Enhancing the 12S rRNA reference library of South African chondrichthyans to aid in eDNA metabarcoding

The rapid expansion of environmental DNA (eDNA)-based biodiversity monitoring has heightened the need for accurate and well-curated taxonomic reference databases. Although mitochondrial cytochrome oxidase I (COI) is widely used, the mitochondrial 12S ribosomal RNA gene (12S rRNA) has increasingly demonstrated strong taxonomic assignment success in chondrichthyan metabarcoding applications. The South African coastline supports one of the most diverse chondrichthyan faunas globally. However, as in many regions, coverage of 12S rRNA reference databases has remained limited. Well-represented reference libraries are critical for improving the accuracy of eDNA-based species detection. Here, we aimed to expand the regional 12S rRNA reference database for South African chondrichthyans, thereby supporting both local and global eDNA applications for widely distributed species.

A total of 36 chondrichthyan species, represented by 69 individuals, were sequenced to expand the 12S rRNA reference library for sharks, rays and chimaeras from South Africa's Indian and Atlantic Ocean coastlines. A newly developed primer set (V05F_898E / teleoR_Elas), designed to overlap with the commonly used Elas02 region, achieved consistent amplification across diverse lineages. The new data increased regional reference coverage from 50.3% to 69.1% and added 18 species globally. Sequence authenticity was confirmed through comparison with GenBank, and Bayesian phylogenetic analyses showed that 97.2% of sequences clustered within their expected family-level lineages, although some uncertainties remained in groups with low mitochondrial divergence or complex taxonomic histories. Percent identity analyses revealed high intraspecific similarity and clear interspecific separation across taxa. However, comparative in silico analyses of the shorter Elas02 fragment demonstrated reduced phylogenetic resolution and less consistent clustering among closely related taxa. While the Elas02 region is suitable for accurate amplicon sequence variant (ASV) matching in eDNA metabarcoding, the longer 12S rRNA fragment generated here provides improved phylogenetic resolution and may aid in the identification of unknown or ambiguous ASVs.

The expanded 12S rRNA reference library and associated phylogenetic analyses expanded genetic resources for South African chondrichthyans and support global applications. The newly developed marker can be incorporated in future eDNA studies dealing with elasmobranchs, since it can have the added benefit of providing more phylogenetic resolution than the short Elas02 fragment.