Subgenome Partitioning and Polyploid Genome Evolution in the Loach Family Botiidae (Order Cypriniformes).

Vertebrates have undergone two rounds of whole-genome duplication (WGD), termed 1R and 2R, with a third, teleost-specific duplication (TSGD or 3R) occurring in ray-finned fishes. In the order Cypriniformes, additional lineage-specific WGDs have further contributed to species diversification. While polyploidy is well characterized in species like common carp and goldfish, other polyploid taxa-particularly loaches-remain understudied. Here, high-quality, chromosome-level genome assemblies are presented for two loach species: Sinibotia superciliaris (Golden Chinese Loach) and Parabotia fasciatus (Yichang Sand Loach). By integrating these genomes into a comparative framework with 20 other cypriniform species, key phylogenetic relationships are reconstructed, and introduce a novel subgenome partitioning method (M3). Unlike previous approaches, M3 uses differential sequence divergence to accurately and rapidly assign subgenomes, completing partitioning within minutes and outperforming existing tools. Applying M3, a markedly reduced subgenome is uncovered in the Golden Chinese Loach, with lineage-specific molecular changes in several candidate genes, suggesting potential adaptive significance. This study offers a comprehensive view of polyploidy and subgenome evolution in loaches, highlighting the genomic complexity shaped by repeated WGDs in Cypriniformes and providing valuable resources for future research on vertebrate genome evolution and adaptation.