Comparison of Environmental DNA Metabarcoding and Underwater Visual Census for Assessing Macrobenthic Diversity

Simple Summary

This study evaluates the efficacy of environmental DNA (eDNA) metabarcoding and underwater visual census (UVC) in assessing the diversity of subtidal macrobenthic communities. We compared water eDNA, sediment eDNA, and traditional UVC methods in the Nanji Islands, China. Sediment eDNA demonstrated superior performance in detecting key benthic phyla such as Annelida and Arthropoda, whereas UVC was more effective for large and active organisms. Integrating these methods provides a more comprehensive biodiversity assessment, highlighting the importance of combining molecular and traditional techniques for effective conservation and management strategies in marine ecosystems.

Abstract

The rapid advancement of environmental DNA (eDNA) technology has transformed ecological research, particularly in aquatic ecosystems. However, the optimal sampling matrix (e.g., water or sediment) and the potential for eDNA to replace or complement traditional underwater visual census (UVC) remain unclear. Here, we integrate water eDNA, sediment eDNA, and UVC approaches to systematically compare the diversity of benthic macrofauna in the subtidal zones of the Nanji Islands, China. Our results show that sediment eDNA samples exhibited the highest species richness, while UVC had the lowest. Each method revealed distinct species profiles, with relatively few shared taxa at the order level and below. Environmental eDNA showed significant advantages in detecting key phyla such as Annelida and Arthropoda. In contrast, traditional UVC was crucial for identifying certain taxa, such as Bryozoa, which were undetectable by eDNA methods. The low overlap in species detected by these methods underscores their complementary nature, highlighting the necessity of integrating multiple approaches to achieve a more comprehensive and accurate biodiversity assessment. Future research should focus on refining eDNA techniques, such as developing more universal primers, to further enhance their applicability in biodiversity monitoring.

1. Introduction

The subtidal zone, a critical interface between intertidal zones and shallow sea, usually harbors a rich diversity of benthic fauna, yet it is one of the areas most severely affected by human activities [1]. Traditional in situ observations through underwater visual census (UVC)—where trained SCUBA divers survey species richness and community assemblages in the field—have long been the predominant method for studying marine organisms [2]. However, this approach is limited by the need for specialized skills, suitable field conditions, and significant resources [3]. Moreover, the complex currents and topography of subtidal zones often pose substantial challenges for UVC surveys, restricting the comprehensive biomonitoring of these habitats [4].

Recent advancements in environmental DNA (eDNA) technology have revolutionized biodiversity assessments in aquatic ecosystems [5]. eDNA metabarcoding allows for the non-invasive detection of species through the analysis of genetic material present in environmental samples such as water and sediment [6]. This approach has been successfully applied to monitor fish and invertebrate communities in various aquatic habitats, including rivers, lakes, and marine environments [5,6,7,8]. However, the optimal sampling matrix (water vs. sediment) and the potential for eDNA to replace or complement the traditional UVC method to access the macrobenthos diversity remain uncertain.

The Nanji Islands, located in the eastern sea area of Zhejiang Province, China, is a biosphere reserve of the United Nations Educational, Scientific and Cultural Organization (UNESCO). This subtropical marine protected area is characterized by complex subtidal zones with high biodiversity [9]. The seabed terrain in this area slopes downwards from northwest to southeast, with a water depth generally between 15 and 25 m. There are two deep-water channels on the northeast and southwest sides of Nanji Island, with a depth of over 30 m and a maximum depth of 45 m. The subtidal areas here include several distinct habitat types, such as steep rocky cliffs, platforms, boulder fields, small area beaches, and sediments mainly composed of silty clay. Due to the fact that the subtidal zone with a depth less than 20 m is mainly composed of rocks, bottom trawl survey is impossible, and box-type mud collection is also limited. While the underwater visual census (UVC) is the remaining available traditional method, a systematic biodiversity survey is still lacking.

Generally, the Nanji Islands provide an ideal setting for comparing the effectiveness of different biodiversity assessment methods. In this study, we integrate water eDNA, sediment eDNA, and UVC methods to systematically compare their efficiency in detecting benthic macrofaunal diversity in the subtidal zone with a depth < 20 m of the Nanji Islands. Our objectives are to quantify the differences in species detection efficiency among the three methods, and explore the complementary nature of these methods across different taxonomic levels.

2. Materials and Methods

2.1. Study Area and Sample Location

The Nanji Islands (27°27′43″ N–27°46′48″ N, 120°02′55″ E–121°07′58″ E) are located in the eastern sea area of Wenzhou City, Zhejiang Province, China. This subtropical marine protected area is characterized by mainly rocky inter- and sub-tidal zones with high biodiversity. Ten subtidal sites representing the broader subtidal environment were selected for this study based on accessibility by boat on 8–10 May 2024 (Figure 1).

Figure 1.

Sampling sites in the subtidal zone of the Nanji Islands (The capitalized names in the right-side figure are abbreviations for each sub-island. “SMA” stands for “Shangmaan”, “XMA” for “Xiamaan”, “PY” for “Poyu”, “JY” for “Jianyu”, “XCY” for “Xiaochaiyu”, “LCJ” for “Longchuanjiao”, “DSJ” for “Dashanjiao”, “SPW” for “Sanpanwei”, “HJS” for “Houjishan”, and “DLS” for “Daleishan”).

2.2. Underwater Visual Census (UVC) and Photo Analysis

UVC survey conducted during high-tide period. The basic unit of UVC was a 50 m long transect line, with macrobenthos surveyed in two 1 m wide by 2 m high bands on either side of the transect line [10]. The proportions of the area examined in relation to the rocky subtidal area of each site ranged from approximately 10% to 30%. Digital photo-quadrats were taken at 2.5 m intervals along the transect line (i.e., 20 per 50 m transect). Due to the extremely low transparency of seawater (<1 m), the SMA, DSJ, HJS, and DLS sites were not suitable for diving. Consequently, the UVC units of only six stations with a depth range of 5–15 m were completely investigated (Figure 1, marked by green color). Two taxonomists (W. Huo and W. Chen) estimated the number of species from the visual data together, without prior knowledge of the eDNA outcomes. Due to the generally low transparency of seawater, many photo-quadrats were not very clear, and only 30 species could be identified to species level (Table A1).

2.3. Environmental DNA Sampling

Water and sediment samples were collected from ten stations in the subtidal zones of the Nanji Islands. Considering that the larvae of benthic organisms may float in surface water layers, 1 L of seawater was collected from both surface and bottom layers by divers to gather as much macrobenthos eDNA as possible. The samples were mixed thoroughly, and 1.5 L seawater was filtered [11] through a 0.22 µm mixed cellulose ester (MCE) membrane, and the filter paper was immediately transferred to a 1.5 mL cryopreservation tube and stored at −20 °C.

Sediment samples were collected using a grab sampler near the water sampling sites. At the sites XMA and XCY, however, the sediment could not be collected by the grab sampler due to the thin sediment layer. Instead, divers collected sediment using 50 mL centrifuge tubes in those sites. Approximately 20 g of surface sediment was placed into a sterile, enzyme-free sampling bag and stored at −20 °C until further analysis.

2.4. DNA Extraction and Sequencing

Environmental DNA was extracted from water sample filters using the DNeasy Power Water Kit (Qiagen, Hilden, Germany) and from sediment samples using the QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany), following standardized protocols to ensure high extraction efficiency and purity. The COI gene fragments were targeted and amplified using universal primers mlCOIintF (5′-GGWACWGGWTGAACWGTWTAYCCYCC-3′) [12] and jgHCO2198 (5′-TAIACYTCIGGRTGICCRAARAAYCA-3′) [13]. The PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min, with a final extension at 72 °C for 5 min. Negative controls (DNA-free samples) were processed in parallel to ensure the reliability of the PCR. Sequence libraries were prepared using the TruSeq^® DNA PCR-Free Kit (Illumina, San Diego, CA, USA) and quantified by Qubit and Q-PCR. The libraries were sequenced as 250 bp paired-end reads on a NovaSeq 6000 platform.

