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Full Research Article

Fish Diversity in Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve based on eDNA Metabarcoding

Y
Yuanyuan Zhao1
J
Jinzhu Meng1
Y
Yaojie Yang1
X
Xiaodong Zhang1
J
Jie Mei1
J
Jiawen Ba1,*
1Guizhou Provincial Key Laboratory for Biodiversity Conservation and Utilization in the Fanjing Mountain Region/Tongren University, Tongren 554300, China.

ABSTRACT

Background: The Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve located in Tongren City, Guizhou Province, China, has abundant fish resources. Accurate monitoring of the composition and diversity of fish communities is of vital importance for assessing the health of ecosystems, protecting biodiversity and endangered species, ensuring the sustainable utilization of fishery resources and managing invasive species.

Methods: We examined the distribution and abundance of fishes in water and sediments of the upstream, midstream and downstream of Xieqiao River using eDNA (environmental DNA) metabarcoding.

Result: We recognized 112 fish species belonging to 16 orders, 28 families and 73 genera. The most abundant fishes in Xieqiao River at order, family, genus and species level were Cypriniformes, Xenocyprididae, Opsariichthys and Opsariichthys bidens, which varied in different habitats. Forty-three dominant fish species were identified, including 3 species of specially protected fish in China, 26 endangered species of China Red List of Biodiversity·Freshwater fish, 17 species with extremely high relative sequence abundance in Guizhou Province and 7 species of endemic fish of the upper Yangtze River and 14 alien species. The Chao1 index of water samples are higher than that of sediments (P<0.05). In addition, the fish communities of water in river were different from those in reservoir. In conclusion, the Xieqiao river is rich in fish resources and diversity, but the invasion of alien species should be vigilant. This study assessed fish resources and provides reference for subsequent management of Xieqiao river endemic fish national aquatic germplasm resources reserve.

INTRODUCTION

Fish constitute a critical component of aquatic ecosystems and their diversity plays a direct role in maintaining ecological balance and aquatic ecosystem health (Shen et al., 2022). Global fish diversity is currently facing severe threats from multiple factors, including water pollution, overfishing, habitat destruction, climate change and invasive alien species (Shen et al., 2022; Ruan et al., 2022). Freshwater fish accounts for over 50% of the global fish species diversity. However, their survival situation is extremely serious: nearly one-third of the existing freshwater fish species are at risk of extinction and more than 30% of the species have been assessed as being critically endangered (Vardakas et al., 2025; Thomson-Laing et al., 2023). Traditional monitoring of freshwater fish mainly relies on capture sampling (such as netting, electrofishing, cage trapping) and morphological identification, but it has multiple limitations: such as low efficiency, high cost, ecological disturbance and applicability restrictions, low accuracy and insufficient data coverage, etc. (Liu et al., 2025). Consequently, the precise monitoring of fish community composition and diversity is essential for evaluating ecosystem health, conserving biodiversity and endangered species, ensuring the sustainable use of fishery resources and managing invasive species (Shen et al., 2022; Yao et al., 2022; Zhang et al., 2024).
       
eDNA metabarcoding represents an emerging and innovative biological monitoring tool that offers a robust means of assessing the diversity of marine and freshwater fish communities. In comparison to traditional fishing and observation methods, eDNA metabarcoding exhibits several advantages, including high sensitivity, non-invasiveness and enhanced efficiency (Hervé et al., 2022; Qian et al., 2023; Saroj et al., 2020; Jesintha et al., 2021; Sit et al., 2021). This approach not only facilitates the detection of species that are challenging to capture using conventional techniques such as gill nets and trawls, thereby providing a more comprehensive species inventory, but also enables a more sensitive reflection of the spatio-temporal variation characteristics of fish communities. Furthermore, it assists scientists in better understanding ecosystem structure and function (Wang et al., 2022; Shen et al., 2023; Zou et al., 2020; Wang et al., 2021).
       
In recent years, eDNA metabarcoding has gained widespread application in the investigation of fish diversity in both marine and freshwater ecosystems. For instance, researchers have employed eDNA metabarcoding to assess fish diversity in various aquatic environments, including Atlantic Sea, the Mediterranean, Caribbean Sea, Cosmonaut Sea in East Antarctica, Sylt Outer Reef in the North Sea of Germany, Baltic Sea and  Black Sea (Roblet et al., 2024; Gonzalez Colmenares et al., 2023; Liao et al., 2023; Kasmi et al., 2024; Ye et al., 2025; Urban et al., 2024; Gelen and Pekmezci, 2023) and Jiangmen coastal waters, Wuzhizhou Island in the South China Sea, the Ma’an Islands Special Conservation Area, the Yangtze River Estuary and the Xinglin Bay Reservoir in Xiamen in China (Zhang et al., 2024; Wang et al., 2024a; Wang et al., 2022; Lyu et al., 2024; Wang et al., 2024b). Furthermore, eDNA metabarcoding has been used to monitor alien species, especially those brought by sea transportation (Schreiber et al., 2023; Aglieri et al., 2023). The diversity of freshwater fish has been studied using eDNA metabarcoding in most parts of China, especially in the Yangtze River basin (Qian et al., 2023; Shen et al., 2023; Qu et al., 2020; Cheng et al., 2024). Guizhou Province situate in the upper reaches of the Yangtze River and the fish diversity of Wujiang River, Chishui River and Dulliujiang River has been reported (Shen et al., 2023; Peng et al., 2025; Yang 2023; Cheng et al., 2023). However, there is a paucity research focusing on rivers surrounding Tongren City, Guizhou Province, China.
       
