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

1. Introduction The Yellow River Delta wetland, located on the Bohai Sea coast in northeastern Shandong Prov- ince, is the broadest, most complete, and youngest wetland ecosystem in the warm temperate zone (with an average annual temperature of 11.7–12.6˚C). Since the Yellow River’s diversion to the Bohai Sea in 1855, the continuous interaction between the river and the ocean has driven geomorphological evolution, creating a diverse ecosystem that supports rich species diversity [1]. It acts as a key indicator of ecological health in the Yellow River Basin and functions as a vital germplasm repository and source of marine biodiversity in the Bohai Sea region [2]. According to the white paper "Biodiversity Conservation in the Yellow River Delta", the coastal wetland hosts 1,145 species of wild plants, 373 species of birds, and 512 species of insects. The wetland is crucial for biodiversity, and supports various fish, invertebrates, and plant species. In addition, it provides essential ecological services, such as water purification, flood regulation, carbon sequestration, and shoreline stabilization. Ongoing conservation efforts focus on pollu- tion control, sustainable water resource management, and habitat protection to ensure the wet- land’s ecological and economic sustainability. Recent studies on macrozoobenthos in coastal ecosystems have demonstrated the utility of eDNA metabarcoding for environmental monitor- ing. He et al. (2021) analyzed benthic impacts from salmon farming, revealing that eDNA meta-

barcoding effectively captures shifts in macrofaunal community structures, including changes in alpha diversity and species composition, under varying environmental stressors. Their research highlights that macrofaunal taxa like polychaetes exhibit distinct responses to organic enrichment, with eDNA data providing enhanced resolution in detecting such changes com- pared to traditional morphological methods [3]. Similarly, Cordier et al. (2017) demonstrated the application of supervised machine learning with eDNA metabarcoding data to predict the ecological status of marine environments. Their study showed that eDNA-derived benthic data not only aligns well with conventional biotic indices but also offers a scalable and cost-effective solution for routine benthic monitoring. This method overcomes challenges associated with unassigned sequences in taxonomic databases, making it a robust tool for future ecological assessments [4]. Despite these advancements, the understanding of aquatic biodiversity, partic- ularly related to zoobenthos, in specific regions such as the Yellow River Delta remains limited. Expanding research efforts using eDNA metabarcoding will be crucial to bridge these knowl- edge gaps and ensure the sustainable management of these ecologically valuable systems. Zoobenthos, aquatic organisms that reside on the bottom of water bodies for most or all of their lives, are critical ecological indicators in coastal wetlands [5,6]. Their presence, abun- dance, and diversity provide valuable insights into sediment and water quality. Zoobenthos

play an essential role in nutrient cycling by decomposing organic matter and recycling nutri- ents like nitrogen and phosphorus, which support primary production and maintain ecological balance [7]. As a fundamental part of the aquatic food web, they are a primary food source for fish and bird species, directly impacting higher trophic levels [8]. Their activities, such as bur- rowing, affect sediment structure and stability, influencing the distribution of other organisms [9]. Additionally, some zoobenthos species can bioaccumulate contaminants, aiding in biore- mediation and pollution management [10]. Different zoobenthic species have distinct ecologi- cal roles in coastal wetlands. Amphipods, such as Gammarus spp., can enhance nutrient availability through detritus consumption and sediment aeration [11]. Polychaetes were observed to increase sediment permeability and support nutrient cycling via bioturbation [12]. Bivalves, like Mytilus edulis, play roles in improving water quality and stabilizing sediments through filtration [13]. These examples indicate that understanding the population structure and distribution characteristics of zoobenthos is crucial for maintaining the health and func- tionality of coastal wetland ecosystems. 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 2 / 19 Regulation (Grant No. 02040192401) [http://www. sdaep.cn/], the National Natural Science Foundation of China (Grant Nos. 52370204,

42107230) [https://www.nsfc.gov.cn/], the Shandong Provincial Natural Science Foundation (Grant No. ZR2023ZD51) [http://kjt.shandong.gov. cn/], the Shandong Provincial Taishan Scholars Youth Expert Program (Grant No. 2021-292) [http://edu.shandong.gov.cn/], the Shandong Provincial Excellent Young Scientists Fund Program (Grant No. 2023HWYQ-078) [http://kjt. shandong.gov.cn/], the Shandong Provincial Youth Innovation Team Development Plan of Colleges and Universities (Grant No. 2022KJ220) [http:// edu.shandong.gov.cn/], and the Dongying Municipal Key Laboratory of Hydrology and Ecology in the Yellow River Delta (Grant No. DYSWSHJK2024030701) [http://dymp.sdswyc. net:8099/bh-mp-vo/]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: Competing interests: The authors have declared that no competing interests exist.