2.5. Bioinformatics Analysis

Raw sequencing data were processed using DADA2 (version 2.15.2) to remove primer sequences and low-quality reads [14]. Amplicon sequence variants (ASVs) were clustered at 97% similarity using VSEARCH, and ASVs with less than 0.01% abundance across all samples were filtered out [15,16]. Taxonomic annotations were performed against the NCBI database, focusing on eukaryotic groups. Non-marine groups (e.g., Arachnida, Insecta, and terrestrial Craniata) were manually excluded from the ASV table [17].

2.6. Statistical Analyses

To comprehensively assess the species diversity based on the three methods, species richness, Shannon–Wiener diversity, and Pielou’s evenness indices were employed. Species richness and the relative ratio of species number at class, order, and family level were compared among UVC, sediment eDNA, and water eDNA methods. Venn diagrams were generated to assess overlapping taxa detected by the different methods. As the species diversity data from sediment and water eDNA did not meet the normal distribution assumption (Shapiro–Wilk test, p < 0.05) or the homogeneity of variance assumption (Bartlett test, p < 0.05), a nonparametric Kruskal–Wallis test was used to analyze intergroup differences in species diversity. Post hoc Dunn tests with Holm–Bonferroni correction for p-values were further conducted to evaluate species diversity differences across different methods.

In the analysis of the community structure, Jaccard distances were calculated based on species composition matrices, and non-metric multidimensional scaling (NMDS) was used to visualize community differences among the three methods. The reliability of the ordination was evaluated by the stress value (the closer the value is to zero, the higher the degree of fit). The clustering of samples was observed in the NMDS plot, and the significance of community structure differences among groups was verified by ANOSIM tests (based on Jaccard distances). All statistical analyses and figures were performed using R version 4.0.3, with plots created using the ggplot2 package (version 3.3.3) [18].

3. Results

3.1. Sequencing and UVC Results

Only eight of ten water and sediment eDNA samples were successfully amplified even after repeating the PCR experiments several times, respectively (Figure 1, successful amplification stations marked by yellow and blue colors, respectively). A total of 657,161 and 752,136 raw reads were obtained from water and sediment eDNA samples, respectively. After bioinformatics processing, 1879 and 7487 amplicon sequence variants (ASVs) were identified for water and sediment eDNA, respectively. The majority of ASVs (79.30% for water eDNA and 64.90% for sediment eDNA) were either taxonomically unassigned or belonged to non-marine groups. The remaining ASVs represented a diverse range of marine taxa, including 11 phyla, 22 classes, 47 orders, 72 families, 72 genera, and 94 species for water eDNA; and 14 phyla, 28 classes, 64 orders, 114 families, 125 genera, and 166 species for sediment eDNA. In contrast, UVC identified a total of nine phyla, 16 classes, 18 orders, 29 families, 29 genera, and 39 species based on photo-quadrat analysis. Overall, the three methods collectively identified 15 phyla, 38 classes, 89 orders, 179 families, 206 genera, and 284 species (Table 1 and Table A1).

Table 1.

Species number of phyla using sediment and water eDNA and UVC methods.

Phylum	Sediment	Water	UVC
Annelida	22	8	1
Arthropoda	48	14	1
Bryozoa	0	0	5
Chaetognatha	1	4	0
Chordata	5	1	1
Cnidaria	26	32	7
Ctenophora	2	0	1
Echinodermata	5	4	4
Gastrotricha	6	1	0
Mollusca	37	19	17
Nematoda	3	0	0
Nemertea	2	2	0
Phoronida	1	0	0
Platyhelminthes	1	2	0
Porifera	7	7	2
total	166	94	39

3.2. Comparison of the Taxonomic Compositions Among the Three Methods

Taxonomic compositions were compared among the three methods at five common stations (Figure 2). Sediment eDNA detected the highest number of phyla (13), classes (27), and species (131), followed by water eDNA (10 phyla, 21 classes, 72 species) and UVC (seven phyla, 14 classes, 15 orders, 32 species). At the phylum level, sediment and water eDNA showed similar compositions, with Mollusca, Arthropoda, and Cnidaria being dominant (Table 1). In contrast, UVC identified Mollusca, Cnidaria, and Bryozoa as the most dominant group, and the Bryozoa was undetectable by eDNA methods (Figure 2a). At the class level, sediment eDNA was dominated by Gastropoda, followed by Malacostraca and Hydrozoa, while water eDNA showed a similar pattern with Gastropoda, Hydrozoa, and Demospongiae as dominant classes. UVC identified Gastropoda, Anthozoa, and Demospongiae as the dominant classes (Figure 2b). At the order level, sediment eDNA showed Anthomedusae, Decapoda, and Stylommatophora as dominant orders, while water eDNA was dominated by Leptothecata, Anthoathecata, and Stylommatophora. UVC identified Neogastropoda, Nudibranchia, and Demospongiae as dominant orders (Figure 2c).

Figure 2.

Stacked bar charts showing the percentage of species richness at the phylum (a), class (b), and order (c) levels for underwater visual census (UVC) and sediment (Sed) and water eDNA methods.

Venn diagrams revealed relatively high taxon overlaps at the phylum and class levels, and low overlaps at the order level and below among the taxonomic compositions from the three methods (Figure 3). Sediment eDNA detected the highest number of species overall, while traditional UVC methods detected fewer but unique species. No common species was found among the three methods. Only 18 common species were found between sediment and water eDNA samples, and only 1 common species was found between traditional UVC and sediment/water eDNA samples.

Figure 3.

Venn diagrams showing the species overlaps at the different taxonomic ranks detected by underwater visual census (UVC) and sediment (Sed) and water eDNA methods at the common stations.

3.3. Alpha and Beta Diversity Among the Three Methods

Species richness was highest in sediment eDNA samples across all stations except LCJ, with UVC showing the lowest species richness (Figure 4a). Overall, sediment eDNA samples exhibited the highest average species richness, while UVC had the lowest (Figure 4b). The Kruskal–Wallis test and post hoc Dunn test indicated significant differences in species richness between sediment eDNA and UVC methods (Figure 4c). Sediment eDNA also consistently showed higher Shannon and Pielou’s evenness indices compared to water eDNA, indicating superior performance in both species abundance and community evenness (Figure 4d,e).

Figure 4.

Alpha and beta diversity analyses based on sediment eDNA, water eDNA, and underwater visual census (UVC) methods. (a) Species richness based on the sediment eDNA, water eDNA, and UVC at the common sampling stations (JY, LCJ, PY, XCY, XMA). (b) The total species richness based on the three methods from all the common stations. (c) Significant richness differences among the three methods conducted by post hoc Dunn tests. *, indicating significant differences. (d) The total Shannon index based on the three methods from all the common stations. (e) The total Pielou’s evenness index based on the three methods from all the common stations. (f) The clustering of samples with non-metric multidimensional scaling (NMDS) plot.

Non-metric multidimensional scaling (NMDS) ordination plots showed distinct clustering of samples from sediment eDNA, water eDNA, and UVC methods, with no overlap among the groups (Figure 4f). The stress value of zero and an ANOSIM test p-value of 0.001 confirmed the high reliability of the ordination results, indicating significant differences in community structure captured by the three methods. This suggests that each method provides unique insights into the biodiversity of the subtidal zone, highlighting the importance of integrating multiple approaches for comprehensive biodiversity assessment.

