Study On Fish Diversity Of The Lake-type Water Bodies Based On Environmental DNA

The lake-type water bodies mainly refer to those still water habitats such as natural lakes and artificial reservoirs,and they are numerous,widely distributed and diverse in China.In this study,four typical lake-type water bodies located at the eastern plains of our country,the Poyang Lake,the Baiyangdian Lake,the Miyun Reservoir and the Yanqi Lake(also called the Beitaishang Reservoir),were selected as the representatives of large/small-medium sized natural lakes and artificial reservoirs,respectively.The fish species composition,α-diversity,β-diversity,functional diversity,and distribution features were studied in depth used environmental DNA(e DNA)method.The e DNA sampling and laboratorial protocols suitable for the lake-type water bodies were recommended,through comparing and analyzing advantages and disadvantages of the protocols applied at four different water bodies,three-replicated 1 L water samples were recommended to be collected at one sampling site,mainly from surface of water bodies,and sampling sites were suggested to cover key micro-habitats of the investigation areas as much as possible.Filtration method with 0.45 μm mixed cellulose acetate and nitrate(MCE)filters was recommended for e DNA collecting and e DNA samples were best stored at-10℃ or lower.The DNeasy Blood and Tissue DNA extraction kit(Qiagen)was recommended for DNA extraction,12 S r RNA was preferred as a target marker for PCR amplification,and Illumina sequencing platform was suggested for high-throughput sequencing.The bioinformatics software such as Trimmomatic 0.32 and QIIME2 were recommended for sequence analysis,and a basic local alignment search tool NCBI-Blast was proposed for fish species alignment.Based on above recommended protocols,specific sampling and experimental protocols such as choice of sampling sites,number of filtration membranes used for e DNA collection,and some key steps of DNA extraction and PCR amplification were improved and optimized according to the environmental conditions of different water bodies,in order to yield higher e DNA concentration and reduce impacts of the inhibitions on PCR results during sampling and experimenting.The results of fish detection based on e DNA indicated that total 5 orders,11 families,35 genera and 57 species were in northern area of the Poyang Lake,3 orders,6 families,21 genera and 24 species were in the Baiyangdian Lake,4 orders,5 families,16 genera and 19 species were in the Miyun Reservoir,and 5 orders,8 families,24 genera and 29 species were in the Yanqi Lake.Results of four water bodies were all close to those of the traditional fishing survey methods,and could detect genera or species that were not detected by traditional fishing methods.According to results of fish α-diversity of four lake-type water bodies based on e DNA,Margalef index performed the Poyang Lake >the Yanqi Lake > the Baiyangdian Lake > the Miyun Reservoir,which was consistent with compared results of species richness;Shannon-Wiener and Simpson indices indicated that the Yanqi Lake > the Miyun Reservoir > the Poyang Lake > the Baiyangdian Lake;Pielou＿evenness index displayed that the Miyun Reservoir > the Yanqi Lake > the Poyang Lake > the Baiyangdian Lake.According to above results,allα-diversity indices of the Poyang Lake were higher than those of the Baiyangdian Lake,the reason might be the Poyang Lake could supply more suitable living conditions for fishes because of its wider water surface and connection with middle reaches of the Yangtze River.The Margalef,Shannon-Wiener and Simpson indices of the Yanqi Lake were higher than those of the Miyun Reservoir,which might be due to the richer aquatic plants and higher habitat heterogeneity of the Yanqi Lake,rather than the Miyun Reservoir.According to analysis results of β-diversity,fish community composition of the Poyang Lake was significantly different from other three water bodies,which might attributed to its belonging to the Yangtze River basin and harboring different geographical locations and climate conditions,while other water bodies all belonging to the Haihe River basin.As the natural lakes,analysis results of functional diversity of fish performed functional richness(FRic)and functional dispersion(FDis)indices of the Poyang Lake were both significantly higher than those of the Baiyangdian Lake,which might be related to the higher fish species richness and richer functional traits of the Poyang Lake,and fish functional communities of the Baiyangdian Lake were relatively simple since it experienced many times of drying up in the history and economic fish species had become the major fish component after its artificial ecological restoration.As the artificial reservoirs,all functional diversity indices of the Yanqi Lake were higher than those of the Miyun Reservoir,and the former represented the higher stability of ecosystem.The correlation results between functional diversity and α-diversity indices of four water bodies indicated that there was a significant positive correlation between FDis index and each α-diversity index,Rao’s quadratic entropy(Rao)index and each α-diversity index,as