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

There are significant differences in the living habits of zoobenthos. For instance, the larvae of Chironomidae are often found in nutrient-poor sediments where they consume detritus, enhancing nutrient availability and supporting their population growth [14]. In contrast, the Capitella capitata complex thrive in organically enriched environments, often associated with pollution, benefiting from high organic matter availability for their feeding and reproductive activities [15]. Salinity also plays a crucial role in the distribution of zoobenthic species. For example, the Baltic clam, Macoma balthica, is adapted to low-salinity environments and is commonly found in brackish waters with varying salinity levels [16]. Conversely, some species of blue mussel are more tolerant of higher salinity levels and are typically found in marine environments [17]. With the replenishment of ecological water, salinity and nutrient elements in the coastal wetlands of the Yellow River Delta show gradient variations [18]. It is speculated that ecological water replenishment lead to spatial differences in the distribution of zoobenthos within the coastal wetland [19]. Traditional benthic surveys, relying on morpho-taxonomic approaches, are labor-intensive, time-consuming, and require significant expertise [20]. These methods face challenges such as low throughput, ambiguities in taxonomic identification, and significant difficulties in sample preservation, which can affect both the accuracy and reliability of the results. In contrast, the

rapid advancement of high-throughput sequencing has positioned molecular taxonomy as a promising tool for biodiversity studies. These DNA-based techniques identify taxa using spe- cific genome fragments, known as "DNA barcode" regions [21]. Metabarcoding employs high- throughput sequencing to detect multiple species from complex samples. Environmental DNA (eDNA) metabarcoding targets environmental samples, offering significant advantages such as high throughput, non-invasiveness, and the ability to detect species at low abundance levels with high specificity [22]. Several studies have shown that eDNA metabarcoding can outper- form traditional methods in terms of detecting species richness and identifying cryptic or rare species that may not be easily detected using morphology-based approaches. For instance, a study in freshwater ecosystems revealed that eDNA detected 1.1 to 2 times more species than traditional sampling methods, especially in environments where physical sampling might be limited due to habitat complexity [23,24]. Similarly, in freshwater bivalve monitoring, eDNA was found to detect species that were missed in conventional surveys, highlighting its potential for early detection of invasive species [25]. Advances in sediment sampling techniques and DNA extraction methods are enhancing the efficiency and reliability of eDNA applications in zoobenthos surveys. This study aimed to investigate the community structure of zoobenthos in the ecological water replenishment area of the coastal wetland in the Yellow River Estuary using eDNA meta-

barcoding. Key environmental factors influencing community structure were identified through correlation analysis. The findings of this study could enhance our understanding of the biodiversity and distribution patterns of zoobenthos in the ecological water replenishment area of Yellow River Estuary Coastal Wetland but also provide valuable insights for biodiver- sity conservation, habitat protection, and ecosystem management. By identifying key environ- mental drivers, the study contributes to more effective conservation strategies, such as targeted habitat restoration and the design of management practices that support the preservation of aquatic biodiversity in this unique ecosystem. 2. Materials and methods 2.1. Study area and sample collection The Yellow River Estuary Coastal Wetland, located on the south coast of Bohai Bay and the west coast of Laizhou Bay in China (between 117˚310-119˚180E and 36˚550-38˚160N), is a 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 3 / 19