4. Discussion

4.1. Environmental DNA Is Essential for Biodiversity Assessment of Rocky Subtidal Zone

Traditional bottom trawling and grab sampling are unsuitable for biodiversity assessments in rocky subtidal zones. Underwater visual census (UVC) is often used for macrobenthic surveys, but its effectiveness is limited by low seawater transparency, as seen in this study. Compared to UVC, the present study demonstrates that eDNA is more effective for assessing benthic macrofaunal diversity, particularly for key phyla such as Annelida and Arthropoda (Table 1; Figure 3). The sediment eDNA detected a significantly higher species richness compared to the UVC method (Figure 4). Generally, both the sediment and water eDNA methods can uncover those relatively smaller benthos (e.g., Chaetognatha, Gastrotricha, Nemertea, Platyhelminthes, and most of Annelida and Arthropoda) that were missed by the UVC survey (Table 1 and Table A1). Furthermore, eDNA showed significant differences in community structure with the UVC (Figure 4f).

In terms of total number, the sediment eDNA detected more species than the water eDNA (166 vs. 94; Table 1). For all the phyla except Cnidaria and Chaetognatha, the sediment eDNA detected no less species than the water eDNA (Table 1). Notably, sediment eDNA failed to detect Octocorallia species, which were uncovered by water eDNA. Most octocorals prefer to live on hard substrates such as rocks, rather than sediments. Consequently, the octocoral eDNA in the sediment is too rare to amplify. In contrast, the eDNA concentration in water samples increases after filtration and concentration, which may contain more octocoral eDNA. For the planktonic Chaetognatha, it is not surprising that the water eDNA detected more species than the sediment eDNA (Table A1).

For the macrobenthos, the superior detection efficiency of sediment eDNA is likely due to the higher concentration and longer persistence of DNA in sediments compared to water samples, which allows for retrospective genetic monitoring and extends the seasonal window for species assessment [19,20,21,22,23,24,25]. For example, the decay rate of fish eDNA in sediment is significantly lower than that in water (0.033%/h vs. 1.9%/h), and eDNA is 8–1800 times more concentrated in sediment than in water. This suggests that sediment eDNA can capture a broader range of species, including those that may be temporarily absent or less active in the water column.

4.2. Limitations of Traditional UVC and eDNA Methods

While the traditional UVC method is effective for detecting large and highly active organisms, they have limited capacity for detecting small or cryptic species [2,26]. In the present study, this limitation is partly due to the difficulty in capturing and identifying relatively small organisms such as polychaetes (belonging to the phylum Annelida), Chaetognatha, Gastrotricha, Nemertea, Platyhelminthes, and Arthropoda, which often require further taxonomic examination under a microscope. Additionally, environmental factors such as water turbidity in the subtidal zones can further reduce the effectiveness of the UVC method. However, traditional UVC remains valuable for certain taxa that may not be well-represented in eDNA samples, such as Bryozoa, which was undetectable by eDNA in our study but was identified through the UVC method.

The missed detection of Bryozoa highlights the need for further refinement of eDNA techniques. The COI primers used in this study failed to detect Bryozoa, indicating potential limitations in primer universality or suboptimal annealing temperature for PCR reactions. Additionally, the incomplete nature of current DNA sequence databases limits the accuracy of species-level identifications. Future research should focus on developing more universal primers and optimal PCR reactions, and expanding reference databases to improve the reliability and resolution of eDNA-based biodiversity assessments.

4.3. Complementary Nature of the Methods and Implications for Biodiversity Monitoring

In general, taxonomic compositions from the three methods shared relatively few taxa at the order level and below, and no common species was found among these methods (Figure 2c and Figure 3). The low overlap in species detected by the three methods underscores their complementary nature. Sediment eDNA provided the most comprehensive species inventory, detecting a significantly higher number of species compared to water eDNA and traditional UVC methods. However, the unique detection of certain taxa by the UVC method highlights the importance of integrating both eDNA and UVC approaches for a more complete assessment of biodiversity. This integration can help overcome the limitations of each method, such as primer bias in eDNA techniques and the challenges of species identification in traditional methods. Future biodiversity monitoring efforts should consider the complementary nature of these methods and the potential for further technological advancements to enhance the accuracy and efficiency of biodiversity assessments. This integrated approach will be crucial for informing effective conservation and management strategies in complex aquatic environments.

5. Conclusions

The present study provides a comprehensive evaluation of environmental DNA (eDNA) metabarcoding of COI and traditional underwater visual census (UVC) methods for assessing benthic macrofaunal diversity in subtidal zones. While taxonomic compositions from the three methods show similar patterns at the phylum level, they share relatively few taxa at the order level and below. Sediment eDNA emerged as a highly effective tool, particularly for detecting key benthic phyla such as Annelida and Arthropoda, due to its enhanced preservation and detection capabilities. However, traditional UVC remains crucial for identifying certain taxa, such as Bryozoa, which were undetectable by eDNA methods. The low overlap in species detected by these methods underscores their complementary nature, highlighting the necessity of integrating multiple approaches to achieve a more comprehensive and accurate biodiversity assessment. Future research should focus on refining eDNA techniques, such as developing more universal primers and expanding reference databases, to further enhance their applicability in biodiversity monitoring.

Appendix A

Table A1.

Species composition based on the sediment and water eDNA and UVC methods.

