Diversification of eight-barbel loaches of Lefua (Cypriniformes: Nemacheilidae) inferred from mitochondrial DNA

Many freshwater organisms are threatened by human activities, requiring urgent biological data to support conservation. We investigated the diversification of the endangered eight-barbel loaches (genus Lefua) by analyzing mitochondrial cytochrome b and D-loop sequences. Phylogenetic reconstruction identified genetically distinct clades corresponding to all recognized species (excluding L. sayu, whose assignment to Lefua remains uncertain) and intraspecific populations. However, the phylogenetic position of the recently described L. nishimurai-originally the Reihoku population of L. torrentis-contradicts its original species description. Our data show L. nishimurai forming a highly supported clade with the Sanyo and Kii-Shikoku populations of L. torrentis. Diversification within the genus began with a primary divergence into two main lineages, followed by subsequent speciation and the differentiation of seven local populations of L. echigonia, two of L. tokaiensis, and two (or three if L. nishimurai is not valid) of L. torrentis. These results provide a refined evolutionary framework and clarify the taxonomic uncertainties surrounding L. nishimurai, facilitating the establishment of evolutionarily significant units (ESUs) and more effective conservation strategies for these endangered lineages.