| With the continuous decline of global species diversity,parallel biomonitoring pattern are urgently performed in freshwater ecosystems to facilitate the formulation of management treaties and the improvement of conservation measures.Macroinvertebrates are ideal indicators for water quality monitoring,and of great value in responding to anthropogenic activities and indicating ecosystem status.However,conventional identification schemes,which cost researchers much efforts,mainly rely on morphological characteristics and remain a major challenge for time-consuming and high professional.Environmental DNA(eDNA)is a signal of organisms shed into environment,which can offer reliable genetic information.Preservative ethanol storing specimens is an effective source of eDNA to obtain mass species data without interfering with morphological analysis.Currently,the applicability of ethanol-derived eDNA metabarcoding is mainly evaluated with low sample size,and lack of large-scale and repetitive demonstration.In addition,high-throughput pipelines supported by water samples have been widely applied in various freshwater ecosystems.The application prospect and detectability of two nondestructive methods still deserve attention and exploration,but no study has been conducted to formally compare their performance on macroinvertebrate detection.In this study,we employed filters to capture eDNA in preservative ethanol from 42 unsorted bulk samples and to verify its feasibility and stability on monitoring macroinvertebrate diversity.On the basis of morphological identification data,we investigated detection performance of preservative ethanol in stream ecosystem from the aspects of community composition and species diversity.Then we evaluated the ability of ethanol samples to detect rare(<1%of the total abundance),moderately abundant(1-5%)and highly abundant(>5%)morphotaxa for ethanol samples.Our results found that all ethanol samples yielded effective molecular data and recovered 19 morphologically identified orders.Ethanol samples detected higher species richness than ES-eDNA(858 vs.229).Pearson correlation analysis implied that the two schemes showed strong consistency in richness of EPT taxa(p<0.001,r=0.677),but weak correlation in benthic communities.The average detection rate of macroinvertebrate morphotaxa in all ethanol samples was 55.42%,and 71.77%in EPT taxa.Overall,detection rates of were higher in moderately and highly abundant taxa,with 74.29%and 97.46%on average,respectively,but weaker in rare taxa(39.16%).Relative sequence abundance and the number of individuals per taxon were significantly correlated in 24 sampling sites.To further explore diversity assessments of preservative ethanol and water samples,we compared community composition,diversity analysis and morphotaxa recovery of 14 sets of eDNA samples to summarize their pros and cons.All eDNA samples were amplified successfully except for two water samples(WSA09 and WSA11).Water samples captured more OTUs than ethanol samples(2,866 vs.2,406),but less macroinvertebrate OTUs(478 vs.548).Among sampling sites,two eDNA approaches exhibited relatively large dissimilarity on inferred community composition(p<0.001).Furthermore,ethanol samples exhibited better detectability for morphological identification approaches than did water samples(24.24%vs.17.63%,p=0.002),especially for highly abundant taxa.Two eDNA technology offered a species list which are not available in morphology data,mainly Chironomidae and Naididae.Our study highlighted an optimized and low-cost scheme of ethanol-eDNA,and verified the detectability and stability of preservative ethanol.Its high coverage can effectively supplement the morphological datasets of macroinvertebrates for local-scale diversity,and be a new option in building molecular measure-based biomonitoring program.To the contrary,water samples are more suitable for describing patterns of diversity of the whole communities as its easier and more convenient way to collect samples. |
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