Freshwater fish biodiversity is quickly decreasing and requires effective monitoring and conservation. Environmental DNA (eDNA)-based methods have been shown to be highly sensitive and cost-efficient for aquatic biodiversity surveys, but few studies have systematically investigated how spatial sampling design affects eDNA-detected fish communities across lentic systems of different sizes. We compared the spatial patterns of fish diversity determined using eDNA in three lakes of small (SL; 3 ha), medium (ML; 122 ha) and large (LL; 4,343 ha) size using a spatially explicit grid sampling method. A total of 100 water samples (including 9, 17, and 18 shoreline samples and 6, 14, and 36 interior samples from SL, ML, and LL, respectively) were collected, and fish communities were analyzed using eDNA metabarcoding of the mitochondrial 12S region. Together, 30, 35, and 41 fish taxa were detected in samples from SL, ML, and LL, respectively. We observed that eDNA from shoreline samples effectively captured the majority of the fish diversity of entire waterbodies, and pooled samples recovered fewer species than individually processed samples. Significant spatial autocorrelations between fish communities within 250 m and 2 km of each other were detected in ML and LL, respectively. Additionally, the relative sequence abundances of many fish species exhibited spatial distribution patterns that correlated with their typical habitat occupation. Overall, our results support the validity of a shoreline sampling strategy for eDNA-based fish community surveys in lentic systems but also suggest that a spatially comprehensive sampling design can reveal finer distribution patterns of individual species.

01 Rawdata.xlsx  Summary of sequence abundance data generated by the ECOTAG program.
Table shows sequence abundance data of samples from Weiming Lake (SL), Kunming Lake (ML) and Guanting Reservoir (LL) generated by the ECOTAG program, retaining low-frequency sequences and negative control sequencing results, including the sample blank (SB), filtration blank (FB), extraction blank (EB), and PCR blank (PB). Also included are: total read counts of each sequence, taxid, taxonomic assignment by the ECOTAG program, taxonomic assignment at the species level by BLAST against GenBank nucleotide databases, the identity scores and accession numbers of the highest matched sequences, and the query sequences. Sequences with an assigned taxon of “NA” and human sequences were removed from the table.

02 Tele02_ngsfilter.txt  File used to assign sequences to samples in the OBITOOLS package.
File used to assign sequences to each sample in the OBITOOLS package, including the Teleo2 primers and primer tags.

03 Tele02_database.fa  Reference database constructed by in silico PCR with the Teleo2 primers using the ECOPCR program.
File shows the in silico PCR results using the Teleo2 primers to extract sequences in the EMBL database (release 138), which was used as the reference database in the ECOTAG program to assign a taxon to each sequence.