Environmental DNA (eDNA) has been used in research relevant to fish ecology such as species diversity and conservation studies, threatened and invasive species monitoring, and analyses of population structure and distribution. How to choose the optimal laboratory protocols on the basis of the research targets is the first question to be considered when conducting an eDNA study. In this review, we searched 554 published articles using the topic subject ((eDNA or environmental DNA) and (fish)) within the time span 2011–2021 via Thompson Reuters Web of Science (WoS) and China National Knowledge Infrastructure (CNKI) literature databases, and screened 371 articles related to eDNA research on fish ecology. These articles were categorized into “article (334)”, “review (36)”, and “letter (1)” based on the type, and “article” was divided into “article (method research)” and “article (eDNA application)” in line with the study objectives. The experimental methods adopted in each study were reviewed, and advantages and disadvantages of the main protocols were analyzed for each step. We recommend a set of optimal protocols for regular eDNA-based fish diversity detection and present the following suggestions for water sample collection and subsequent sample processing and experiments. Sample size is suggested to be 2 L regardless of the type of water bodies; three water replicates are recommended per sampling site, and water collection sites should be designed to cover various water layers and micro-habitats within research areas. Filtration is the best method for collecting eDNA from the larger water samples; 0.45 μm glass fiber/glass microfiber (GF) filters and mixed cellulose acetate and nitrate (MCE) filters are recommended for use, and MCE filters are suitable for use in turbid waters; pre-filtration (>10 μm filtering membranes) can be used to prevent clogging. Freezing temperature storage can slow eDNA degradation, and this is the optimal way to store DNA no matter what filtering method is applied. The Qiagen DNeasy Blood and Tissue DNA extraction kit was the most economical and efficient DNA extraction method compared to other commercial kits. The 12S rRNA gene is the first choice for detecting interspecies variation in fishes, and five 12s primer sets, Ac12S, MDB07, Mi-Fish, Vert-12SV5, and Teleo, are recommended. The TruSeq DNA PCR-free LT Sample Prep kit and NEBNext DNA Library Prep Master Mix Set for the 454 kit can be chosen. The Illumina HiSeq platform can obtain sufficient data depth for fish species detection. QIIME and OBITools are independent software packages used for eDNA sequences analysis of fishes, and bioinformatic analyses include several indispensable steps such as filtering raw reads, clustering filtered reads into molecular operational taxonomic units (MOTUs) or amplicon sequence variants (ASVs), and completing taxon annotation. Contamination, inhibition, lack of reference DNA data, and bioinformatic analysis are key challenges in future eDNA research, and we should develop effective experimental techniques and analysis software regarding these aspects. This review intends to help eDNA beginners to quickly understand laboratory protocols applied in fish ecological research; the information will be useful for the improvement and development of eDNA techniques in the future.

West Island is the second largest island in the Hainan Province, China and its surrounding sea area has a well-preserved coral reef ecosystem and high species diversity of coral reef fishes. Undoubtedly, coral reef fishes and coral reefs have complex symbiotic relationships and fish species diversity should reflect the healthy status of coral reef ecosystems. Environmental DNA (eDNA) is a useful and sensitive tool to detect fish species and causes less environmental damage than traditional fish survey methods. This paper investigated coral reef fish species of West Island, Hainan Province, China, based on eDNA and provided scientific data for understanding and protection of the coral reef ecosystem of the South China Sea. The sea area surrounding West Island is the most important component of the coral reef ecosystem in the northern part of the South China Sea, which is also an essential part of the largest National Coral Reef Reserve in China. However, complete data of fish species distributed in this region have been a long-term gap. This study provides information on 41 fish species belonging to 28 genera, 16 families and three orders in this sea area and is the first complete record of coral reef fishes surrounding the West Island. In addition, the information of Molecular Operational Taxonomic Units (MOTUs) for taxon identification were also provided and it could contribute to building specific eDNA taxonomy database of coral reef fishes of the South China Sea. The study includes three datasets, with aspects of fish taxon-occurrences, MOTUs sequences and information of environmental indicators surrounding West Island, Hainan Province, China. The “fish taxon occurrences” dataset presents records involving taxonomic, distribution, habitat condition, latitude and longitude of 41 coral reef fish species detected, based on eDNA, the “MOTUs sequences” dataset provides MOTUs sequences and their abundance of 31 species detected and the “information of environmental indicators” dataset presents records of transparency, temperature, water pressure, dissolved oxygen, electrical conductivity, hydrogen and redox potential measured from five sampling localities.