The Xieqiao river endemic fish national aquatic germplasm resources reserve located in Tongren City, Guizhou Province, China, with a total river length of 25.8 km and geographical coordinates between 108°59'25"~ 109°08'44" east longitude and 27°35'12"~ 27°39'34" north latitude. The main protected fishes are Silurus asotus (Linnaeus, 1758), Onychostoma lini (H. W. Wu, 1939) and Siniperca chuatsi (Basilewsky, 1855), while other protected fishes include Pelteobagrus fulvidraco (Richardso, 1846), Pseudogyrincheilus prochilu (Sauvage et Dabry, 1874), Hemibarbus maculatus (Bleeker, 1871), Rhinogobio cylindricus (Gunther, 1888), Sarcocheilichthys sinensis (Bleeker, 1871), Saurogobio dabryi (Bleeker, 1871), Zacco platypus (Temminck and Schlegel, 1846) and Opsariichthys bidens (Günther, 1873). To investigate the structure and diversity of fish communities in the reserve, we examined the distribution and abundance of fish in water and sediments of the upstream, midstream and downstream of Xieqiao River using eDNA metabarcoding, which will assess fish resources and provide reference for the reserve management.

MATERIALS AND METHODS

eDNA sampling and processing

In this study, we set 3 sampling points (upstream: 109°03'49.0090", 27°36'41.5639"; midstream: 109°04' 44.3035", 27°36'41.3588"; downstream: 109°08' 18.4637", 27°38'58.4685") (Fig 1), a total of 18 samples (3 water samples and 3 sediment samples at each sampling point) were collected in October 2023. Water samples (2 L) from the middle and sides of the river were collected using clean disposable bottle disinfected with a 10% bleach solution. About 5 g sediments were also collected at the same locations as the water samples. Three replicates were collected at each sampling point. The collected water samples and sediment samples were stored under 4! until returned to the laboratory. Every 2 L of water was filtered by a vacuum pump through a mixed cellulose filter membrane with a pore size of 0.45 μm (Whatman). The filtering equipment was disinfected and washed with ultrapure water before each filtration to avoid cross contamination between samples. Distilled water (2 L) was used as a negative control to evaluate the presence of exogenous DNA contamination. Three membranes from the same sampling point were stored in the same cryostorage tube at -80°C for DNA extraction. The sediment samples from the same sampling point were mixed to cover as many species as possible and stored at -80°C. Water samples of the upstream, midstream and downstream in Xieqiao River are labeled W1-3 and sediment samples are labeled S1-3.

Fig 1: Information on sampling locations by ArcGIS 10.7. S1, S2 and S3 represent upstream, midstream and downstream of Xieqiao River, respectively.


 
DNA extraction and metabarcoding
 
E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) was used to extract DNA from the sediment samples and filter membranes following the manufacturer’s instructions. The membrane filtered distilled water was used as the negative control. Tele02 primers were used to amplify A hypervariable fragment of the mitochondrial 12S rRNA gene (Taberle et al., 2018). PCR was performed using TransStart®FastPfu DNA polymerase (TransGen Biotech Co., Beijing, China) with a DNA template of 10 ng and three replicates. The negative control was also amplified. 2% agarose gel electrophoresis was used to detect PCR products. As a result, all samples except negative controls produced detectable PCR products. According to the preliminary quantitative results of electrophoresis, PCR products were detected and quantified using the QuantiFluor™ -ST blue fluorescence quantification system (Promeg, Madison, USA). The pooled DNA product was used to construct Illumina Pair-End library and was paired-end sequenced (2×250) on an Illumina MiSeq platform (Biozeron Co., Ltd, Shanghai, China).
 
Bioinformatics and statistical analyses
 
Poor-quality sequences were eliminated using Trimmomatic v.0.36. The remaining paired reads were combined into a single sequence by FLASH (v.1.2.11). Chimeric sequences were identified and eliminated using Usearch software (version 10, http://drive5.com/uparse/) compared with the reference sequences of GOLD database. The primers were removed with Cutadapt (v4.0). Valid tags of 12S rRNA were aligned to amplicon sequence variants (ASVs) and generated a feature table by DADA2. All the valid tags were then searched remotely against the Mitofish (http://mitofish.aori.u-tokyo.ac.jp/download.html), NCBI-NT-euk and local nucleotide reference databases (FishCOI) with BLASTn. We make a slight modification to the classification assignment criteria set by Zhang et al., (2020): (a) if the query sequence matches a single locally present species recorded in the databasee≥98%, the species is assigned; (b) if the query matches more than one native species with ≥98% identity, the lowest taxonomic level that included all these species was assigned; (c)Sequences with homology <98% but ≥95% belong to genus and <95% but ≥92% belong to family (Zhang et al., 2020).
 
Fish diversity analysis
 
Freshwater fish were screened according to FishBase database, false positive species were removed (reads with an abundance ≤5 at species level of a single sample were considered false positive) and all ASVs with an abundance of 0 were removed and subsequent analysis was conducted on this basis at the species level. The sequence abundance of the species was analyzed and Alpha diversity among different habitats was calculated using the R programming language (Auckland, New Zealand). The Shannon–Wiener, Simpson index, Chao1 index and Pielou_J index were evaluated and presented in box plot. To explore the similarity of fish composition in different samples, principal coordinate analysis (PCoA) was constructed using Bray-Curtis distance matrix which also used to evaluate non-metric multidimensional scales (NMDS) representing beta diversity between different habitats. Overlaping and unique fish taxa of three sampling points were shown in Venn diagram using the R software package pheatmap (https://CRAN.R-project.org/package= pheatmap).
       
