taxonomic recognition. Overall, our findings (i) reveal the presence of two freshwater endemic species in the , and pending further investigation, hypothesize the presence of a third cryptic species; (ii) revise and document a narrow distributional range and low diversity for , in contrast to a wider distributional range and high diversity for ; (iii) suggest that the climatic oscillations of the Pleistocene were associated with the cladogenesis within the ; and (iv) allowed for the recognition of conservation units and proposition of management measures.