Rapid speciation of Chinese hypogean fishes driven by paleogeoclimatic and morphological adaptations

Major geoclimatic events trigger clade divergence, shaping diversification patterns. However, the influence of historical geoclimatic events on the diversification of subsurface biota remains poorly understood. This study investigates the phylogeny and evolutionary history of under-recognized hypogean fishes in the Nemacheilidae family (HFN) in southwestern China, using mitogenome and nuclear gene sequencing. Our phylogeny supports the current genus-level classification but reveals conflicts between mitochondrial and nuclear gene topologies, suggesting past hybridization events. The ancestor of the HFN originated in eastern China-Korean Peninsula-Japanese Islands, north of northwest China-Mongolian Plateau, the Qinghai-Xizang (Tibetan) Plateau-Hengduan Mountains during the late Eocene (~36 million years ago [Mya]) and early Miocene (~16 Mya), and dispersed twice into the karst region of southwestern China. An ancient radiation event occurred from 22.44 Mya to 12.25 Mya. In situ diversification is the major speciation event, originating around 30 Mya and increasing sharply at ~11 Ma, with 3 peaks at ~7 Mya, ~3 Mya, and 1 Mya, and 2 valleys at ~5 Mya and ~2 Mya. Ancestral state reconstruction suggests at least 4 independent origins for the colorless, eye-blind, and troglobitic species morphs, as opposed to 2 events for the caudal adipose keel, and that these traits have undergone multiple reversals. These results highlight the role of geological processes and climatic events in the evolution of hypogean fishes and provide insights for conservation efforts, particularly in specialized cave habitats.