Statistical analysis and figures were performed using R version 4.0.3 (https://www.r-project.org/) (R Core Team, 2020). T-test and Tukey ‘HSD test were used to detect the differences between two groups and multiple groups.

RESULTS AND DISCUSSION

Sequence information and taxonomic assignment
 
High throughput sequencing analysis of 6 samples resulted in 3438615 clean sequences with a total base number of 589877142 bp. The average length was 171.54 bp. Following BLAST and manual refinement, we recognized 112 fish species belonging to 16 orders, 28 families and 73 genera in Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve.
 
Fish community among different river sections
 
Among all fish species, the most abundant species at order, family, genus and species level were Cypriniformes, Xenocyprididae, Opsariichthys and Opsariichthys bidens (Fig 2). But the dominant species and abundance were different in different habitats. At order level, Cypriniformes was the most dominant species, its proportion in water was 95.54%, while its proportion in sediments was 62.04%. Corresponding, Gobiiformes, Cichliformes and Osteoglossiformes occupied about 12.08%, 12.34% and 12.32% in sediments, but very few in water. At family level, Xenocyprididae were the most dominant species (66.25% in water, 49.38% in sediments), followed by Cyprinidae (27.80%) in water and Cichlidae (12.34%), Notopteridae (12.32%), Gobiidae (12.08%) in sediments. At genus level, the difference was more obvious. Opsariichthys and Carassius were the most dominant species in water, accounting for 56.33% and 25.51% respectively, while there were more dominant species in sediments, accounting for a slightly smaller proportion of each species, Hypophthalmichthys, Sarotherodo, Mugilogobius, Cyprinus and Hemiculter accounting for 31.73%, 12.34%, 12.05%, 8.83% and 8.24%, respectively (Fig 3). Besides, the dominant species of reservoir and river were not exactly the same. At order and family level, the most dominant species were Cypriniformes and Xenocyprididae, which was similar in different habitats. However, there were more dominant species in river (N=6 and N=10) and more concentrated in reservoir (N=3 and N=4). At genus and species level, the dominant species in reservoir were Hypophthalmichthys (29.96%), Carassius (24.13%), Hemiculter (19.18%) and Hypophthalmichthys molitrix (Valenciennes, 1844) (29.94%), Carassius auratus (Linnaeus, 1758) (24.05%), Hemiculter leucisculus (Basilewsky, 1855) (19.18%), While the dominant species in river were Opsariichthys (37.41%) and Opsariichthys bidens (37.41%) (Fig 4). Finally, at order and family level, the most dominant species in different reaches were the same, but at genus level, the dominant species were Opsariichthys (30.20%), Hypophthalmichthys (22.39%), Carassius (14.14%), Chitala (12.47%), Cyprinus (13.42%) in upstream, Opsariichthys (44.75%), Sarotherodon (18.51%), Mugilogobius (18.07%) in midstream and Hypophthalmichthys (29.96%), Carassius (24.13%), Hemiculter (19.18%) in downstream. The distribution of dominant species at species level is similar to that at genus level (Fig 5). The fish composition and abundance of each sample are shown in Fig 6.

Fig 2: Species composition of each sample at order, family, genus and species level.



Fig 3: Species composition of water and sediments at order, family, genus and species level.



Fig 4: Species composition of reservoir and river at order, family, genus and species level.



Fig 5: Species composition of different river sections in Xieqiao River at order, family, genus and species level.



Fig 6: Heatmap analysis of fish community.


       
After removing false positive species (reads£5), screening fresh water fish and reviewing biological classification information, 43 dominant fish species belonging to 6 orders, 12 families, 33 genera were identified, including 3 species of specially protected fish in China, 26 endangered species of China Red List of Biodiversity· Freshwater fish, 17 species with extremely high relative sequence abundance in Guizhou Province and 7 species of endemic fish of the upper Yangtze River and 14 alien species (Table 1).

Table 1: Species list and sequence number of fish detected by eDNA in xieqiao river endemic fish national aquatic germplasm resources reserve.


 
Differences in fish community among different river sections
 
In the six samples from the three sampling points, the Chao1 index of water samples were generally higher than that of sediments and the difference was significant (P<0.05). While, the differences of Shannon index, Simpson index and Pielou_J index between water samples and sediment samples were not significant (Fig 7A-D). PC1 and PC2 axes in PCoA (Principal co-ordinates analysis) explained 36% and 28% of the changes in the composition of the overall fish community diversity, respectively, with a total coverage of more than 60%. The most similar fish communities were those of W1 and W2, followed by S1 and S3 (Fig 7E). The fish communities in water were clearly differentiated from those in sediments, but the difference is not significant (ANOSIM: R=0.5926, P=0.1>0.05). In addition, the fish communities of water in river were different from those in reservoir (Fig 7F).

Fig 7: The boxplots showing the alpha diversity index for water and sediments and the PCoA and NMDS showing the beta diversity index for each sample.


