Abstract
Estuaries are important and productive ecosystems situated 1 at the boundary between
2 freshwater and marine environments. These ecosystems face various anthropogenic
3 threats, including pollution, habitat fragmentation, and water abstraction, which
4 endanger their biodiversity and ecosystem health. Therefore, effective biomonitoring
5 is crucial for their conservation and management. Traditional biomonitoring methods
6 for estuaries are labour intensive, time-consuming, and usually require taxonomic
7 expertise. Hence, alternative methods are needed to identify species efficiently and
8 accurately for estuarine biomonitoring. Two molecular tools that use genetic variation
9 between species to identify and classify species are DNA barcoding and eDNA
10 metabarcoding. This study utilized these tools to generate a reference sequence
11 database for estuarine benthic invertebrate fauna in KwaZulu-Natal, to assess the
12 presence of species using sediment samples. The results demonstrate that eDNA
13 metabarcoding is a fast and efficient method for detecting the presence of benthic
14 invertebrates in South African estuaries, and DNA barcoding accurately identifies
15 species. However, the study encountered limitations, such as primer inefficiency and
16 incomplete reference databases, which may have contributed to the detection of a
17 lower number of species compared to previous studies that utilized traditional
18 methods. Despite these limitations, our findings suggest that DNA barcoding and
19 eDNA metabarcoding can complement traditional methods in biomonitoring South
20 African estuarine fauna. Moreover, increasing barcoding efforts can lead to more
21 comprehensive databases that can enhance the detection of species by eDNA
22 metabarcoding.