Insights into the representativeness of biodiversity assessment in large reservoir through eDNA metabarcoding

resulting in a gradient-like distribution that becomes more uniform over time. These processes effectively blur the boundaries of the originally distinct organismal clusters, illustrating the dis- tinct dynamics between direct ecological observations and molecular ecological data. Here, eDNA could also detect species never reported for the study area, compensating for some of the limitations of the traditional monitoring methods, such as erroneous identification, dedi- cated time, and seasonal changes [66]. These findings support eDNA metabarcoding as an effective method when evaluating fish communities, as already affirmed in previous studies [67]. For this reason, the eDNA can be employed for rapid and large-scale biodiversity assess- ments for monitoring programs [17], reducing costs and minimizing interventions in aquatic environments. The use of multivariate analysis combined with rarefaction curves allowed us to assess whether the eDNA-based diversity was robust and to identify potential redundant sampling sites while highlighting the most diverse locations. Our findings show that even though the water samples were collected at the exact same location within a few minutes’ interval, the eDNA obtained was different enough to distinguish these water samples. Both alpha diversity analysis and PCA showed that many samples within the same stations are highly divergent, presenting distinct numbers of taxa and do not contain similar variance regarding community composition (Figs 4 and 5A). The PCA revealed that station C01 contributed the most to the

observed variation (Fig 2A). This overrepresentation appears to result from species exclusive to this compartment, as supported by the frequency heatmaps (Fig 3). The C01 station alone accounted for 37 of 79 non-fish MOTUs (~46%) and 32 (80%) of all fish species. However, the three samples from C01 presented higher variation than expected from replicates, as observed in the DAPC clustering analysis. Due to potential differences in composition and abundance, two out of the three samples from C01 are distinct enough to be considered a separate ecologi- cal group (Fig 5D). For C01, a potential explanation for these results is the higher current speed (0.5 m/s compared to 0.05 m/s in other stations). Because eDNA behaves like fine partic- ulate organic matter, the concentration of eDNA can be influenced by the hydrological profile, being transported according to the velocity of water movement (65). Consequently, the eDNA obtained during each sampling would substantially differ from each other. However, it is also possible that the ichthyofauna habitat preferences are shaping these communities’ differences. As previously demonstrated in the literature, the ichthyofauna may exhibit distinct preferences regarding water velocity, temperature and depth, for example, in lotic systems [68, 69]. It is worth noting that in the PCA, all pilot sampling was very close to all stations except C01 (Fig 2A). This indicates that despite the one-year difference between the samples, this time gap did not result in significant differences in the composition of local communities.

However, the different sample sizes from the two samplings, plus the short duration of the study, highlights the need for more comprehensive spatial analyses to confirm the time-scale effects on fish communities in the TI. Previous studies have indicated significant changes in community composition after a few weeks [70]. In some cases, temporal dynamics per se are not the most determinant factors; rather, environmental factors such as seasonality, salinity, or precipitation can have a greater influence on species distribution and occurrence [66]. Following station C01, stations C10 and C11 demonstrated significant contributions to the clustering analysis of groups from DAPC (Fig 2D). Interestingly, these samples are at the boundaries (C01), and at the center arm (C10 and C11) of the TI reservoir (Fig 1A). Hydrody- namic modeling revealed that water velocity at station C01 (0.05–0.1 m/s) is higher than at other stations, which show velocities of 0.0–0.05 m/s (S2 Fig). This elevated water flow in C01 likely contributes to the increased diversity observed among replicates, as faster flow enhances mixing and eDNA transport. Furthermore, the water renewal rate at C01 is substantially higher, with 50% of the water being renewed every 5–10 days. In contrast, stations C10 and PLOS ONE eDNA metabarcoding reservoir PLOS ONE | https://doi.org/10.1371/journal.pone.0314210 January 24, 2025 16 / 22