       
The fish communities of rivers, lakes and reservoirs in Guizhou Province are well known, especially Wujiang River (Cheng et al., 2023). However, the research mainly focuses on the master stream of each river system and the research on tributaries is less. In particular, the report on the fish communities of Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve has not been systematically discussed. Moreover, it has been nearly four years since the 10-year fishing ban in the Yangtze River was implemented in 2020 and the recovery effect of fish populations needs to be evaluated urgently. Therefore, in this study, eDNA high-throughput sequencing was used to investigate the fish communities in Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve.
       
In this study, we identified 6 orders, 12 families, 33 genera and 43 dominant fish species in Xieqiao river, the 5 most abandant orders were Cypriniformes, Osteoglossiformes, Gobiiformes, Cichliformes, Siluriformes and Centrarchiformes, which was similar to that of Wujiang river, Chishui river and Duliujiang River in Guizhou. Cypriniformes and Siluriformes were dominant orders in the Wujiang, Chishui River and the Duliujiang River (Yang, 2023; Cheng et al., 2023; Guo, 2023). Osteoglossiformes were detected in Yangtze River (Qian et al., 2023). Cichliformes, Gobiiformes and Centrarchiformes tend to be grouped in the order Perciformes (Yang, 2023; Wang, 2022). Xenocyprididae is a subfamily of Cyprinidae and they are often grouped together in a single group. Therefore, Cyprinidae is the most dominant fish family in Xieqiao River, which is consistent with the dominant fish families in the Yangtze River, Wujiang River, Chishui River and Duliujiang River (Shen et al., 2023; Yang, 2023; Cheng et al., 2023; Guo, 2023). The dominant genus Opsariichthys, Hypophthalmichthys, Carassius and Hemiculter in Xieqiao River were common species in other rivers and lakes (Shen et al., 2023; Yan et al., 2023). Opsariichthys bidens, Carassius auratus, Sarotherodon galilaeus, Mugilogobius myxodermus, Hypophthalmichthys molitrix were dominant fishes in Xieqiao River. In most studies Opsariichthys bidens were detected, while in a few studies Opsariichthys uncirostris were detected, which might be attributed to their close genetic relationship (Qian et al., 2023; Zhang et al., 2023; Chen et al., 2008). Sarotherodon galilaeus, an invasive tilapia species, has been dominant fishes in the Shanmei Reservoir and Jiulong River Basin of Southeast China (Yang et al., 2024; Feng et al., 2025). Mugilogobius myxodermus has been detected in the Yangtze River, the Pearl River and Dianchi Lake in Yunnan, but its distribution in Guizhou waters has not been reported (Shen et al., 2023; Chen et al., 2022; Yue et al., 2018). Carassius auratus and Hypophthalmichthys molitrix distributed widely in China. Finally, three protected fish species were detected in Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve, including Silurus asotus, Zacco platypus and Opsariichthys bidens. Other protected fish species, most of which were demersal fish, were not detected in this study, possibly was due to insufficient sampling sites and failure to collect deep water from reservoir.
       
Three species of National wildlife under special protection, 26 endangered species of China Red List of Biodiversity·Freshwater fish, 17 species with extremely high relative sequence abundance in Guizhou Province and 7 species of endemic fish of the upper Yangtze River were detected, which indicated that the ecological environment of Xieqiao River was suitable for the survival of protected fish and it was necessary to establish Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve. In addition, 14 alien species were detected in Xieqiao River, accounting for 9.24% of the total resource, among which Sarotherodon galilaeus, Chitala ornata and Micropterus salmoides were the most abundant. Chitala ornata was found as early as May 2007 on the upper Mekong in Menglun (21°56'20"N, 101°15'18"E) in Xishuangbanna, China (Kang, 2013). Micropterus salmoides has invaded many natural waters in China, including Beipan River, Wujiang River in Guizhou and Dianchi Lake in Yunnan (Qiao et al., 2020). The presence of these invasive species warns us to pay close attention to them and take effective measures to control them and prevent them from endangering the survival of local aquatic organisms.
       
Alpha diversity analysis showed that the Chao1 index of water samples was generally higher than that of sediments and the difference was significant (P<0.05), While, the differences of Shannon index, Simpson index and Pielou_J index between water samples and sediment samples were not significant, indicating that fish diversity in water was significantly higher than that in sediments, but there were no significant differences in species richness and evenness between two samples. The β-diversity indicated that fish diversity in sediments of midstream was quite different from that of upstream and downstream, which might be related to the presence of duck in this section. Fish diversity in water of downstream was quite different from that of upstream and midstream, which may be due to the fact that the downstream was reservoir, whereas the upstream and midstream were rivers. River was suitable for rheophilic fishes and reservoir for limnophilic fishes (Yang, 2023). Moreover, the reservoir was more susceptible to human activities and dam barriers and altitude changes could also affect fish community structure (Peng et al., 2025; Wang, 2022).

CONCLUSION

The Xieqiao river is rich in fish resources and diversity. There are 112 species of fish belonging to 16 orders, 28 families and 73 genera in Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve, among which there are 43 dominant fish species. But the dominant species and abundance were different in different habitats. In addition, the alien species account for 9.24% of the total resource, especially Sarotherodon galilaeus, Chitala ornata and Micropterus salmoides, which should be vigilant. This study assessed fish resources and provides reference for subsequent management of Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve.

