Reconstructing the phylogeny and evolutionary history of freshwater fishes (Nemacheilidae) across Eurasia since early Eocene

Eurasia has undergone substantial tectonic, geological, and climatic changes throughout the Cenozoic, primarily associated with tectonic plate collisions and a global cooling trend. The evolution of present-day biodiversity unfolded in this dynamic environment, characterised by intricate interactions of abiotic factors. However, comprehensive, large-scale reconstructions illustrating the extent of these influences are lacking. We reconstructed the evolutionary history of the freshwater fish family Nemacheilidae across Eurasia and spanning most of the Cenozoic on the base of 471 specimens representing 279 species and 37 genera. Molecular phylogeny using 6 genes uncovered six major clades within the family, along with numerous unresolved taxonomic issues. Dating of cladogenetic events and ancestral range estimation traced the origin of Nemacheilidae to Indochina around 48 million years ago. Subsequently, one branch of Nemacheilidae colonised eastern, central, and northern Asia, as well as Europe, while another branch expanded into the Burmese region, the Indian subcontinent, the Near East, and northeast Africa. These expansions were facilitated by tectonic connections, favourable climatic conditions, and orogenic processes. Conversely, aridification emerged as the primary cause of extinction events. Our study marks the first comprehensive reconstruction of the evolution of Eurasian freshwater biodiversity on a continental scale and across deep geological time.

Methods
471 samples of more than 250 species from 37 genera of the family Nemacheilidae were examined, including the set of sequences of 364 specimens analysed in our laboratory and sequences of 107 specimens obtained from GenBank. We have sequenced one mitochondrial and five nuclear genes. We have performed various phylogenetic analyses, including maximum likelihood analyses in IQ-TREE, Bayesian inference in MrBayes, species tree was constructed using ASTRAL-III. The divergence time estimations were performed in BEAST2.