Species ID	Sediment	Water	UVC	Phylum	Class	Order	Family	Genus	Species
OTU_1958	1	1	0	Annelida	Polychaeta				Polychaeta sp. NB-Po509
OTU_1601	1	0	0	Annelida	Clitellata	Enchytraeida	Enchytraeidae	Enchytronia	Enchytronia christenseni
OTU_4793	1	0	0	Annelida	Polychaeta	Terebellida	Flabelligeridae	Treadwellius	Treadwellius bifidus
OTU_16546	1	0	0	Annelida	Polychaeta		Maldanidae	Asychis	Asychis chilensis
OTU_17049	1	0	0	Annelida	Clitellata	Crassiclitellata	Megascolecidae	Perionyx	Perionyx rufulus
OTU_5756	1	0	0	Annelida	Polychaeta	Phyllodocida	Nereididae	Neanthes	Neanthes sp. RP2011-N
OTU_7431	1	0	0	Annelida	Polychaeta	Phyllodocida	Nereididae	Neanthes	Neanthes sp. 1
OTU_4848	1	0	0	Annelida	Polychaeta		Orbiniidae	Scoloplos	Scoloplos acmeceps
OTU_2403	1	0	0	Annelida		Phascolosomatiformes	Phascolosomatidae	Phascolosoma	Phascolosoma esculenta
OTU_13606	1	0	0	Annelida	Polychaeta	Sabellida	Serpulidae	Serpula	Serpula vermicularis
OTU_14440	1	0	0	Annelida	Polychaeta	Sabellida	Serpulidae	Pileolaria	Pileolaria sp. 11BIOAK-0893
OTU_3016	1	0	0	Annelida	Polychaeta	Sabellida	Serpulidae	Crucigera	Crucigera sp. CMC01
OTU_331	1	0	0	Annelida	Polychaeta	Sabellida	Serpulidae	Serpula	Serpula columbiana
OTU_7545	1	0	0	Annelida	Clitellata	Lumbriculida	Sparganophilidae	Sparganophilus	Sparganophilus sp. MAC_SRS_S115
OTU_14076	1	0	0	Annelida	Polychaeta	Spionida	Spionidae	Polydora	Polydora cornuta
OTU_2533	1	0	0	Annelida	Polychaeta	Spionida	Spionidae	Paraprionospio	Paraprionospio patiens
OTU_16980	1	0	0	Annelida	Polychaeta	Terebellida	Terebellidae	Thelepus	Thelepus sp. CMC01
OTU_2402	1	0	0	Annelida	Polychaeta	Terebellida	Terebellidae	Lysilla	Lysilla pacifica
OTU_15614	1	0	0	Annelida	Polychaeta	Terebellida	Trichobranchidae	Terebellides	Terebellides stroemii
OTU_17438	1	0	0	Annelida	Polychaeta	Xenopneusta	Urechidae	Urechis	Urechis unicinctus
OTU_13481	1	0	0	Annelida	Polychaeta				Polychaeta sp. EBS61o-Po36
OTU_3837	1	0	0	Annelida	Polychaeta				Polychaeta sp. OPC-2015
OTU_12617	0	1	0	Annelida	Polychaeta	Sabellida	Serpulidae	Protolaeospira	Protolaeospira eximia
OTU_1436	0	1	0	Annelida	Clitellata	Rhynchobdellida	Glossiphoniidae	Torix	Torix tukubana
OTU_1486	0	1	0	Annelida	Clitellata	Crassiclitellata	Megascolecidae	Amynthas	Amynthas exiguus
OTU_2537	0	1	0	Annelida	Polychaeta	Terebellida	Terebellidae	Loimia	Loimia arborea
OTU_3294	0	1	0	Annelida	Polychaeta	Sabellida	Serpulidae	Serpula	Serpula vermicularis
OTU_518	0	1	0	Annelida	Polychaeta	Spionida	Trochochaetidae	Trochochaeta	Trochochaeta multisetosa
OTU_6200	0	1	0	Annelida	Polychaeta	Phyllodocida	Nereididae	Nereis	Nereis sp. CMC01
UVC_01	0	0	1	Annelida	Polychaeta	sabellida	Sabellidae		Sabellidae sp.
UVC_02	0	0	1	Arthropoda	Malacostraca	Decapoda	Paguridae	Pagurus	Pagurus hedleyi
OTU_2085	1	1	0	Arthropoda	Collembola	Entomobryomorpha	Entomobryidae	Entomobrya	Entomobrya unostrigata
OTU_13808	1	0	0	Arthropoda	Hexanauplia	Harpacticoida	Aegisthidae		Aegisthidae sp. DZMB479
OTU_8881	1	0	0	Arthropoda	Hexanauplia	Harpacticoida	Aegisthidae		Aegisthidae sp. 1
OTU_14428	1	0	0	Arthropoda	Malacostraca	Decapoda	Aeglidae	Aegla	Aegla longirostri complex sp. UFSM SG15
OTU_7458	1	0	0	Arthropoda	Hexanauplia	Sessilia	Archaeobalanidae	Conopea	Conopea basicuneata
OTU_11323	1	0	0	Arthropoda	Ichthyostraca	Arguloida	Argulidae	Argulus	Argulus japonicus
OTU_13718	1	0	0	Arthropoda	Malacostraca	Isopoda	Armadillidae	Spherillo	Spherillo sp. Kanzaki
OTU_5774	1	0	0	Arthropoda	Branchiopoda	Anostraca	Branchipodidae	Parartemia	Parartemia serventyi
OTU_8686	1	0	0	Arthropoda	Branchiopoda	Anostraca	Branchipodidae	Parartemia	Parartemia sp. 1
OTU_6592	1	0	0	Arthropoda	Malacostraca	Decapoda	Cambaridae	Faxonius	Faxonius validus
OTU_14411	1	0	0	Arthropoda	Malacostraca	Amphipoda	Chiltoniidae		Chiltoniidae sp. CU1
OTU_5711	1	0	0	Arthropoda	Hexanauplia	Sessilia	Chthamalidae	Chthamalus	Chthamalus panamensis
OTU_13431	1	0	0	Arthropoda	Ostracoda	Podocopida	Cyprididae	Strandesia	Strandesia velhoi
OTU_14178	1	0	0	Arthropoda	Ostracoda	Podocopida	Cyprididae	Cyprideis	Cyprideis torosa
OTU_9379	1	0	0	Arthropoda	Ostracoda	Podocopida	Darwinulidae	Darwinula	Darwinula stevensoni
OTU_14387	1	0	0	Arthropoda	Hexanauplia	Calanoida	Diaptomidae	Hesperodiaptomus	Hesperodiaptomus arcticus
OTU_4874	1	0	0	Arthropoda	Collembola	Symphypleona	Dicyrtomidae		Dicyrtomidae sp. BIOUG21696-E03
OTU_14442	1	0	0	Arthropoda	Collembola	Entomobryomorpha	Entomobryidae	Entomobrya	Entomobrya decemfasciata
OTU_5455	1	0	0	Arthropoda	Collembola	Entomobryomorpha	Entomobryidae	Lepidocyrtus	Lepidocyrtus sp. COLNO287-09
OTU_8097	1	0	0	Arthropoda	Collembola	Entomobryomorpha	Entomobryidae	Entomobrya	Entomobrya sp. HB0852_3
OTU_4780	1	0	0	Arthropoda	Malacostraca	Amphipoda	Eusiridae		Eusiridae sp. 06 MK-2021
OTU_4668	1	0	0	Arthropoda	Malacostraca	Decapoda	Grapsidae	Grapsus	Grapsus albolineatus
OTU_2814	1	0	0	Arthropoda	Malacostraca	Isopoda	Haploniscidae	Mastigoniscus	Mastigoniscus sp. NB-Iso41
OTU_14314	1	0	0	Arthropoda	Collembola	Poduromorpha	Hypogastruridae		Hypogastruridae sp. BIOUG23106-B05
OTU_7317	1	0	0	Arthropoda	Malacostraca	Isopoda	Ischnomesidae	Ischnomesus	Ischnomesus sp. lineage 7d JB-2020
OTU_7631	1	0	0	Arthropoda	Ostracoda	Podocopida	Loxoconchidae	Microloxoconcha	Microloxoconcha ikeyai
OTU_13364	1	0	0	Arthropoda	Malacostraca	Amphipoda	Lysianassidae	Eurythenes	Eurythenes gryllus