ACKNOWLEDGEMENT

This study was supported by National Natural Science Foundation of China (32060274), Doctoral Talents project of Science and Technology Bureau of Tongren, Guizhou Province ([2022]1).
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors  and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

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    Fish Diversity in Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve based on eDNA Metabarcoding

    Y
    Yuanyuan Zhao1
    J
    Jinzhu Meng1
    Y
    Yaojie Yang1
    X
    Xiaodong Zhang1
    J
    Jie Mei1
    J
    Jiawen Ba1,*
    1Guizhou Provincial Key Laboratory for Biodiversity Conservation and Utilization in the Fanjing Mountain Region/Tongren University, Tongren 554300, China.

    ABSTRACT

    Background: The Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve located in Tongren City, Guizhou Province, China, has abundant fish resources. Accurate monitoring of the composition and diversity of fish communities is of vital importance for assessing the health of ecosystems, protecting biodiversity and endangered species, ensuring the sustainable utilization of fishery resources and managing invasive species.

    Methods: We examined the distribution and abundance of fishes in water and sediments of the upstream, midstream and downstream of Xieqiao River using eDNA (environmental DNA) metabarcoding.

    Result: We recognized 112 fish species belonging to 16 orders, 28 families and 73 genera. The most abundant fishes in Xieqiao River at order, family, genus and species level were Cypriniformes, Xenocyprididae, Opsariichthys and Opsariichthys bidens, which varied in different habitats. Forty-three dominant fish species were identified, including 3 species of specially protected fish in China, 26 endangered species of China Red List of Biodiversity·Freshwater fish, 17 species with extremely high relative sequence abundance in Guizhou Province and 7 species of endemic fish of the upper Yangtze River and 14 alien species. The Chao1 index of water samples are higher than that of sediments (P<0.05). In addition, the fish communities of water in river were different from those in reservoir. In conclusion, the Xieqiao river is rich in fish resources and diversity, but the invasion of alien species should be vigilant. This study assessed fish resources and provides reference for subsequent management of Xieqiao river endemic fish national aquatic germplasm resources reserve.

    INTRODUCTION

    Fish constitute a critical component of aquatic ecosystems and their diversity plays a direct role in maintaining ecological balance and aquatic ecosystem health (Shen et al., 2022). Global fish diversity is currently facing severe threats from multiple factors, including water pollution, overfishing, habitat destruction, climate change and invasive alien species (Shen et al., 2022; Ruan et al., 2022). Freshwater fish accounts for over 50% of the global fish species diversity. However, their survival situation is extremely serious: nearly one-third of the existing freshwater fish species are at risk of extinction and more than 30% of the species have been assessed as being critically endangered (Vardakas et al., 2025; Thomson-Laing et al., 2023). Traditional monitoring of freshwater fish mainly relies on capture sampling (such as netting, electrofishing, cage trapping) and morphological identification, but it has multiple limitations: such as low efficiency, high cost, ecological disturbance and applicability restrictions, low accuracy and insufficient data coverage, etc. (Liu et al., 2025). Consequently, the precise monitoring of fish community composition and diversity is essential for evaluating ecosystem health, conserving biodiversity and endangered species, ensuring the sustainable use of fishery resources and managing invasive species (Shen et al., 2022; Yao et al., 2022; Zhang et al., 2024).
           
    eDNA metabarcoding represents an emerging and innovative biological monitoring tool that offers a robust means of assessing the diversity of marine and freshwater fish communities. In comparison to traditional fishing and observation methods, eDNA metabarcoding exhibits several advantages, including high sensitivity, non-invasiveness and enhanced efficiency (Hervé et al., 2022; Qian et al., 2023; Saroj et al., 2020; Jesintha et al., 2021; Sit et al., 2021). This approach not only facilitates the detection of species that are challenging to capture using conventional techniques such as gill nets and trawls, thereby providing a more comprehensive species inventory, but also enables a more sensitive reflection of the spatio-temporal variation characteristics of fish communities. Furthermore, it assists scientists in better understanding ecosystem structure and function (Wang et al., 2022; Shen et al., 2023; Zou et al., 2020; Wang et al., 2021).
           
    In recent years, eDNA metabarcoding has gained widespread application in the investigation of fish diversity in both marine and freshwater ecosystems. For instance, researchers have employed eDNA metabarcoding to assess fish diversity in various aquatic environments, including Atlantic Sea, the Mediterranean, Caribbean Sea, Cosmonaut Sea in East Antarctica, Sylt Outer Reef in the North Sea of Germany, Baltic Sea and  Black Sea (Roblet et al., 2024; Gonzalez Colmenares et al., 2023; Liao et al., 2023; Kasmi et al., 2024; Ye et al., 2025; Urban et al., 2024; Gelen and Pekmezci, 2023) and Jiangmen coastal waters, Wuzhizhou Island in the South China Sea, the Ma’an Islands Special Conservation Area, the Yangtze River Estuary and the Xinglin Bay Reservoir in Xiamen in China (Zhang et al., 2024; Wang et al., 2024a; Wang et al., 2022; Lyu et al., 2024; Wang et al., 2024b). Furthermore, eDNA metabarcoding has been used to monitor alien species, especially those brought by sea transportation (Schreiber et al., 2023; Aglieri et al., 2023). The diversity of freshwater fish has been studied using eDNA metabarcoding in most parts of China, especially in the Yangtze River basin (Qian et al., 2023; Shen et al., 2023; Qu et al., 2020; Cheng et al., 2024). Guizhou Province situate in the upper reaches of the Yangtze River and the fish diversity of Wujiang River, Chishui River and Dulliujiang River has been reported (Shen et al., 2023; Peng et al., 2025; Yang 2023; Cheng et al., 2023). However, there is a paucity research focusing on rivers surrounding Tongren City, Guizhou Province, China.
           