well as functional divergence(FDiv)index and three α-diversity indices except Margalef index,respectively in the Poyang Lake and the Baiyangdian Lake,and there was an extremely significant positive correlation between FRic and Margalef indices in the Baiyangdian Lake.Only FRic index had a significantly positive correlation with Shannon-Wiener,Simpson and Pielou＿evenness indices,respectively in the Miyun Reservoir.The FDis and Rao’s indices both had extremely significant positive correlations with Shannon-Wiener,Simpson and Pielou＿evenness indices,respectively in the Yanqi Lake.And there was no correlation between the FEve index and each α-diversity indices in four water bodies.The in-depth analysis of fish diversity in northern area of the Poyang Lake,fish fauna was dominated by Cyprinidae,which was the common characteristics of freshwater-fish fauna in the Yangtze River basin and most of rivers in China.Only one endemic species of the Yangtze River Gobiobotia filife was detected.Total 34 sedentary fish species were detected,accounting for nearly 60% of total number of fish species,which were main components of fish in the Poyang Lake.There were six lake-river migratory fish species.Small-sized fish species were dominated in the research area,and total 35 species occupied more than 60% of total number of fish species.In terms of spawn type,the fish species which lay sinking eggs account for one-third of total number.In order to understand distribution characteristics of fish diversity in northern area of the Poyang Lake,three zones including lake(sampling sites 1-21),above the dam(sampling sites 22-32)and under the dam(sampling sites 33-45)were divided according to location of the proposed Poyang Lake Water Conservancy Project.Based on GIS results of fish species density in each zone,species density in zone of lake was the highest,and that in zones above and under the dam were little difference,and the reason might be that zone of lake could provide more suitable living conditions for lake-type fish species since its more open water and closer to environment of lake.According to species composition,a total of 48 fish species were distributed in all three zones,accounting for 84.21% of total number.The fish species which spawned drifting eggs were mainly distributed in zone of lake.According to results of α-diversity,Margalef index displayed zone under the dam >zone of lake > zone above the dam,and this trend was consistent with that of species richness.The Shannon-Wiener,Simpson and Pielou＿evenness indices performed zone above the dam > zone of lake > zone under the dam,and this trend was opposite to that of species richness,as well as consistent with results of traditional fishing methods.According to results of β-diversity,there were no significant differences in composition of fish communities between three zones,and it might due to no obvious habitat differences between three zones,and continuous changes of environments from the lake to the river along zone of lake to zone under the dam.The FRic index of zone under the dam and zone of lake were higher than that of zone above the dam,since number of fish species in the former two zones were higher than the latter one,so that more functional traits of fish lead to higher functional richness.The FDis and FEve indices of zone above the dam were higher than that of other two zones,because the fish occupying different ecological niches in the former dam were relatively balanced and competitions between fish species were weaker,so that its ecosystem was more stable,as well as sequence relative abundance of large carnivorous and omnivorous fishes in zone above the dam was higher,so that relatively weak competitions for prey organisms with small omnivorous fishes and the community structure of fish was more balanced.Considering that northern area of the Poyang Lake is the only waterway connected to the Yangtze River,the proportion of fish species is relatively high in the whole lake.It is the main habitat area for migratory fishes as well as those spawning drifting eggs.Therefore,we recommended to strengthen protection of fish resources in this region.With promulgation of the ten-year fishing ban policy in the Poyang Lake,the routine monitoring of aquatic organisms should be strengthened as soon as possible,and e DNA method should be used as a routine method to solve the question that traditional fishing investigations cannot be implemented since fishing ban.The construction of the dam will change natural environment of northern area of the Poyang Lake,which may bring rise of water level and blocking of migratory channels in the lake,and it will have different degree of impacts on the sedentary and migratory fishes,as well as those spawning drifting eggs inhabited in the lake,so it is suggested systematic and in-depth researches of impacts from construction of the Poyang Lake Water Conservancy Project on aquatic biological resources and ecosystem should be carried out,the influence of the project on the ecological environments of the lake should be scientifically evaluated,in order to provide scientific basis and useful suggestions for the rational planning of the project construction.