OTU_4425	1	0	0	Arthropoda	Malacostraca	Amphipoda	Lysianassidae	Eurythenes	Eurythenes sp. 1
OTU_4697	1	0	0	Arthropoda	Malacostraca	Amphipoda	Lysianassidae	Eurythenes	Eurythenes sp. 2A2 HR-2015
OTU_17634	1	0	0	Arthropoda	Hexanauplia	Harpacticoida	Miraciidae	Psammotopa	Psammotopa sp. n. SR-2019
OTU_6939	1	0	0	Arthropoda	Malacostraca	Mysida	Mysidae	Paramesopodopsis	Paramesopodopsis rufa
OTU_1240	1	0	0	Arthropoda	Collembola	Poduromorpha	Neanuridae	Morulina	Morulina mackenziana
OTU_2412	1	0	0	Arthropoda	Malacostraca	Decapoda	Oregoniidae	Pleistacantha	Pleistacantha kannu
OTU_1556	1	0	0	Arthropoda	Malacostraca	Decapoda	Palaemonidae	Macrobrachium	Macrobrachium naiyanetri
OTU_5707	1	0	0	Arthropoda	Malacostraca	Decapoda	Pandalidae	Plesionika	Plesionika semilaevis
OTU_9864	1	0	0	Arthropoda	Malacostraca	Decapoda	Pandalidae	Pandalus	Pandalus montagui
OTU_13528	1	0	0	Arthropoda	Diplopoda	Polyxenida	Polyxenidae		Polyxenidae sp. Biologic-POLX001
OTU_12157	1	0	0	Arthropoda	Malacostraca	Decapoda	Porcellanidae	Petrolisthes	Petrolisthes rufescens
OTU_13725	1	0	0	Arthropoda	Malacostraca	Decapoda	Portunidae	Portunus	Portunus ventralis
OTU_1312	1	0	0	Arthropoda	Collembola	Entomobryomorpha	Tomoceridae	Tomocerus	Tomocerus ocreatus
OTU_1352	1	0	0	Arthropoda	Malacostraca	Isopoda	Tylidae	Tylos	Tylos ponticus
OTU_17543	1	0	0	Arthropoda	Malacostraca	Amphipoda	Typhlogammaridae	Typhlogammarus	Typhlogammarus sp. ZH-2014
OTU_14472	1	0	0	Arthropoda	Hexanauplia	Sessilia	Verrucidae	Metaverruca	Metaverruca recta
OTU_13506	1	0	0	Arthropoda	Malacostraca	Amphipoda			Amphipoda sp. LPdivOTU157
OTU_17242	1	0	0	Arthropoda	Hexanauplia	Copepoda			Copepoda environmental sample
OTU_2669	1	0	0	Arthropoda	Hexanauplia	Copepoda			Copepoda sp. RR-2014
OTU_6511	1	0	0	Arthropoda					Arthropoda environmental sample
OTU_7414	1	0	0	Arthropoda	Hexanauplia	Harpacticoida			Harpacticoida sp. 10BIOBC-00681
OTU_11071	0	1	0	Arthropoda	Hexanauplia	Calanoida	Diaptomidae	Allodiaptomus	Allodiaptomus mirabilipes
OTU_11156	0	1	0	Arthropoda	Ostracoda	Podocopida	Cyprididae	Cypretta	Cypretta campechensis
OTU_15398	0	1	0	Arthropoda					Arthropoda sp. NZAC 03011539
OTU_15775	0	1	0	Arthropoda	Ostracoda	Podocopida	Candonidae	Pseudocandona	Pseudocandona hartwigi
OTU_17132	0	1	0	Arthropoda	Malacostraca	Euphausiacea	Euphausiidae	Pseudeuphausia	Pseudeuphausia sinica
OTU_1993	0	1	0	Arthropoda	Collembola	Symphypleona	Bourletiellidae	Heterosminthurus	Heterosminthurus sp. 2 JRD-2015
OTU_4032	0	1	0	Arthropoda	Malacostraca	Amphipoda	Stenothoidae	Metopa	Metopa boeckii
OTU_4984	0	1	0	Arthropoda	Hexanauplia	Siphonostomatoida			Siphonostomatoida sp. DZMB115
OTU_6169	0	1	0	Arthropoda	Branchiopoda	Diplostraca	Limnadiidae	Limnadopsis	Limnadopsis cf. parvispinus
OTU_6178	0	1	0	Arthropoda	Ostracoda	Podocopida	Cyprididae	Ngarawa	Ngarawa sp. 950.5
OTU_6301	0	1	0	Arthropoda	Malacostraca	Isopoda	Desmosomatidae		Desmosomatidae sp. NB-Iso234
OTU_8282	0	1	0	Arthropoda	Malacostraca	Isopoda	Ligiidae	Ligia	Ligia baudiniana
OTU_8283	0	1	0	Arthropoda	Malacostraca	Amphipoda	Niphargidae	Niphargus	Niphargus parenzani
UVC_03	0	0	1	Bryozoa	Gymnolaemata	Cheilostomatida	Membraniporidae	Biflustra	Biflustra grandicella
UVC_04	0	0	1	Bryozoa					Bryozoa sp.
UVC_05	0	0	1	Bryozoa	Gymnolaemata	Cheilostomatida	Bitectiporidae	entapora	entapora fascialis
UVC_31	0	0	1	Bryozoa	Stenolaemata	Cyclostomatida	Crisiidae	Crisia	Crisia elongata
UVC_33	0	0	1	Bryozoa	Cheilostomatida		Candidae	Caberea	Caberea lata
OTU_10240	1	1	0	Chaetognatha	Sagittoidea	Aphragmophora	Sagittidae	Sagitta	Sagitta sp. ZP304
OTU_36	0	1	0	Chaetognatha	Sagittoidea	Aphragmophora	Sagittidae	Zonosagitta	Zonosagitta nagae
OTU_93	0	1	0	Chaetognatha					Chaetognatha environmental sample
OTU_971	0	1	0	Chaetognatha					Chaetognatha environmental sample
OTU_14459	1	0	0	Chordata	Ascidiacea	Aplousobranchia	Didemnidae	Didemnum	Didemnum candidum
OTU_13738	1	0	0	Chordata	Thaliacea	Pyrosomata	Pyrosomatidae	Pyrostremma	Pyrostremma spinosum
OTU_13999	1	0	0	Chordata	Thaliacea	Salpida	Salpidae	Cyclosalpa	Cyclosalpa sp. USNM IZ 1449879
OTU_13033	1	0	0	Chordata	Ascidiacea	Stolidobranchia	Styelidae	Cnemidocarpa	Cnemidocarpa verrucosa
OTU_2807	1	0	0	Chordata	Ascidiacea	Stolidobranchia	Styelidae	Cnemidocarpa	Cnemidocarpa verrucosa
OTU_121	0	1	0	Chordata	Ascidiacea	Stolidobranchia	Styelidae	Cnemidocarpa	Cnemidocarpa sp. 1
UVC_06	0	0	1	Chordata	Ascidiacea				Ascidiacea sp.
UVC_30	0	0	0	Chordata	Ascidiacea	Stolidobranchia	Styelidae	styela	styela canopus
OTU_8074	1	1	0	Cnidaria	Hydrozoa	Leptothecata	Aequoreidae	Aequorea	Aequorea sp. USNM IZ 1450497
OTU_17794	1	1	0	Cnidaria	Hydrozoa	Anthoathecata	Candelabridae	Candelabrum	Candelabrum cocksii
OTU_1173	1	1	0	Cnidaria	Hydrozoa	Leptothecata	Clytiidae	Clytia	Clytia paulensis
OTU_1784	1	1	0	Cnidaria	Hydrozoa	Leptothecata	Clytiidae	Clytia	Clytia gracilis
OTU_16831	1	1	0	Cnidaria	Hydrozoa	Anthoathecata	Corynidae	Coryne	Coryne sp. JRH-2014
OTU_17154	1	0	0	Cnidaria	Anthozoa	Scleractinia	Acroporidae	Acropora	Acropora sp. IP0357
OTU_17856	1	0	0	Cnidaria	Hydrozoa	Anthoathecata	Bougainvilliidae	Bougainvillia	Bougainvillia muscus
OTU_2568	1	0	0	Cnidaria	Anthozoa	Scleractinia	Caryophylliidae	Dasmosmilia	Dasmosmilia cf. lymani MVK-2010
OTU_2663	1	0	0	Cnidaria	Anthozoa	Scleractinia	Caryophylliidae	Rhizosmilia	Rhizosmilia robusta
OTU_9057	1	0	0	Cnidaria	Scyphozoa	Rhizostomeae	Cassiopeidae	Cassiopea	Cassiopea sp. AUQLCBC