    The Xieqiao river endemic fish national aquatic germplasm resources reserve located in Tongren City, Guizhou Province, China, with a total river length of 25.8 km and geographical coordinates between 108°59'25"~ 109°08'44" east longitude and 27°35'12"~ 27°39'34" north latitude. The main protected fishes are Silurus asotus (Linnaeus, 1758), Onychostoma lini (H. W. Wu, 1939) and Siniperca chuatsi (Basilewsky, 1855), while other protected fishes include Pelteobagrus fulvidraco (Richardso, 1846), Pseudogyrincheilus prochilu (Sauvage et Dabry, 1874), Hemibarbus maculatus (Bleeker, 1871), Rhinogobio cylindricus (Gunther, 1888), Sarcocheilichthys sinensis (Bleeker, 1871), Saurogobio dabryi (Bleeker, 1871), Zacco platypus (Temminck and Schlegel, 1846) and Opsariichthys bidens (Günther, 1873). To investigate the structure and diversity of fish communities in the reserve, we examined the distribution and abundance of fish in water and sediments of the upstream, midstream and downstream of Xieqiao River using eDNA metabarcoding, which will assess fish resources and provide reference for the reserve management.

    MATERIALS AND METHODS

    eDNA sampling and processing

    In this study, we set 3 sampling points (upstream: 109°03'49.0090", 27°36'41.5639"; midstream: 109°04' 44.3035", 27°36'41.3588"; downstream: 109°08' 18.4637", 27°38'58.4685") (Fig 1), a total of 18 samples (3 water samples and 3 sediment samples at each sampling point) were collected in October 2023. Water samples (2 L) from the middle and sides of the river were collected using clean disposable bottle disinfected with a 10% bleach solution. About 5 g sediments were also collected at the same locations as the water samples. Three replicates were collected at each sampling point. The collected water samples and sediment samples were stored under 4! until returned to the laboratory. Every 2 L of water was filtered by a vacuum pump through a mixed cellulose filter membrane with a pore size of 0.45 μm (Whatman). The filtering equipment was disinfected and washed with ultrapure water before each filtration to avoid cross contamination between samples. Distilled water (2 L) was used as a negative control to evaluate the presence of exogenous DNA contamination. Three membranes from the same sampling point were stored in the same cryostorage tube at -80°C for DNA extraction. The sediment samples from the same sampling point were mixed to cover as many species as possible and stored at -80°C. Water samples of the upstream, midstream and downstream in Xieqiao River are labeled W1-3 and sediment samples are labeled S1-3.

    Fig 1: Information on sampling locations by ArcGIS 10.7. S1, S2 and S3 represent upstream, midstream and downstream of Xieqiao River, respectively.


     
    DNA extraction and metabarcoding
     
    E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) was used to extract DNA from the sediment samples and filter membranes following the manufacturer’s instructions. The membrane filtered distilled water was used as the negative control. Tele02 primers were used to amplify A hypervariable fragment of the mitochondrial 12S rRNA gene (Taberle et al., 2018). PCR was performed using TransStart®FastPfu DNA polymerase (TransGen Biotech Co., Beijing, China) with a DNA template of 10 ng and three replicates. The negative control was also amplified. 2% agarose gel electrophoresis was used to detect PCR products. As a result, all samples except negative controls produced detectable PCR products. According to the preliminary quantitative results of electrophoresis, PCR products were detected and quantified using the QuantiFluor™ -ST blue fluorescence quantification system (Promeg, Madison, USA). The pooled DNA product was used to construct Illumina Pair-End library and was paired-end sequenced (2×250) on an Illumina MiSeq platform (Biozeron Co., Ltd, Shanghai, China).
     
    Bioinformatics and statistical analyses
     
    Poor-quality sequences were eliminated using Trimmomatic v.0.36. The remaining paired reads were combined into a single sequence by FLASH (v.1.2.11). Chimeric sequences were identified and eliminated using Usearch software (version 10, http://drive5.com/uparse/) compared with the reference sequences of GOLD database. The primers were removed with Cutadapt (v4.0). Valid tags of 12S rRNA were aligned to amplicon sequence variants (ASVs) and generated a feature table by DADA2. All the valid tags were then searched remotely against the Mitofish (http://mitofish.aori.u-tokyo.ac.jp/download.html), NCBI-NT-euk and local nucleotide reference databases (FishCOI) with BLASTn. We make a slight modification to the classification assignment criteria set by Zhang et al., (2020): (a) if the query sequence matches a single locally present species recorded in the databasee≥98%, the species is assigned; (b) if the query matches more than one native species with ≥98% identity, the lowest taxonomic level that included all these species was assigned; (c)Sequences with homology <98% but ≥95% belong to genus and <95% but ≥92% belong to family (Zhang et al., 2020).
     
    Fish diversity analysis
     
    Freshwater fish were screened according to FishBase database, false positive species were removed (reads with an abundance ≤5 at species level of a single sample were considered false positive) and all ASVs with an abundance of 0 were removed and subsequent analysis was conducted on this basis at the species level. The sequence abundance of the species was analyzed and Alpha diversity among different habitats was calculated using the R programming language (Auckland, New Zealand). The Shannon–Wiener, Simpson index, Chao1 index and Pielou_J index were evaluated and presented in box plot. To explore the similarity of fish composition in different samples, principal coordinate analysis (PCoA) was constructed using Bray-Curtis distance matrix which also used to evaluate non-metric multidimensional scales (NMDS) representing beta diversity between different habitats. Overlaping and unique fish taxa of three sampling points were shown in Venn diagram using the R software package pheatmap (https://CRAN.R-project.org/package= pheatmap).
           
