Assessing Fish Diversity In Plateau Lakes In Central Yunnan Province Using Environmental DNA

The plateau lake in central Yunnan is classified as one of the five major lake types in China.It has become a research hotspot and attracted more and more attention of researchers from China and other countries,due to its special habitat,high altitude,low latitude,low lake runoff,isolation between lakes,simple fish flora and strong species differentiation.At present,monitoring of fish biodiversity in plateau lakes is mainly based on artificial fishing and fishing household surveys,which are time-consuming and laborious.Additionally,the reliability of the collected data is not high,especially when the number of endangered fish stocks is low.Environmental DNA refers to the DNA fragments released into the environment by organisms,from dandruff,exfoliated cells,excreta,mucus,etc.Previous studies have shown that environmental DNA in combination with modern metabarcoding technique can be used to quickly identify species and monitor the biodiversity level of water environment.However,it has not been reported whether this method is suitable for plateau lakes.In this study,this simple and quick method was employed to study fish biodiversity in four plateau lakes in the central Yunnan,namely Fuxian Lake,Xingyun Lake,Yangzonghai and Dianchi Lake The main aims of this study are 1)to monitor the biodiversity of lake fishes in the central Yunnan Plateau;2)to compare the species distribution maps inferred by the method with the historical literature data.In this study,a total of 28 environmental DNA samples were collected from Fuxian Lake,Yangzonghai Lake,Dianchi Lake and Xingyun Lake in July 2018.In order to reduce the errors caused by nonspecific amplification and sequencing,three pairs of twin tag MiFish primers were used for each environmental DNA sample in three independent PCR amplifications.The sublibraries were constructed by mixing the PCR products of each plate.High throughput sequencing was performed on Miseq sequencing platform of Illumina.The softwares(AdapterRemoval 2.2.0,sickle 1.33,SPAdes 3.10.1,PandaSeq 2.11)were used to correct the errors and control the quality of the original sequence.The software DAMe was used to analyze the tagged sequence.The process of clustering is based on sumaclust.The main results of this study are as follows:1.The environmental DNA combined with metabarcoding technique can be used to identify the main fish in the central Yunnan plateau lakes quickly and efficiently.Additionally,the optimized environmental DNA technique for monitoring fish diversity in the central Yunnan plateau could be used as a reference for similar studies.2.The environmental DNA technique was used to investigate the fish diversity in four lakes in the central yunnan plateau,and 39 OTUs were obtained.By excluding non-fish OTUs and positive control,a total of 30 fish OTUs were obtained from four lakes.The 30 fish OTUs belonging to six orders and 25 genera were detected in the four studied lakes.Among them,12 OTUs can match the endemic species information with more than 97%similarity in NCBI,including the indigenous fish(Carassius auratus)and the rare fish(Sinocyclocheilus grahami).Accordingly,we have mapped the distribution of fish species composition in the four lakes.The above results indicate that environmental DNA combined with metabarcoding technique can be used to monitor the fish diversity in high-altitude lakes in central Yunnan,but it needs to be optimized in many procedures to improve its applicability,including field sampling DNA extraction,PCR amplification and bioinformatic analysis.The results of this study are believed to be potentially useful for further studies of aquatic organisms in other high-altitude lakes in central Yunnan.In addition,it also provides technical supports for the investigation of biodiversity in other nature reserves and environmental monitoring bureau.