OTU_3574	1	0	0	Cnidaria	Hydrozoa	Anthoathecata	Corynidae	Stauridiosarsia	Stauridiosarsia cliffordi
OTU_13582	1	0	0	Cnidaria	Staurozoa	Stauromedusae	Craterolophidae	Craterolophus	Craterolophus convolvulus
OTU_3839	1	0	0	Cnidaria	Hydrozoa	Anthoathecata	Eudendriidae	Eudendrium	Eudendrium carneum
OTU_5607	1	0	0	Cnidaria	Hydrozoa	Leptothecata	Lafoeidae	Lafoea	Lafoea dumosa
OTU_10300	1	0	0	Cnidaria	Hydrozoa	Leptothecata	Laodiceidae	Laodicea	Laodicea undulata
OTU_13976	1	0	0	Cnidaria	Staurozoa	Stauromedusae	Lucernariidae	Lucernaria	Lucernaria quadricornis
OTU_17088	1	0	0	Cnidaria	Staurozoa	Stauromedusae	Lucernariidae		Lucernariidae sp. CCSMA208-10
OTU_10965	1	0	0	Cnidaria	Hydrozoa	Leptothecata	Obeliidae	Obelia	Obelia dichotoma
OTU_13515	1	0	0	Cnidaria	Hydrozoa	Leptothecata	Obeliidae	Obelia	Obelia sp. DNZ116
OTU_3127	1	0	0	Cnidaria	Hydrozoa	Anthoathecata	Oceaniidae	Turritopsis	Turritopsis lata
OTU_14409	1	0	0	Cnidaria	Hydrozoa	Anthoathecata	Pandeidae	Leuckartiara	Leuckartiara sp. PS-2018
OTU_13700	1	0	0	Cnidaria	Hydrozoa	Siphonophorae	Rhodaliidae	Dendrogramma	Dendrogramma enigmatica
OTU_4766	1	0	0	Cnidaria	Hydrozoa	Leptothecata	Sertulariidae	Fraseroscyphus	Fraseroscyphus hozawai
OTU_3775	1	0	0	Cnidaria	Anthozoa	Scleractinia	Siderastreidae	Psammocora	Psammocora profundacella
OTU_11341	1	0	0	Cnidaria	Scyphozoa	Semaeostomeae	Ulmaridae	Aurelia	Aurelia sp. IDPAJGB
OTU_8311	1	0	0	Cnidaria	Hydrozoa	Anthoathecata	Zancleidae	Zanclea	Zanclea sango
OTU_10172	0	1	0	Cnidaria	Hydrozoa	Leptothecata	Eirenidae	Helgicirrha	Helgicirrha malayensis
OTU_104	0	1	0	Cnidaria	Hydrozoa	Leptothecata	Clytiidae	Clytia	Clytia hemisphaerica
OTU_10944	0	1	0	Cnidaria	Hydrozoa	Anthoathecata	Corymorphidae	Euphysa	Euphysa aurata
OTU_11207	0	1	0	Cnidaria	Anthozoa	Actiniaria	Phymanthidae	Phymanthus	Phymanthus sp. IP0414
OTU_117	0	1	0	Cnidaria	Anthozoa	Alcyonacea	Plexauridae	Euplexaura	Euplexaura sp. A CSM-2013
OTU_15386	0	1	0	Cnidaria	Staurozoa	Stauromedusae	Haliclystidae	Haliclystus	Haliclystus inabai
OTU_15496	0	1	0	Cnidaria	Hydrozoa	Anthoathecata	Corynidae		Corynidae sp. BCnid2010-002
OTU_15730	0	1	0	Cnidaria	Hydrozoa	Anthoathecata	Asyncorinidae	Asyncoryne	Asyncoryne ryniensis
OTU_15819	0	1	0	Cnidaria	Hydrozoa	Siphonophorae	Prayidae	Praya	Praya reticulata
OTU_15894	0	1	0	Cnidaria	Scyphozoa	Rhizostomeae	Rhizostomatidae	Rhopilema	Rhopilema hispidum
OTU_15897	0	1	0	Cnidaria	Hydrozoa	Anthoathecata	Pandeidae	Amphinema	Amphinema sp. JRH-2012
OTU_16864	0	1	0	Cnidaria	Hydrozoa	Leptothecata	Plumulariidae	Plumularia	Plumularia virginiae
OTU_16960	0	1	0	Cnidaria	Hydrozoa	Anthoathecata	Bougainvilliidae	Bougainvillia	Bougainvillia muscus
OTU_17210	0	1	0	Cnidaria	Hydrozoa	Siphonophorae	Agalmatidae	Marrus	Marrus sp. BO-2021
OTU_17266	0	1	0	Cnidaria	Hydrozoa	Anthoathecata	Corynidae	Coryne	Coryne sp. JRH-2014
OTU_1954	0	1	0	Cnidaria	Hydrozoa	Leptothecata	Aglaopheniidae	Aglaophenia	Aglaophenia tubulifera
OTU_1962	0	1	0	Cnidaria	Hydrozoa	Leptothecata	Obeliidae	Obelia	Obelia sp. BFHL 2016
OTU_2471	0	1	0	Cnidaria	Hydrozoa	Anthoathecata	Pandeidae	Amphinema	Amphinema sp. PS-2017
OTU_2502	0	1	0	Cnidaria	Hydrozoa	Anthoathecata	Zancleidae	Zanclea	Zanclea giancarloi
OTU_3269	0	1	0	Cnidaria	Scyphozoa	Semaeostomeae	Cyaneidae	Cyanea	Cyanea sp. RUNNPEC
OTU_473	0	1	0	Cnidaria	Hydrozoa	Anthoathecata	Corynidae	Coryne	Coryne sp. JRH-2014
OTU_5038	0	1	0	Cnidaria	Hydrozoa	Leptothecata	Obeliidae	Obelia	Obelia bidentata
OTU_8072	0	1	0	Cnidaria	Anthozoa	Alcyonacea	Victorgorgiidae	Victorgorgia	Victorgorgia sp. 2 KMM-2015
OTU_8128	0	1	0	Cnidaria	Anthozoa	Scleractinia	Acroporidae	Astreopora	Astreopora myriophthalma
OTU_8132	0	1	0	Cnidaria	Scyphozoa	Semaeostomeae	Pelagiidae	Chrysaora	Chrysaora lactea
OTU_8177	0	1	0	Cnidaria	Scyphozoa	Coronatae	Nausithoidae	Nausithoe	Nausithoe sp. NHM_353
OTU_969	0	1	0	Cnidaria	Hydrozoa	Leptothecata	Obeliidae	Obelia	Obelia sp. MZUSP 3356
UVC_36	0	0	1	Cnidaria	Hexacorallia	Actiniaria	Actiniidae	Paracondylactis	Paracondylactis sinensis
UVC_07	0	0	1	Cnidaria	Octocorallia	Malacalcyonacea	Melithaeidae	Melithaea	Melithaea formosa
UVC_08	0	0	1	Cnidaria	Hexacorallia	Ceriantharia	Cerianthidae		Cerianthidae sp.
UVC_09	0	0	1	Cnidaria	Octocorallia	malacalcyonacea	Isididae	Hicksonella	Hicksonella guishanensis
UVC_10	0	0	1	Cnidaria	Octocorallia				Octocorallia sp.
UVC_11	0	0	1	Cnidaria	Hexacorallia	Actiniaria	Actiniidae	Anthopleura	Anthopleura japonica
UVC_12	0	0	1	Cnidaria	Hexacorallia	Actiniaria	Actiniidae	Anthopleura	Anthopleura inornata
OTU_13644	1	0	1	Ctenophora	Nuda	Beroida	Beroidae	Beroe	Beroe ovata
OTU_14325	1	0	0	Ctenophora	Nuda	Beroida	Beroidae	Beroe	Beroe sp. 2
UVC_34	0	0	1	Echinodermata	Holothuroidea	Dendrochirotida	Cucumariidae	Actinia	Actinia equina
OTU_8133	0	0	1	Echinodermata	Asteroidea	Forcipulatida	Asteriidae	Stichaster	Stichaster striatus
UVC_13	0	0	1	Echinodermata	Echinoidea	Camarodonta	Echinometridae	Anthocidaris	Anthocidaris crassispina
UVC_14	0	0	1	Echinodermata	Echinoidea	Camarodonta	Temnopleuridae	Temnopleurus	Temnopleurus hardwickii
OTU_4816	1	0	0	Echinodermata	Ophiuroidea	Euryalida	Gorgonocephalidae	Astrodendrum	Astrodendrum sp. NSMT:E-6273
OTU_324	1	0	0	Echinodermata	Ophiuroidea	Amphilepidida	Ophiactidae	Ophiactis	Ophiactis perplexa
OTU_2974	1	0	0	Echinodermata	Ophiuroidea	Amphilepidida	Ophiolepididae	Ophiolepis	Ophiolepis irregularis