Wuzhizhou Island (WZZ) is located in Haitang Bay in the northern region of Sanya, Hainan Island. The sea area surrounding WZZ represents a typical tropical marine ecosystem, characterised by diverse and complex habitats. Therefore, there is a rich variety of marine fish species at WZZ. The marine ecosystem of WZZ was seriously destroyed initially in the 1970s-1980s and recovered in the 1990s, then constructed as the first national tropical marine ranch demonstration area of China in 2019. As fish is an important high trophic vertebrate in the marine ecosystem, understanding the composition and distribution of fish species could help us to recognise the status of the ecosystem of WZZ and supply scientific data for construction of the national marine ranch demonstration area. This study used eDNA technology to investigate the composition of fish community surrounding WZZ and provided a scientific basis for realising and protecting the marine ecosystem of the South China Sea. The WZZ is an offshore island in the South China Sea, harbouring abundant marine fish resources. Although previous research investigated fish species of WZZ, the data were, however, still incomplete due to limitation of sampling methods and survey seasons. In this study, we intended to take advantage of eDNA and supplement data of fish species at WZZ as much as possible. Based on eDNA, this study provided the data on 188 fish species (including nine undetermined species denoted by genus sp.) belonging to 17 orders, 63 families and 124 genera and they were the more comprehensive records of fish species surrounding WZZ. In addition, the information on Molecular Operational Taxonomic Units (MOTUs) for taxon identification was also provided, aiming to contribute to the establishment of a specific eDNA taxon database for fish of the South China Sea. This study included two datasets, which were occurrences of fish taxa at WZZ, as well as MOTUs sequences and geographical coordinate information of sampling sites. The “fish taxon occurrences” dataset presented records on taxonomic, distribution and habitat conditions of 188 fish species detected using eDNA, as well as the latitude and longitude information of the sampling sites, the "MOTUs information" dataset provided the MOTUs sequences, source of sequences, abundance of sequences for 188 fish species, also included the species matched in NCBI and the best NCBI BLAST sequence similarity.

The Dingqu River Basin, a major tributary of the upper Yangtze River, is located at southeast edge of the Qinghai-Tibet Plateau of China. The fishes of this plateau constitute a major faunal component of this basin, particularly Schizothoracinae (Cypriniformes: Cyprinidae) and Triplophysa (Cypriniformes: Nemacheilidae). Hydropower development is an impact that affects natural habitats and biological resources of the upper Yangtze River and this has led to a decrease in biodiversity. This study investigated the species diversity of fishes of the Dingqu River Basin and accumulated basic data for conservation of biodiversity and assessment of ecological health of the upper Yangtze River. The upper streams of the Jinshajiang River harbours numerous endemic fish species in China. Most of them belong to the Qianghai-Tibet Plateau fish fauna. However, while the fish species of the Jinshajiang River have been well studied, there is still a gap in the research on fish species diversity of the Dingqu River Basin tributary. This study provides information for 18 native fish species belonging to two orders, four families, three subfamilies and eight genera, and is the first complete record of fishes in the Dingqu River Basin, a primary tributary of the Yangtze River. Supplemental information of species diversity in the Jinshajiang River is also provided. The study includes two datasets, which present taxonomic, distribution, habitat condition, endemism and threat information for each species collected from the Dingqu River Basin and historical literature, respectively. In particular, these fish species all have limited distribution within the south-eastern Qinghai-Tibet Plateau areas of China and could determine the importance of habitat protection for the upper Yangtze River.