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

sequence 26 specimens. In addition, the fact that our rarefaction/extrapolation analysis showed a lack of saturation for most of the sites indicates that substantially more research is needed in this region. Overall, however, our results are in line with recent work showing high haplotype endemicity among isolated watersheds across the Isthmus of Panama [25]. In fact, despite low sample size, we found at least 153 (~91%) novel haplotypes within the PCW. Thus, we expand on this previous work by highlighting the possibility that endemicity of Neotropical macroin- vertebrates can be substantial even within a single watershed. Typically, diversification in freshwater organisms is marked by a strong geographic signa- ture, where genetic divergence is facilitated by spatial isolation among populations [26,27]. However, the contribution of geographic isolation to the diversification of Neotropical fresh- water macroinvertebrates has received little attention to date [25]. In addition, the fact that most macroinvertebrates are semiaquatic, and are likely to disperse during the adult stages [15,16] could limit genetic isolation among nearby stream communities. Yet, the possibility of high endemicity even within a single watershed suggests that spatial isolation, habitat variabil- ity, and perhaps, local adaptation are important drivers of macroinvertebrate diversity. Another interesting finding was that macroinvertebrate diversity appeared to increase at sites located in Central Panama, specifically within the PCW (e.g., Frijoles, Frijolito, Trinidad).

However, additional research is needed to confirm this pattern and to explore the underlying drivers. We encourage the application of more efficient tools such as DNA metabarcoding to facilitate this endeavor. Our finding of high endemicity at a small geographic scale is also relevant in the face of increasing anthropogenic disturbances [31,32,71–73]. Specifically, it suggests that small-scale local disturbances could have drastic consequences for the maintenance of a unique freshwater biodiversity–but this diversity is still unknown. We therefore predict that the current rate of species loss in freshwater ecosystems might be surpassing the rate of species discovery in Neo- tropical environments. Overall, however, further work is clearly needed to disentangle the con- tribution of other factors such as genetic drift, local adaptation, and environmental disturbance to persistence and diversification of Neotropical freshwater macroinvertebrates. Taken together, our results confirm the expectation that the diversity of Neotropical macro- invertebrates remains under-studied. They also indicate that uncovering this hidden diversity is crucial to our understanding of the local and regional processes that shape biodiversity in Neotropical freshwater environments. Supporting information S1 Fig. Molecular diversity in freshwater macroinvertebrates from Central Panama. The Bayesian inference tree shows species delimitation analyses based on generalized mixed Yule coalescent (GMYC) and the automatic barcode gap discovery (ABGD). Black and grey blocks

represent putative molecular species, with taxa sharing the same block corresponding to simi- lar species. The numbers next to the nodes represent Bayesian posterior probability values. (PDF) S2 Fig. Rarefaction and extrapolation curves for molecular diversity (MOTUs) at each site. Number at the top represent fit curves for the first three Hill numbers: species richness (q = 0), the exponential of Shannon entropy (“Shannon diversity”, q = 1), and the inverse Simpson concentration (“Simpson diversity”, q = 2), using individual-based abundance data. Sites are: Alema´n (ALE), Chorro (CHO), Blanco (BLA), Guabal (GUA), Capira (CAP), Frijolito (FRI), Cerro Azul (CAZ), and Chucantı´ (CHR) from Mu´rria et al. 2015; and Trinidad (TRI), Frijolito (FTO), Frijoles (FES) and Indio (IND) (from the present study). (PDF) PLOS ONE Hidden biodiversity in Neotropical streams PLOS ONE | https://doi.org/10.1371/journal.pone.0231683 August 7, 2020 9 / 13