Biodiversity and distribution of zoobenthos in the ecological water replenishment area of the Yellow River estuary coastal wetland revealed by eDNA metabarcoding

eDNA studies in aquatic systems [40]. These primers vary in specificity and range, with 18S rRNA and COI being more universally applicable across zoobenthos, and 12S tailored for spe- cific groups. In the coastal wetland of the Yellow River Estuary, zoobenthos from 174 families were iden- tified using 18S rRNA gene primers, with 307 species identified at the generic level. In contrast, traditional morpho-taxonomic approaches in previous studies identified only 60 zoobenthic families in the Yellow River Estuary Coastal Wetland, with further categorization at the genus or species level hindered by the need for specialized expertise in morphological classification [41]. Based on the bioinformation provided by eDNA metabarcoding, the zoobenthic genera were categorized according to their connectivity within and among modules. This analysis identified several key species as connectors: Eldenia, Chironomus, Tholymis, Gomphonema, Caenis, Proales, Strombidium, Sinistrostrombidium, Mononchus, Paracerior, Tubulanus, Micrura, and Robbea (S2 Fig). These genera exhibit high among-module connectivity, indicat- ing their crucial role in linking different modules within the network. Chironomus is a genus of midges in the family Chironomidae, commonly known as non-biting midges. These insects inhabit a variety of freshwater environments and can thrive in both clean and polluted water bodies. The larvae are a significant food source for fish and other aquatic organisms. Due to their sensitivity to pollutants and ease of culture in the laboratory, Chironomus species are fre-

quently used in environmental monitoring and toxicological studies. The presence of Chiro- nomidae in the Yellow River Estuary Coastal Wetland has been confirmed using traditional morpho-taxonomic approaches, whereas the research does not provide identification at the generic level [19]. For example, Eldenia, a genus of flatworms in the family Provorticidae, pri- marily inhabits brackish water environments, particularly in marine benthic zones such as shores and intertidal areas [42]. This genus has been documented in regions such as Germany, specifically in the brackish waters of the Northern Hemisphere. The presence of Eldenia and Chironomus species, which thrive in brackish environments, highlights the adaptability of cer- tain zoobenthos to fluctuating salinity conditions in estuarine systems. Although eDNA metabarcoding has proven to be a powerful tool for biodiversity assess- ment, significant potential remains for optimizing this technology for monitoring benthic organisms. The identified zoobenthic families in the present study were compared not only with those identified by traditional morpho-taxonomic approaches in the same study area, but also with the classifications in the Chinese Nearshore Benthos Classification System Book [43], which provide the most comprehensive introduction to zoobenthos in China (Fig 5). On the one hand, eDNA metabarcoding revealed 118 unique zoobenthic families in the Yellow River Estuary Coastal Wetland, demonstrating that this technique offers a new perspective for

understanding zoobenthic diversity. On the other hand, traditional morpho-taxonomic approaches identified 60 zoobenthic families, whereas eDNA metabarcoding detected only 11 of these families. Additionally, out of the 771 zoobenthic families documented in Chinese zoo- benthos literature, eDNA metabarcoding identified 49 families in the Yellow River Estuary Coastal Wetland, while traditional morpho-taxonomic methods identified 22 families. These findings suggest that a significant amount of biodiversity information remains undetected by eDNA metabarcoding. This phenomenon can be partially attributed to the inefficiency of biological information collection. The specific survival patterns of different aquatic organisms influence their ecologi- cal niches and the spatial distribution of eDNA. Extraction of eDNA involves subsampling small volumes, often measured in grams. At this scale, the uneven distribution of eDNA, along with small organisms in the sediment, introduces considerable random sampling variability [27,44]. In contrast, morphological analysis typically involves sieving large volumes of sedi- ment at specific mesh sizes. For example, Yang [19] collected benthic invertebrates from the PLOS ONE Biodiversity and zoobenthos distribution in yellow river estuary wetlands revealed by eDNA metabarcoding PLOS ONE | https://doi.org/10.1371/journal.pone.0315346 December 18, 2024 11 / 19