OTU_5418	1	0	0	Echinodermata	Ophiuroidea	Amphilepidida	Ophiotrichidae	Ophiothrix	Ophiothrix spiculata
OTU_14449	1	0	0	Echinodermata	Crinoidea	Cyrtocrinida	Sclerocrinidae	Neogymnocrinus	Neogymnocrinus richeri
OTU_11136	0	1	0	Echinodermata	Crinoidea	Comatulida	Antedonidae	Antedon	Antedon loveni
OTU_16102	0	1	0	Echinodermata	Ophiuroidea	Euryalida	Gorgonocephalidae	Astrothrombus	Astrothrombus chrysanthi
OTU_466	0	1	0	Echinodermata	Crinoidea	Comatulida	Antedonidae	Antedon	Antedon loveni
OTU_8133	0	1	0	Echinodermata	Asteroidea	Forcipulatida	Asteriidae	Stichaster	Stichaster striatus
OTU_17372	1	0	0	Gastrotricha		Chaetonotida	Chaetonotidae	Chaetonotus	Chaetonotus aff. gelidus MK-2019
OTU_3701	1	0	0	Gastrotricha		Chaetonotida	Chaetonotidae	Aspidiophorus	Aspidiophorus sp. n. MK-2019
OTU_4126	1	0	0	Gastrotricha		Chaetonotida	Chaetonotidae	Aspidiophorus	Aspidiophorus sp. 1
OTU_4761	1	0	0	Gastrotricha		Chaetonotida	Chaetonotidae	Chaetonotus	Chaetonotus sp. 2 MK-2019
OTU_6356	1	0	0	Gastrotricha		Chaetonotida	Chaetonotidae	Chaetonotus	Chaetonotus sp. 1
OTU_7757	1	0	0	Gastrotricha		Chaetonotida	Chaetonotidae	Aspidiophorus	Aspidiophorus sp. n. MK-2019
OTU_15575	0	1	0	Gastrotricha		Macrodasyida	Thaumastodermatidae	Tetranchyroderma	Tetranchyroderma sp. 3 TK-2011
OTU_1292	1	1	0	Mollusca	Gastropoda	Cycloneritida	Neritidae	Neritina	Neritina sp. Suji
OTU_1469	1	1	0	Mollusca	Gastropoda	Systellommatophora	Onchidiidae	Melayonchis	Melayonchis tillieri
OTU_3647	1	1	0	Mollusca	Gastropoda		Pachychilidae	Sulcospira	Sulcospira paludiformis
OTU_94	1	1	0	Mollusca	Gastropoda		Planorbidae	Biomphalaria	Biomphalaria straminea
OTU_6172	1	1	0	Mollusca	Gastropoda	Neogastropoda	Terebridae	Myurella	Myurella affinis
OTU_10351	1	0	0	Mollusca	Gastropoda	Architaenioglossa	Ampullariidae	Pomacea	Pomacea catemacensis
OTU_14521	1	0	0	Mollusca	Gastropoda	Architaenioglossa	Ampullariidae	Pomacea	Pomacea canaliculata
OTU_7625	1	0	0	Mollusca	Gastropoda	Aplysiida	Aplysiidae	Aplysia	Aplysia fasciata
OTU_13638	1	0	0	Mollusca	Gastropoda	Trochida	Calliostomatidae	Calliostoma	Calliostoma iridium
OTU_14338	1	0	0	Mollusca	Gastropoda	Stylommatophora	Camaenidae	Xanthomelon	Xanthomelon interpositum
OTU_14515	1	0	0	Mollusca	Gastropoda	Stylommatophora	Camaenidae	Satsuma	Satsuma jacobii
OTU_2134	1	0	0	Mollusca	Gastropoda	Stylommatophora	Camaenidae	Satsuma	Satsuma hemihelvus
OTU_5901	1	0	0	Mollusca	Gastropoda	Stylommatophora	Camaenidae	Satsuma	Satsuma sp. SPW-2007
OTU_9949	1	0	0	Mollusca	Gastropoda	Stylommatophora	Clausiliidae	Montenegrina	Montenegrina helvola
OTU_736	1	0	0	Mollusca	Gastropoda	Neogastropoda	Costellariidae	Pusia	Pusia savignyi
OTU_6464	1	0	0	Mollusca	Gastropoda	Nudibranchia	Cuthonellidae	Cuthonella	Cuthonella punicea
OTU_4617	1	0	0	Mollusca	Gastropoda	Architaenioglossa	Diplommatinidae	Diplommatina	Diplommatina sp. nov. AG MS-2010
OTU_6514	1	0	0	Mollusca	Gastropoda	Nudibranchia	Dotidae	Doto	Doto coronata
OTU_10095	1	0	0	Mollusca	Gastropoda	Ellobiida	Ellobiidae	Ellobium	Ellobium scheepmakeri
OTU_3788	1	0	0	Mollusca	Gastropoda	Lepetellida	Fissurellidae	Fissurella	Fissurella sp. SWA-2011
OTU_2686	1	0	0	Mollusca	Gastropoda	Stylommatophora	Geomitridae	Xerocrassa	Xerocrassa ponsi
OTU_4632	1	0	0	Mollusca	Gastropoda	Littorinimorpha	Hydrobiidae	Marstonia	Marstonia lustrica
OTU_14429	1	0	0	Mollusca	Gastropoda	Neogastropoda	Muricidae	Concholepas	Concholepas concholepas
OTU_9579	1	0	0	Mollusca	Gastropoda	Neogastropoda	Muricidae	Drupa	Drupa morum
OTU_14543	1	0	0	Mollusca	Gastropoda	Cycloneritida	Neritidae	Nerita	Nerita atramentosa
OTU_1864	1	0	0	Mollusca	Gastropoda	Cycloneritida	Neritidae	Neritina	Neritina dilatata
OTU_5511	1	0	0	Mollusca	Cephalopoda	Teuthida	Ommastrephidae	Dosidicus	Dosidicus gigas
OTU_10283	1	0	0	Mollusca	Gastropoda	Systellommatophora	Onchidiidae	Paromoionchis	Paromoionchis daemelii
OTU_13627	1	0	0	Mollusca	Gastropoda	Systellommatophora	Onchidiidae	Onchidella	Onchidella marginata
OTU_14456	1	0	0	Mollusca	Gastropoda		Pachychilidae	Sulcospira	Sulcospira paludiformis
OTU_1546	1	0	0	Mollusca	Bivalvia	Unionida	Unionidae	Middendorffinaia	Middendorffinaia mongolica
OTU_4294	1	0	0	Mollusca	Bivalvia	Unionida	Unionidae	Pleuronaia	Pleuronaia barnesiana
OTU_4892	1	0	0	Mollusca	Bivalvia	Unionida	Unionidae	Toxolasma	Toxolasma lividus
OTU_7039	1	0	0	Mollusca	Bivalvia	Unionida	Unionidae	Aculamprotula	Aculamprotula fibrosa
OTU_10224	1	0	0	Mollusca	Gastropoda	Littorinimorpha	Vermetidae	Dendropoma	Dendropoma gregarium
OTU_5629	1	0	0	Mollusca	Gastropoda	Littorinimorpha	Vermetidae	Dendropoma	Dendropoma platypus
OTU_14128	1	0	0	Mollusca	Bivalvia				Bivalvia environmental sample
OTU_12851	0	1	0	Mollusca	Gastropoda	Stylommatophora	Camaenidae	Satsuma	Satsuma hemihelvus
OTU_15803	0	1	0	Mollusca	Bivalvia	Galeommatida	Galeommatidae	Galeomma	Galeomma turtoni
OTU_1980	0	1	0	Mollusca	Gastropoda	Stylommatophora	Geomitridae	Candidula	Candidula sp. AR_BC7338
OTU_3248	0	1	0	Mollusca	Gastropoda	Neogastropoda	Terebridae	Hastula	Hastula matheroniana
OTU_3959	0	1	0	Mollusca	Gastropoda		Physidae	Physella	Physella acuta
OTU_4408	0	1	0	Mollusca	Gastropoda	Stylommatophora	Helicidae	Eobania	Eobania vermiculata
OTU_470	0	1	0	Mollusca	Gastropoda		Plakobranchidae	Thuridilla	Thuridilla vatae
OTU_6184	0	1	0	Mollusca	Gastropoda	Cycloneritida	Phenacolepadidae	Plesiothyreus	Plesiothyreus newtoni
OTU_6194	0	1	0	Mollusca	Bivalvia	Unionida	Unionidae	Hyriopsis	Hyriopsis bialatus complex sp. 2 PP-2017
