Hidden biodiversity in Neotropical streams: DNA barcoding uncovers high endemicity of freshwater macroinvertebrates at small spatial scales

accumulation curves for most of the sites (Fig 3), and this pattern was consistent across the first three Hill numbers (S3 Fig). Similar results were found when looking at diversity Hill across the Isthmus of Panama using the compiled barcoding dataset (Fig 3). In particular, we observed substantial diversity of both MOTUs and unique haplotypes across sites, but the accumulation curves did not reach saturation for most of the sites (Fig 3). In addition, both molecular species and haplotype diversity tended to increase at sites within the PCW, in con- trast to sites located in the eastern and western portion of the country (Fig 3). Data on assign- ment and diversity of MOTUs across study sites are available in the supplementary material (S1 Table). Discussion Using DNA-barcoding, we examined the diversity of freshwater macroinvertebrates at a small spatial scale, among four streams within the Panama Canal watershed (PCW). We also com- piled existing barcoding data [25] to contrast macroinvertebrate diversity at a broader spatial scale, across eight streams along the Isthmus of Panama. Overall, we found high lineage diver- sity across sites within the PCW (Table 1; S1 Fig), and a large portion of these lineages appear to be unique to each site (Fig 2). In addition, our rarefaction/extrapolation approach showed that this diversity is still under-sampled across sites both within the PCW and along the Isth- mus of Panama (Fig 3). These findings confirm that the diversity of freshwater macroinvertebrates in Neotropical

environments is largely under-studied [33,69,70], and could be much higher than previously thought. In particular, these findings highlight the fact that there is limited published research using genetic methods to study macroinvertebrate diversity in this region. This was reflected by one of our sites (Frijolito), which despite being sampled by a previous barcoding study [25], still showed a substantial number of novel haplotypes. In addition, the fact that only a small number of our specimens matched available sequences in public databases further highlights the potential for biodiversity discovery in Neotropical freshwater environments. This seems particularly relevant for taxa such as Hydropsychidae, Gerridae, Chironomidae, Leptophlebii- dae, Libellulidae and Notonectidae, which showed high lineage/haplotype diversity across sites Fig 2. Distribution of molecular diversity in freshwater macroinvertebrates among streams within Panama Canal Watershed. Venn diagrams show the number of shared and unique haplotypes (A) and MOTUs (B) across four streams: Trinidad (pink), Frijolito (blue), Indio (yellow), and Frijoles (green). https://doi.org/10.1371/journal.pone.0231683.g002 PLOS ONE Hidden biodiversity in Neotropical streams PLOS ONE | https://doi.org/10.1371/journal.pone.0231683 August 7, 2020 7 / 13