    Statistical analysis and figures were performed using R version 4.0.3 (https://www.r-project.org/) (R Core Team, 2020). T-test and Tukey ‘HSD test were used to detect the differences between two groups and multiple groups.

    RESULTS AND DISCUSSION

    Sequence information and taxonomic assignment
     
    High throughput sequencing analysis of 6 samples resulted in 3438615 clean sequences with a total base number of 589877142 bp. The average length was 171.54 bp. Following BLAST and manual refinement, we recognized 112 fish species belonging to 16 orders, 28 families and 73 genera in Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve.
     
    Fish community among different river sections
     
    Among all fish species, the most abundant species at order, family, genus and species level were Cypriniformes, Xenocyprididae, Opsariichthys and Opsariichthys bidens (Fig 2). But the dominant species and abundance were different in different habitats. At order level, Cypriniformes was the most dominant species, its proportion in water was 95.54%, while its proportion in sediments was 62.04%. Corresponding, Gobiiformes, Cichliformes and Osteoglossiformes occupied about 12.08%, 12.34% and 12.32% in sediments, but very few in water. At family level, Xenocyprididae were the most dominant species (66.25% in water, 49.38% in sediments), followed by Cyprinidae (27.80%) in water and Cichlidae (12.34%), Notopteridae (12.32%), Gobiidae (12.08%) in sediments. At genus level, the difference was more obvious. Opsariichthys and Carassius were the most dominant species in water, accounting for 56.33% and 25.51% respectively, while there were more dominant species in sediments, accounting for a slightly smaller proportion of each species, Hypophthalmichthys, Sarotherodo, Mugilogobius, Cyprinus and Hemiculter accounting for 31.73%, 12.34%, 12.05%, 8.83% and 8.24%, respectively (Fig 3). Besides, the dominant species of reservoir and river were not exactly the same. At order and family level, the most dominant species were Cypriniformes and Xenocyprididae, which was similar in different habitats. However, there were more dominant species in river (N=6 and N=10) and more concentrated in reservoir (N=3 and N=4). At genus and species level, the dominant species in reservoir were Hypophthalmichthys (29.96%), Carassius (24.13%), Hemiculter (19.18%) and Hypophthalmichthys molitrix (Valenciennes, 1844) (29.94%), Carassius auratus (Linnaeus, 1758) (24.05%), Hemiculter leucisculus (Basilewsky, 1855) (19.18%), While the dominant species in river were Opsariichthys (37.41%) and Opsariichthys bidens (37.41%) (Fig 4). Finally, at order and family level, the most dominant species in different reaches were the same, but at genus level, the dominant species were Opsariichthys (30.20%), Hypophthalmichthys (22.39%), Carassius (14.14%), Chitala (12.47%), Cyprinus (13.42%) in upstream, Opsariichthys (44.75%), Sarotherodon (18.51%), Mugilogobius (18.07%) in midstream and Hypophthalmichthys (29.96%), Carassius (24.13%), Hemiculter (19.18%) in downstream. The distribution of dominant species at species level is similar to that at genus level (Fig 5). The fish composition and abundance of each sample are shown in Fig 6.

    Fig 2: Species composition of each sample at order, family, genus and species level.



    Fig 3: Species composition of water and sediments at order, family, genus and species level.



    Fig 4: Species composition of reservoir and river at order, family, genus and species level.



    Fig 5: Species composition of different river sections in Xieqiao River at order, family, genus and species level.



    Fig 6: Heatmap analysis of fish community.


           
    After removing false positive species (reads£5), screening fresh water fish and reviewing biological classification information, 43 dominant fish species belonging to 6 orders, 12 families, 33 genera were identified, including 3 species of specially protected fish in China, 26 endangered species of China Red List of Biodiversity· Freshwater fish, 17 species with extremely high relative sequence abundance in Guizhou Province and 7 species of endemic fish of the upper Yangtze River and 14 alien species (Table 1).

    Table 1: Species list and sequence number of fish detected by eDNA in xieqiao river endemic fish national aquatic germplasm resources reserve.


     
    Differences in fish community among different river sections
     
    In the six samples from the three sampling points, the Chao1 index of water samples were generally higher than that of sediments and the difference was significant (P<0.05). While, the differences of Shannon index, Simpson index and Pielou_J index between water samples and sediment samples were not significant (Fig 7A-D). PC1 and PC2 axes in PCoA (Principal co-ordinates analysis) explained 36% and 28% of the changes in the composition of the overall fish community diversity, respectively, with a total coverage of more than 60%. The most similar fish communities were those of W1 and W2, followed by S1 and S3 (Fig 7E). The fish communities in water were clearly differentiated from those in sediments, but the difference is not significant (ANOSIM: R=0.5926, P=0.1>0.05). In addition, the fish communities of water in river were different from those in reservoir (Fig 7F).

    Fig 7: The boxplots showing the alpha diversity index for water and sediments and the PCoA and NMDS showing the beta diversity index for each sample.


