Abstract
South Africa has approximately 300 functional estuaries that are categorized into estuarine lakes, lagoons, and bays; predominantly open estuaries; large and small temporarily closed estuaries; large and small fluvially dominated estuaries; predominantly arid estuaries, and micro-estuaries. Being subjected to both freshwater and marine input, estuaries are inhabited by macroinvertebrate species that are endemic to these systems and are absent from both rivers and the ocean. Despite their ecological importance to aquatic life, South African estuaries are highly threatened, with many now being considered functionally degraded due to an increase of anthropogenic activities in the surrounding areas making it increasingly important to conserve estuarine habitats. One of the key factors that can contribute to the conservation and management of these estuarine habitats is understanding the community assemblages of macroinvertebrates in estuaries. Many typical estuarine macroinvertebrates are missing from DNA repositories despite their ecological importance. This study made use of traditional DNA barcoding to generate a reference library of macroinvertebrate species and used eDNA metabarcoding to assess the completeness of the reference library. DNA barcoding was successful in generating novel sequence submissions and eDNA highlighted that many species were still undocumented in DNA libraries. DNA barcoding was successful in generating 11 novel sequence submissions to BOLD and eDNA metabarcoding managed to generate 80 sequences of 59 species, however the eDNA results showed the classification of several sister taxa that occur outside of the range of South Africa. Since the CRUX database blasts sequences against known taxa on GenBank, the identification of sister taxa that commonly occur outside of South Africa is possibly a result of missing data on South African species in DNA repositories as opposed to actual incidence of these species in South African waters, highlighting that many South African species are still undocumented in DNA libraries. The findings of this study serve as a baseline for future surveys and provide a rationale for more studies that are aimed at documenting the DNA barcodes of macroinvertebrate species that occur in South African estuaries.