Guizhou is an important ecological barrier in the upper reaches of the Yangtze River and the Pearl River basins with abundant fish species. However, fish from these regions are threatened by anthropogenic activities, including overfishing and habitat destruction. Here, we assessed the fish diversity including more than half of the species from the region using DNA barcoding (partial sequence of cytochrome c oxidase subunit I (COI) gene). We obtained 800 mitochondrial COI barcode sequences from 82 genera, 18 families and 8 orders of fishes. The average Kimura two-parameter (K2P) distances within species and genera were 0.35% and 5.44%, respectively. The average interspecific distance was 15.54 times higher than the mean intraspecific distance. Moreover, DNA barcodes revealed 175 operational taxonomic units (OTUs) based on consensus demarcation schemes. Barcoding gaps were detected in 94.81% of morphospecies. Three fish species (Schistura fasciolata, Vanmanenia pingchowensis, and Misgurnus dabryanus) have considerable intraspecific variability, and each was divided into multiple molecular operational taxonomic units (MOTUs) using molecular definition methods (Automatic Barcode Gap Discovery, Refined Single Linkage, General Mixed Yule Coalescent, and Poisson Tree Processes), possibly indicating the occurrence of cryptic species. Altogether, our study reveals the complex diversity of fish species in Guizhou Province, serving as a reference for the conservation and monitoring of fish species in this region.

Sinocyclocheilus multipunctatus is a second-class nationally protected wild animal in China. As one of the cavefish, S. multipunctatus has strong adaptability to harsh subterranean environments. In this study, we used PacBio SMRT sequencing technology to generate a first representative full-length transcriptome for S. multipunctatus. Sequence clustering analysis obtained 232,126 full-length transcripts. Among all transcripts, 40,487 were annotated in public databases, while 70,300 microsatellites, 2384 transcription factors, and 16,321 long non-coding RNAs were identified. The phylogenetic tree showed that S. multipunctatus shows a closer relationship to Carassius auratus and Cyprinus carpio, phylogenetically diverging from the common ancestor ~14.74 million years ago (Mya). We also found that between 15.6 and 17.5 Mya, S. multipunctatus also experienced an additional whole-genome duplication (WGD) event, which may have promoted the species evolution of S. multipunctatus. Meanwhile, the overall rates of evolutionary of polyploid S. multipunctatus were significantly higher than those of the other cyprinids, and 220 positively selected genes (PSGs) were identified in two sub-genomes of S. multipunctatus. These PSGs are likely to fulfill critical roles in the process of adapting to diverse cave environments. This study has the potential to facilitate future investigations into the genomic characteristics of S. multipunctatus and provide valuable insights into revealing the evolutionary history of polyploid S. multipunctatus.