1. Introduction
A sharp decline of biodiversity has been one of the most critical challenges all over the world since the 20th century [
1]. Worldwide species extinction rate during this period exceeds that of prehuman periods [
2,
3]. This severely threatens the sustainability of ecosystems and human health [
1,
4]. Specifically, in aquatic ecosystems, a rapid decrease of fish diversity is one of the major problems in the management of fishery resources. The decrease is caused by overfishing, water pollution, habitat degradation, genetic pollution, climate change, and invasive species [
5,
6,
7]. Therefore, it is necessary to take effective measures of fish protection. These measures are based on detailed information of fish distribution and population characteristics, as well as the physiological characteristics and ecological niche of different fish. However, due to the complexity of aquatic ecosystems and the diversity of fish migration routes [
8], it is extremely difficult for an accurate assessment of fish diversity. Traditional methods of investigating fish diversity, such as trawling, seining, electric fishing [
9], underwater acoustic [
10], and visual methods, may underestimate fish diversity, since some rare fishes, e.g., endangered species and invasive taxa, are extremely difficult to detect [
11,
12].
In terms of the abovementioned issues, environmental DNA (eDNA) provides an alternative way of detecting various types of fish. The application of the eDNA method in freshwater ecosystems started in 2008, through the way of detecting the existence of American bullfrogs in a pond [
13]. After that, the eDNA method was increasingly used for monitoring aquatic macro-organisms in both freshwater and marine environments [
14]. This method is highly efficient, accurate, and nondestructive [
12]. The eDNA method is defined as the sum of DNA fragments directly extracted from environmental components, like soil, sediments, or water. The DNA fragments contain the intracellular genetic material released into water by biological cells, and the extracellular DNA released into water after cell structure lysis or death [
15]. The eDNA method is based on metagenomics and DNA sequencing technology. DNA fragments extracted from environmental components are identified by molecular biological means and specific gene detection of target fish species. This can be used to analyze the distributional characteristics of target fish species. The eDNA method has progressed in aspects of fish resource management, like gathering the information of endangered fish [
16,
17], investigating invasive species [
18], and evaluating fish diversity [
19] and fish prey diversity [
20]. Despite advantages of the eDNA method compared to conventional fish survey methods, it should be very careful for an interpretation of eDNA data [
21].
Until now, existing knowledge is still insufficient for examining the feasibility and the reliability of the eDNA method in terms of fish diversity survey. That is, what settings of the eDNA method are suitable for specific regions is still not clear. Different sampling methods, laboratory procedures, and bioinformatics were gradually improved to increase the accuracy of the eDNA method and to promote its application in ecology and environment [
22]. Different eDNA extraction reagents result in various extraction effects and experimental costs. These may lead to differences in the extraction efficiency of DNA and a weak contrast among operations. The eDNA metabarcoding detected more fish species than that captured by traditional surveys, like electrofishing [
23] and underwater visual censuses [
24]. In addition, the eDNA metabarcoding was proved to be an efficient bio-monitoring tool for surveying fish diversity, especially in regions often ignored or difficult to access [
25].
This study optimized the eDNA method through comparing different sampling amounts, pretreatment steps, DNA extraction, DNA primers, and DNA sequencing by controlled experiments in the laboratory. An optimized eDNA method was thereafter applied in an aquatic reserve to assess the fish diversity. The assessment of the fish diversity provides a scientific reference for improving the local species conservation list and monitoring invasive species. The following questions were addressed: (1) What are the optimal settings of the eDNA method in the laboratory? (2) How does the optimized eDNA method work for the fish diversity survey in the aquatic reserve?
4. Conclusions
Adopting the environmental DNA (eDNA) method is beneficial for improving the reliability of fish diversity surveys. We firstly simulated necessary steps of the eDNA method in the laboratory through controlled experiments and obtained the optimized settings. Under the same extraction conditions, the DN easy Tissue and Blood DNA extraction kit has the highest concentration and purity of DNA. The DNA purity of the extracts obtained by the filtration method is higher than the centrifugal precipitation method. The sampling amount of 1 L is enough to capture eDNA information of the majority fish taxa. High-throughput sequencing provides more possibilities for the detection of unknown fish species, but clonal sequencing can meet the needs of fish diversity survey with low costs. The 16s primer was considered as the most effective primer for evaluating the fish diversity. These results highlight the necessity of optimizing the eDNA method based on regional characteristics and study objectives. However, data generated during the procedure of optimizing the eDNA method should be interpreted cautiously. Moreover, quantifying effects of environmental factors (e.g., temperature and water quality) on the optimization of the eDNA should be addressed in future researches. The optimized eDNA method was thereafter applied in an aquatic life reserve of Beijing. The results show that there are 29 fish species. The fish species are mainly composed of Cypriniformes and Perciformes. The Shannon–Wiener diversity index of the fish species is the highest in the Huairou Reservoir. Moreover, the diversity index shows seasonal changes. The accuracy rate of detecting fish taxa is positively correlated with the eDNA concentration, based on an evaluation of the fish species Zacco platypus and Odontobutis potamophila. This study provides valuable data for the local fish gene pool, and demonstrates the reliability and high efficiency of the eDNA method in surveying and estimating the biodiversity of aquatic species. In addition, we suggest that the eDNA method and traditional methods of fish survey should be complementary. This allows us to obtain detailed information about demographic and morphologic characteristic of fish species.