OTU_6526	0	1	0	Mollusca	Gastropoda	Stylommatophora	Clausiliidae	Montenegrina	Montenegrina umbilicata
OTU_7189	0	1	0	Mollusca	Gastropoda	Stylommatophora	Camaenidae	Rhagada	Rhagada angulata
OTU_8077	0	1	0	Mollusca	Gastropoda	Siphonariida	Siphonariidae	Siphonaria	Siphonaria lessonii
OTU_86	0	1	0	Mollusca	Gastropoda	Littorinimorpha	Hydrobiidae	Hauffenia	Hauffenia subpiscinalis
OTU_965	0	1	0	Mollusca	Bivalvia	Galeommatida	Galeommatidae	Galeomma	Galeomma turtoni
UVC_37	0	0	1	Mollusca	Bivalvia	Mytilida	Mytilidae	Perna	Perna viridis
UVC_38	0	0	1	Mollusca	Bivalvia	Mytilida	Modiolidae	Vignadula	Vignadula atrata
UVC_15	0	0	1	Mollusca	Cephalopoda	Sepiida	Sepiidae	Sepiella	Sepiella maindroni de Rochebrune
UVC_16	0	0	1	Mollusca	Gastropoda	Trochidae	Calliostomatidae	Tristichotrochus	Tristichotrochus unicum
UVC_17	0	0	1	Mollusca	Gastropoda	Trochidae	Trochidae	Monodonta	Monodonta labio
UVC_18	0	0	1	Mollusca	Gastropoda	Littorinimorpha	Ovulidae		Ovulidae sp.
UVC_19	0	0	1	Mollusca	Gastropoda	Littorinimorpha	Ovulidae	Cuspivolva	Cuspivolva formosa
UVC_20	0	0	1	Mollusca	Gastropoda	Neogastropoda	Buccinidae	Cantharus	Cantharus cecillei
UVC_21	0	0	1	Mollusca	Gastropoda	Neogastropoda	Cymatiidae	Gyrineum	Gyrineum natator
UVC_22	0	0	1	Mollusca	Gastropoda	Neogastropoda	Muricidae	Chicoreus	Chicoreus asianus
UVC_23	0	0	1	Mollusca	Gastropoda	Neogastropoda	Muricidae	Reishia	Reishia clavigera
UVC_24	0	0	1	Mollusca	Gastropoda	Neogastropoda	Muricidae	Reishia	Reishia luteostoma
UVC_25	0	0	1	Mollusca	Gastropoda	Neogastropoda	Nassariidae	Nassarius	Nassarius hepaticus
UVC_26	0	0	1	Mollusca	Gastropoda	Nudibranchia			Nudibranchia sp.
UVC_27	0	0	1	Mollusca	Gastropoda	Nudibranchia	Facelinidae	Sakuraeolis	Sakuraeolis sakuracea
UVC_28	0	0	1	Mollusca	Gastropoda	Nudibranchia	Dendrodorididae	Doriopsilla	Doriopsilla sp.
UVC_32	0	0	1	Mollusca	Gastropoda		Pyramidellidae	Odostomia	Odostomia subangulata
OTU_7430	1	0	0	Nematoda	Chromadorea	Strongylida	Ancylostomatidae	Ancylostoma	Ancylostoma ceylanicum
OTU_7275	1	0	0	Nematoda	Chromadorea	Desmodorida	Desmodoridae	Metachromadora	Metachromadora sp. 2AMA
OTU_13801	1	0	0	Nematoda	Chromadorea	Rhabditida	Rhabditidae	Caenorhabditis	Caenorhabditis inopinata
OTU_8772	1	0	0	Nemertea	Pilidiophora	Heteronemertea	Baseodiscidae	Baseodiscus	Baseodiscus sp. 2 SA-2011
OTU_2470	1	0	0	Nemertea	Palaeonemertea		Tubulanidae	Tubulanus	Tubulanus polymorphus
OTU_17313	0	1	0	Nemertea	Enopla	Monostilifera			Monostilifera sp. MCZ IZ 45646
OTU_502	0	1	0	Nemertea	Enopla	Monostilifera	Amphiporidae	Amphiporus	Amphiporus cruentatus
OTU_17155	1	0	0	Phoronida					Phoroniformea sp. larval OTU P1
OTU_435	1	1	0	Platyhelminthes	Trematoda	Diplostomida	Diplostomidae	Crassiphiala	Crassiphiala sp. Lineage 2
OTU_16856	0	1	0	Platyhelminthes	Rhabditophora	Tricladida	Geoplanidae	Microplana	Microplana sp. MOTU37
OTU_897	1	0	0	Porifera	Demospongiae	Bubarida	Dictyonellidae	Scopalina	Scopalina lophyropoda
OTU_7359	1	0	0	Porifera	Demospongiae	Suberitida	Halichondriidae	Ciocalypta	Ciocalypta sp. DE-2012
OTU_8918	1	0	0	Porifera	Demospongiae	Haplosclerida	Niphatidae	Amphimedon	Amphimedon queenslandica
OTU_7393	1	0	0	Porifera	Homoscleromorpha	Homosclerophorida	Plakinidae	Plakina	Plakina coerulea
OTU_9873	1	0	0	Porifera	Homoscleromorpha	Homosclerophorida	Plakinidae	Plakina	Plakina sp. 1
OTU_7613	1	0	0	Porifera	Demospongiae	Axinellida	Raspailiidae		Raspailiidae sp. Po.25592
OTU_9606	1	0	0	Porifera	Demospongiae	Tetractinellida	Tetillidae	Cinachyrella	Cinachyrella alloclada
OTU_10893	0	1	0	Porifera	Demospongiae	Suberitida	Suberitidae	Aaptos	Aaptos nuda
OTU_15892	0	1	0	Porifera	Demospongiae	Suberitida	Halichondriidae		Halichondriidae sp. UCMPWC1015
OTU_16058	0	1	0	Porifera	Demospongiae	Poecilosclerida	Mycalidae	Mycale	Mycale mirabilis
OTU_16093	0	1	0	Porifera					Porifera sp. Sp08
OTU_17151	0	1	0	Porifera	Demospongiae	Tetractinellida	Tetillidae	Cinachyrella	Cinachyrella australiensis
OTU_2563	0	1	0	Porifera	Demospongiae	Tethyida	Hemiasterellidae	Adreus	Adreus fascicularis
OTU_5032	0	1	0	Porifera	Demospongiae	Haplosclerida	Chalinidae	Haliclona	Haliclona sp. P2MK
UVC_35	0	0	1	Porifera	Demospongiae	Tethyida	Tethyidae	Tethya	Tethya aurantium
UVC_29	0	0	1	Porifera	Demospongiae	Tethyida	Tethyidae	Tethya	Tethya sp. 1

Author Contributions

Conceptualization, Z.Z., Y.L., and K.X.; methodology, W.H., S.X., W.C., and X.L.; software, W.H. and Z.Z.; validation, Z.Z. and K.X.; formal analysis, W.H., S.X., Z.Z., and W.C.; investigation, X.L., Z.Z., S.X., and W.C.; writing—review and editing, Z.Z. and K.X.; visualization, W.H. and Z.Z.; supervision, Z.Z.; project administration, K.X.; funding acquisition, Y.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw sequence reads detected by eDNA are deposited in the NCBI Sequence Read Archive database with the BioProjects PRJNA1248872 and PRJNA1248901.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by National Key R&D Program of China, grant number 2022YFC2803800, the National Natural Science Foundation of China, grant number 42176128, the Strategic Priority Research Program of the Chinese Academy of Sciences, grant number XDB42000000, and Science and Technology Program of Nanji Islands National Marine Nature Reserve Administration, grant numbers and ZJYY-PYCG-2024092001 and JJZB-PYCG-2021112901. The APC was funded by No. 2022YFC2803800.