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The Formosa Ridge, also named Site F, is an active cold seep marine ecosystem site that has been studied since it was discovered on the continental slope of the northeast South China Sea (SCS). However, few studies have focused on the eukaryotic diversity at Site F. Environmental DNA (eDNA) technology is a non-invasive method applied in biodiversity surveys with a high species detection probability. In the present study, we identified multi-trophic biodiversity using eDNA metabarcoding combined with multiple ribosomal RNA gene (rDNA) markers. We detected 142 phytoplankton, 90 invertebrates, and 64 fish species by amplifying the 18S rRNA gene V4 region, the 18S rRNA gene V9 region, and the 12S rRNA gene. The results elucidated dissimilar trends of different assemblages with depth. The diversity of phytoplankton and invertebrate assemblages markedly decreased with depth, whereas little change was observed within the fish assemblage. We comprehensively assessed the relationship between the three assemblages and environmental factors (temperature, salinity, depth, dissolved oxygen, and chlorophyll a). These factors strongly impacted on phytoplankton and invertebrates, but only slightly on fish. We inferred the finding might be due to fish having a strong migration capacity and wide distribution. This study indicates that eDNA metabarcoding with multiple markers is a powerful tool for marine biodiversity research that is able to provide technical support and knowledge for resource management and biodiversity protection efforts. • eDNA technology effectively revealed the eukaryotic diversity in the cold seep. • Distribution and diversity of multi-trophic levels varied in the vertical direction. • Environmental factors were markedly correlated with assemblage structures. [ABSTRACT FROM AUTHOR]

The South China Sea (SCS) is an important part of the Indo-Pacific convergence zone, with high biodiversity and abundant marine resources. Traditional methods are primarily used to monitor biodiversity. However, a few studies have used environmental DNA (eDNA) metabarcoding to research the assemblage structure of the SCS. This study used eDNA metabarcoding to survey the SCS assemblage and its relationship with environmental factors over a month-long time-series (August 30th to September 30th, 2020) of seawater samples from the central part of the SCS (9°–20°86′ N, 113°–118°47′ E). 32 stations were divided into six groups (A, B, C, D, E, F) according to longitude. We collected water samples, extracted eDNA, and amplified 18S rRNA gene V4 region (18S V4), 18S rRNA gene V9 region (18S V9), and 12S rRNA gene (12S). Krona diagrams were used to show species composition. We identified 192 phytoplankton, 104 invertebrate, and 61 fish species from 18S V4, 18S V9, and 12S, respectively. Generally, the three assemblage structures exhibited an increase in species diversity with increasing longitude. Group E had the highest fish diversity. Groups F and C had the highest phytoplankton and invertebrate diversity, respectively. Canonical correspondence analysis showed that four factors (chlorophyll a , depth, salinity, and temperature) were correlated with assemblage structure. Chlorophyll a was the main environmental factor that affected fish, phytoplankton, and invertebrate assemblage structures; salinity was strongly correlated with fish and invertebrate assemblage structures; temperature was a key factor that impacted fish and invertebrate assemblage structures; and depth was strongly correlated with invertebrate assemblage structure. Our results revealed that eDNA metabarcoding is a powerful tool for improving detection rate and using multiple markers is an effective approach for monitoring biodiversity. This study provided information that can be used to enhance biodiversity protection efforts in the SCS. • eDNA metabarcoding is sensitive to reveal biodiversity in the SCS. • Multiple trophic levels were identified effectively from one eDNA sample. • eDNA abundance reflected the assemblage variations across longitude. • Environmental factors were significantly correlated with assemblage structures. [ABSTRACT FROM AUTHOR]