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

significant ecological region with a semi-humid and semi-arid warm-temperate continental monsoon climate. This area experiences four distinct seasons, an average annual temperature of 12.1˚C, and annual rainfall between 530 and 630 mm, predominantly in the summer months. Prevailing winds from the south-southeast and east influence its weather patterns. In October 2023, sediment and water samples were collected from 16 sites within the eco- logical water replenishment area (Fig 1). Two sites were managed by the One Thousand and Two Management Station of the Yellow River Delta, covering the old course of the Yellow River. The remaining 14 sites were within the Yellow River Delta National Nature Reserve. The longitude and latitude coordinates of the 16 sampling sites were shown in S1 Table. Three parallel samples were taken at each site: approximately 1.0 kg of surface sediment and 1.0 L of water from the same points [4]. The sediment weight was estimated by collecting a known vol- ume of sediment, which was later confirmed by weighing in the laboratory. The sediment was collected using a bottom sampler (LB-TC1001, Lubo, Qingdao, China). Pebbles, twigs, shells, and other debris were carefully removed from the sediment, and the sample was thoroughly mixed before being placed in a sterile sampling bag. Water samples were collected using steril- ized high-density polyethylene (HDPE) bottles. The bottles were rinsed with site water three times before collecting the final 1.0 L sample at the same locations. To prevent cross-contami-

nation, sterile gloves were worn during both the sampling and analysis processes, and gloves were changed between each sample. All samples were kept on ice during transport and ana- lyzed immediately upon arrival at the laboratory. 2.2. Ethical statement This study utilized environmental DNA (eDNA) technology and did not involve the capture or experimentation of animal subjects. All field sampling was conducted in accordance with relevant legal and ethical guidelines and with approval from the Shandong Yellow River Delta National Nature Reserve Administration Committee. Fig 1. Geographic location (A) and elevation (B) of the Yellow River delta. locations of 2 sampling sites in the One Thousand and Two Management Station (C) and 14 sites in the Shandong Yellow River Delta National Nature Reserve (D); habitat conditions during sample collection (E) and (F). (Base map from USGS; url: https://www.usgs.gov/tools/national-map-viewer). https://doi.org/10.1371/journal.pone.0315346.g001 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 4 / 19