           
    The fish communities of rivers, lakes and reservoirs in Guizhou Province are well known, especially Wujiang River (Cheng et al., 2023). However, the research mainly focuses on the master stream of each river system and the research on tributaries is less. In particular, the report on the fish communities of Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve has not been systematically discussed. Moreover, it has been nearly four years since the 10-year fishing ban in the Yangtze River was implemented in 2020 and the recovery effect of fish populations needs to be evaluated urgently. Therefore, in this study, eDNA high-throughput sequencing was used to investigate the fish communities in Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve.
           
    In this study, we identified 6 orders, 12 families, 33 genera and 43 dominant fish species in Xieqiao river, the 5 most abandant orders were Cypriniformes, Osteoglossiformes, Gobiiformes, Cichliformes, Siluriformes and Centrarchiformes, which was similar to that of Wujiang river, Chishui river and Duliujiang River in Guizhou. Cypriniformes and Siluriformes were dominant orders in the Wujiang, Chishui River and the Duliujiang River (Yang, 2023; Cheng et al., 2023; Guo, 2023). Osteoglossiformes were detected in Yangtze River (Qian et al., 2023). Cichliformes, Gobiiformes and Centrarchiformes tend to be grouped in the order Perciformes (Yang, 2023; Wang, 2022). Xenocyprididae is a subfamily of Cyprinidae and they are often grouped together in a single group. Therefore, Cyprinidae is the most dominant fish family in Xieqiao River, which is consistent with the dominant fish families in the Yangtze River, Wujiang River, Chishui River and Duliujiang River (Shen et al., 2023; Yang, 2023; Cheng et al., 2023; Guo, 2023). The dominant genus Opsariichthys, Hypophthalmichthys, Carassius and Hemiculter in Xieqiao River were common species in other rivers and lakes (Shen et al., 2023; Yan et al., 2023). Opsariichthys bidens, Carassius auratus, Sarotherodon galilaeus, Mugilogobius myxodermus, Hypophthalmichthys molitrix were dominant fishes in Xieqiao River. In most studies Opsariichthys bidens were detected, while in a few studies Opsariichthys uncirostris were detected, which might be attributed to their close genetic relationship (Qian et al., 2023; Zhang et al., 2023; Chen et al., 2008). Sarotherodon galilaeus, an invasive tilapia species, has been dominant fishes in the Shanmei Reservoir and Jiulong River Basin of Southeast China (Yang et al., 2024; Feng et al., 2025). Mugilogobius myxodermus has been detected in the Yangtze River, the Pearl River and Dianchi Lake in Yunnan, but its distribution in Guizhou waters has not been reported (Shen et al., 2023; Chen et al., 2022; Yue et al., 2018). Carassius auratus and Hypophthalmichthys molitrix distributed widely in China. Finally, three protected fish species were detected in Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve, including Silurus asotus, Zacco platypus and Opsariichthys bidens. Other protected fish species, most of which were demersal fish, were not detected in this study, possibly was due to insufficient sampling sites and failure to collect deep water from reservoir.
           
    Three species of National wildlife under special protection, 26 endangered species of China Red List of Biodiversity·Freshwater fish, 17 species with extremely high relative sequence abundance in Guizhou Province and 7 species of endemic fish of the upper Yangtze River were detected, which indicated that the ecological environment of Xieqiao River was suitable for the survival of protected fish and it was necessary to establish Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve. In addition, 14 alien species were detected in Xieqiao River, accounting for 9.24% of the total resource, among which Sarotherodon galilaeus, Chitala ornata and Micropterus salmoides were the most abundant. Chitala ornata was found as early as May 2007 on the upper Mekong in Menglun (21°56'20"N, 101°15'18"E) in Xishuangbanna, China (Kang, 2013). Micropterus salmoides has invaded many natural waters in China, including Beipan River, Wujiang River in Guizhou and Dianchi Lake in Yunnan (Qiao et al., 2020). The presence of these invasive species warns us to pay close attention to them and take effective measures to control them and prevent them from endangering the survival of local aquatic organisms.
           
    Alpha diversity analysis showed that the Chao1 index of water samples was generally higher than that of sediments and the difference was significant (P<0.05), While, the differences of Shannon index, Simpson index and Pielou_J index between water samples and sediment samples were not significant, indicating that fish diversity in water was significantly higher than that in sediments, but there were no significant differences in species richness and evenness between two samples. The β-diversity indicated that fish diversity in sediments of midstream was quite different from that of upstream and downstream, which might be related to the presence of duck in this section. Fish diversity in water of downstream was quite different from that of upstream and midstream, which may be due to the fact that the downstream was reservoir, whereas the upstream and midstream were rivers. River was suitable for rheophilic fishes and reservoir for limnophilic fishes (Yang, 2023). Moreover, the reservoir was more susceptible to human activities and dam barriers and altitude changes could also affect fish community structure (Peng et al., 2025; Wang, 2022).

    CONCLUSION

    The Xieqiao river is rich in fish resources and diversity. There are 112 species of fish belonging to 16 orders, 28 families and 73 genera in Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve, among which there are 43 dominant fish species. But the dominant species and abundance were different in different habitats. In addition, the alien species account for 9.24% of the total resource, especially Sarotherodon galilaeus, Chitala ornata and Micropterus salmoides, which should be vigilant. This study assessed fish resources and provides reference for subsequent management of Xieqiao River Endemic Fish National Aquatic Germplasm Resources Reserve.

    ACKNOWLEDGEMENT

    This study was supported by National Natural Science Foundation of China (32060274), Doctoral Talents project of Science and Technology Bureau of Tongren, Guizhou Province ([2022]1).
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors  and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

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