The status of COI and 12S rRNA DNA barcode reference libraries for freshwater fish in South Africa: Implications for future eDNA projects

Environmental DNA metabarcoding (eDNA) is a rapidly emerging field in which high-throughput sequencing is used to catalogue the biodiversity of ecosystems through the amplification of DNA extracted from environmental samples (water, air, faeces and soil). Although eDNA has strong links to DNA barcoding, the molecular marker most often used to detect vertebrates in eDNA studies is a portion of the mitochondrial 12S ribosomal RNA (12S rRNA) and not the standard cytochrome oxidase I (COI) marker used in traditional DNA barcoding. eDNA methods rely on a comprehensive reference library to link sequence data to species, which are often lacking in hyper-diverse countries such as South Africa. In this study, we review the present state of DNA barcode reference databases for both 12S rRNA and COI for freshwater fish (native and introduced) found in South African aquatic systems. Analysis of DNA records available on GenBank and the Barcode of Life Database (BOLD) revealed incomplete records of the examined taxa for both markers. Our findings showed that 34 species, 6 genera and 0 families of native South African freshwater fish lack COI barcode records, while 86 species, 22 genera and 8 families lack 12S rRNA records. Unlike the native freshwater fish, the non-native fish all had barcode records available for both COI and 12S rRNA. Producing comprehensive reference libraries for both markers is an important first step in developing an eDNA protocol for the non-invasive monitoring of native and non-native freshwater fish in South Africa.