Butterfly hillstream loach (Beaufortia kweichowensis), a benthic fish in torrential mountain streams, possesses a totally flat ventrum, flattened craniofacial and body skeletons, and enlarged paired fins covered by substantially small keratinous structures. However, little is known about the genetic basis of these specialized morphological adaptations. Here we present a 448.52 Mb genome assembly with contig N50 length of 5.53 Mb by integrating Illumina short-read sequencing, Nanopore long-read sequencing and HiC-based chromatin map. Demographic history reconstruction of the butterfly hillstream loach reveals that the population dynamics is correlated with the different stages of uplifting of the Tibetan Plateau. Comparative genomic analysis finds evidence of 6 keratin genes in butterfly hillstream loach evolving under positive selection. Within these genes, 2 keratin genes exhibit species-specific and divergent amino acid changes, suggesting a role in the formation of the unculi. Additionally, a series of positively selected genes, rapid evolving genes, specific variant genes and expanded gene families are found, including genes related to Hedgehog, Notch, and BMP pathways, which may be involved in craniofacial development. These findings may have important implications for understanding the genetic basis of phenotypic adaptation to torrential mountain stream life.

In the present study, a chromosome-level reference genome for the butterfly hillstream loach was generated using a hybrid strategy of Illumina short-read sequencing, long-read Oxford Nanopore sequencing and HiC-based chromatin map.