High-Resolution Integrative Delimitation of Intertidal Limpets via Multi-Locus Barcodes and SEM Morphology

Limpets are marine gastropod molluscs well adapted to intertidal rocky environments, yet their taxonomic resolution remains challenging due to extensive morphological convergence and the presence of cryptic species. In this study, we applied an integrative taxonomic framework combining multi-locus DNA barcoding and fine-scale morphological characterization to clarify species boundaries within three families of limpets—Nacellidae, Lottiidae, and Siphonariidae. A total of 132 individuals collected from six coastal sites in Shenzhen and adjacent areas of southern China were analyzed using four mitochondrial and nuclear markers (COI, 16S rRNA, Cytb, and 28S rRNA), together with scanning electron microscopy (SEM) observations of radular morphology. Molecular analyses identified nine distinct species across five genera. Kimura two-parameter (K2P) distance analyses revealed clear barcode gaps in 16S rRNA, Cytb, and 28S rRNA genes, particularly among Cellana and Nipponacmea, whereas COI exhibited stronger discriminatory power within Siphonaria. Moreover, our study provides newly generated Cytb sequences for Lottia luchuana, Siphonaria atra, Siphonaria sp. and Siphonaria sirius, enriching the public genetic reference database and explaining the lack of corresponding records in previous BLAST searches. SEM-based radular features displayed consistent interspecific variation that corroborated molecularly defined clades, offering reliable morphological evidence for species delimitation. Collectively, our findings highlight the value of integrating lineage-specific molecular markers with detailed morphological analyses to resolve taxonomic ambiguities in morphologically conservative marine gastropods. Furthermore, this approach strengthens molecular reference resources essential for future biodiversity and evolutionary research on intertidal limpets.