Integrative Barcoding of Marine Mollusks

Abstract

Marine mollusks constitute one of the most species-rich and ecologically significant invertebrate phyla in benthic and pelagic ecosystems, yet their taxonomic diversity remains substantially undercharacterised due to cryptic species complexes, widespread shell polymorphism, and inadequate reference sequence coverage in public databases. This study applied integrative barcoding -- combining COI, 16S rRNA, and H3 nuclear markers -- to 1,642 marine mollusk specimens from 28 genera representing six classes (Gastropoda, Bivalvia, Cephalopoda, Polyplacophora, Scaphopoda, Aplacophora) sampled from 32 sites across the North Sea, Mediterranean Sea, and northeastern Atlantic (Sweden, Spain, Italy). COI barcoding resolved 94.2% of morphologically identified species (n = 218), with barcode gaps confirmed in 89.4% of species pairs. Integrative analysis combining all three markers identified 34 candidate cryptic species across 22 genera, of which 12 were corroborated by shell morphometric analysis (MANOVA: Wilks' l < 0.58, p < 0.05 in all 12 cases). COI identification success was significantly lower for Bivalvia (88.4%) than Gastropoda (96.8%) due to reference database gaps, particularly in deepwater families. Network-based biogeographic analysis revealed significant genetic breaks at the Strait of Gibraltar (FnST = 0.28-0.62 across 14 species) and between the North Sea and Atlantic (FnST = 0.14-0.38 across 9 species), consistent with known oceanographic barriers. Database contribution of 3,284 new COI sequences substantially improves BOLD coverage for Mediterranean and Nordic mollusks. These results advance the molecular reference framework essential for eDNA-based bioassessment and biodiversity monitoring of European coastal marine ecosystems.