| Global biodiversity decline is an increasingly prominent ecological crisis in the 21st century;therefore,the establishment of an efficient,sensitive,and responsive method to strengthen biodiversity monitoring and conservation is urgently required.Fish diversity is an important component of aquatic biodiversity.Effective fish monitoring can help to accurately evaluate fish diversity.Traditional fish surveys are usually time-consuming and labor-intensive and suffer from low capture rate of rare species,difficulty in morphological identification,high ecological destruction due to the use of fishing methods,and low accuracy of detection by non-fishing methods.Alternatively,environmental DNA(eDNA)technology is non-invasive,cost-effective,and highly sensitive compared to traditional methods.Therefore,eDNA technology has become a popular new method for monitoring fish diversity in recent years.As eDNA-based biomonitoring is a new approach,the application of this technology in fish monitoring still faces issues.For example,the issues of the eDNA signals in fish embryonic development,the performances of fish eDNA metabarcoding primers under controlled conditions,and the fish diversity and spatial-temporal distribution in large lakes based on eDNA.To explore the above scientific issues,this study was conducted from three aspects:eDNA dynamics in fish embryonic development,primers evaluation for fish eDNA metabarcoding,and fish diversity monitoring in the Erhai Lake.The main results of this study are as follows:1.eDNA dynamics in fish embryonic developmentWe used a model aquatic species,rare minnow(Gobiocypris rarus),to assess the dynamics of mitochondrial and nuclear eDNA during fish embryonic development,including the origin,decay,and release of eDNA,in a culture dish experiment.Mitochondrial cytochrome b(Cytb)and nuclear internal transcribed spacer-1(ITS 1)were used as representative DNA fragments amplified using qPCR.The released sperm and hatched larvae were the two main sources of eDNA during embryonic development.In the culture dish experiment,the eDNA concentration of mitochondrial Cytb was consistently higher than that of nuclear ITS 1;the decay rate of nuclear ITS 1 was faster than that of mitochondrial Cytb.This suggests that mitochondrial Cytb is a more appropriate marker for eDNA detection in early fish life than nuclear ITS1.A modest but positive relationship between eDNA concentration and the number of hatched larvae illustrated that the eDNA signals released during larval hatching of fish may potentially be used to monitor the hatching events of fish.2.Primers evaluation for fish eDNA meta bar codingWe evaluated the performance of 18 primer pairs for mitochondrial cytochrome oxidase subunit I(COI),Cytb,12S rRNA,and 16S rRNA genes,all of which were used in fish eDNA metabarcoding.Ribosomal gene markers performed better than the proteincoding gene markers during in silico screening,resulting in higher taxonomic coverage and appropriate barcode lengths.Four primer pairs,AcMDB07,MiFish-U,Ve16S1,and Ve16S3,designed for various regions of the 12S and 16S rRNA genes were screened for tank metabarcoding in a case study targeting six freshwater fish species,including Carassius auratus,Cyprinus carpio,Gambusia affinis,Hypophthalmichthys molitrix,Misgurnus anguillicaudatus,and Pseudorasbora parva.The four primer pairs could accurately detect all six species in different tanks,showing the good performance of the multispecies qualitative detection for the four primer pairs.However,species read count for each primer pair was not correlated with species abundance.MiFish-U,Ve16S1,and Ve16S3 revealed a significant positive relationship between species read count and biomass for the pooled tank data.A positive relationship was not observed for all species within the tanks.Additionally,primer efficiency differed according to species,and the primer preferential species varied in different fish assemblages.These results illustrated the uncertainty of fish abundance/biomass estimation based on eDNA sequence abundance.3.Fish diversity monitoring in the Erhai LakeWe used eDNA metabarcoding to monitor fish diversity and spatial-temporal distribution in the Erhai Lake.Water samples were collected from the Erhai Lake in the summer and autumn of 2020-2021.Each sample included water samples from the shore,nearshore,and midline of the lake.36 fish taxa were detected in four samplings,including 5 endemic taxa(C.auratus,Homatula spp.,M.anguillicaudatus,Schizothorax spp.,Schizothorax taliensis)and 31 exotic taxa.Carassius auratus,C.carpio,Hemiculter leucisculus,Hypomesus olidus,Rhinogobius cliffordpopei,and Rhinogobius giurinus were the dominat species.The site occupancy or relative read abundance of 25 historicalrecorded species based on eDNA detection was significantly correlated with species rank abundance based on traditional monitoring in recent years,demonstrating that eDNA metabarcoding can accurately estimate fish rank abundance in the Erhai Lake.In addition,we detected one endangered endemic(S.taliensis)and five unreported exotic species(Micropterus salmoides,Misgurnus dabryanus,Silurus spp.,Siniperca spp.,and Squaliobarbus curriculus),demonstrating the potential of eDNA metabarcoding to detect endangered and exotic species in large lakes.eDNA data showed that the fishes in the Erhai lake were mainly cyprinid fishes(accounting for 50%of the total number of fish taxa),and exotic economic fishes and small fishes were the main dominant taxa(accounting for 86.11%of the total number of fish taxa).The spatial and temporal variations in fish eDNA revealed significant seasonal and spatial differences in fish community structure of the Erhai Lake.The seasonal distribution of fish communities was mainly influenced by the water temperature and nutrient status:cyprinid fishes were abundant in the summer with high water temperature,and fishes with strong pollution resistance or plankton-feeding were abundant in the autumn with high nutrient status.The spatial distribution of fish communities was mainly influenced by the water depth:small fishes were abundant in the shore,whereas large fishes were abundant in the interior.These results were consistent with that monitored via traditional fish surveys in recent years,demonstrating that eDNA metabarcoding technology can aid in monitoring fish diversity and spatial-temporal distribution in the Erhai Lake.In addition,the spatial differences of eDNA data revealed that fish eDNA exhibits patchy distribution in large lakes,and demonstrated that the fine spatial sampling can reveal the spatial distribution of lake fish;the shore sampling can detect most fish species in the lake,which is a costsaving sampling strategy for fish diversity monitoring.In summary,this study was conducted from three aspects:eDNA dynamics in fish embryonic development,fish eDNA metabarcoding primers evaluation,and fish diversity monitoring in the Erhai Lake.The findings provide a solid theoretical basis and good examples for the application and development of eDNA technology in fish diversity monitoring;specifically,this study(1)provides basic information on eDNA dynamics during fish embryonic development,revealing the potential of species-specific eDNA detection to monitor the spawning and hatching events of fish;(2)screens the best performing four pairs of primers from 18 pairs of primers,providing candidate primers for fish eDNA metabarcoding detection;and(3)shows the effectiveness of eDNA metabarcoding for assessing fish diversity and spatial-temporal distribution in the Erhai Lake,revealing the application prospects of eDNA metabarcoding to assess fish diversity and spatial-temporal distribution in large lakes. |
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