COI Barcoding Unveils Hidden Fish Diversity and Potential Cryptic Lineages in Inner Mongolia, China

ABSTRACT The freshwater fish fauna of the Inner Mongolia Plateau remains poorly characterized at the molecular level despite its unique biogeographic setting, which combines the Yellow River (Huang He) drainage with multiple isolated endorheic basins. To address this knowledge gap, we conducted systematic sampling at 99 sites across major river systems in Inner Mongolia from 2021 to 2025 and generated 984 cytochrome c oxidase, subunit 1 gene (COI) barcode sequences, which were combined with 96 publicly available sequences to assemble a dataset of 1080 sequences. Using morphological identification together with two tree‐based species delimitation methods (mPTP and GMYC), we recovered 70 molecular operational taxonomic units (OTUs), corresponding to 62 morphologically identified species and three unidentified lineages. The detected species represent only 54.39% of historically recorded species in the region, while multiple widespread species (e.g., Abbottina rivularis , Barbatula toni , and Rhinogobius cliffordpopei ) exhibited substantial genetic differentiation and were partitioned into multiple independent OTUs, revealing overlooked cryptic diversity. This study demonstrates the effectiveness of COI barcoding for rapid regional fish diversity assessment and cryptic species discovery, while also highlighting the limitations of single‐locus markers in resolving recently diverged lineages. The barcode reference dataset and the cryptic lineages identified here provide critical baseline data for aquatic biodiversity conservation, fisheries management, and environmental impact assessments in this ecologically fragile region.