6 115 agarose gel and purified with the enzymes Exonuclease I (EXO) and Shrimp Alkaline 116 Phosphatase (SAP) [43] , before sequencing using an Applied Biosystems Genetic Analyzer (ABI 117 3130xl, Applied Biosystems, Carlsbad, California). Sequences were aligned using Geneious 118 V7.03 [44] . Sequence alignments were also inspected by eye to confirm overall sequence quality.
119 Project sequences are available in GenBank (accession numbers: KX039451-KX039650, 120 KU980966-KU981004). 121 122 Data analysis 123 To confirm morphological identification for our sequenced specimens, we performed 168 were used as input. All analyses were run using a relative barcoding gap width (X value) set to 169 1.0. Only the recursive results were used because they allowed for different gap thresholds , and 4 from the present study). One site (Frijolitos) was sampled during both studies, but 183 we analyzed them separately to preserve independence between the two studies. We then applied 184 the same rarefaction/extrapolation approach described above to generate rarefaction curves as 185 function of the number of individuals sampled. Although there might be important 186 methodological differences between the two studies, our objective here is to provide a general 9 187 overview molecular diversity across the sites, rather than a precise estimate within each site.
189 Results 190 We collected approximately 300 specimens across the four sites; however, our analysis focused 191 on the 224 individuals that were successfully barcoded (Table 1 ; S1 Table) . We were able to 192 identify nearly 70% of individuals to genus level using the morphological approach, but species-193 level identification was only possible for 56 individuals (25%). Some of the most numerous taxa 194 across sites included Leptophlebiidae (11.2 % of individuals), Libellulidae (11.2 %), Naucoridae 195 (6.7 %), Notonectidae (6.3 %), Chironomidae (5.4%), Gerridae (4.9 %), Hydropsychidae (4.0 196 %), Perlidae (3.6 %), and Baetidae (3.6 %).
197 The 224 COI sequences revealed a total of 168 haplotypes (S1 Table) . 225 Both ML and BI inference trees for all specimens showed well-defined clades at the level of 226 order and family, with some differences in the topology, but overall support was higher for the 227 BI tree, which we used to represent the number of molecular species (Fig 2; S1 Fig) . (Fig 4) , and this pattern was consistent across the first three Hill numbers 247 (Fig S2) . Similar results were found when looking at diversity Hill across the Isthmus of Panama 248 using the compiled barcoding data set. In particular, we observed substantial diversity of both 249 MOTUs and unique haplotypes across sites (Fig 4) , but the accumulation curves did not reach 250 saturation (Fig 4) . In addition, both MOTUs and haplotype diversity tended to increase at sites 251 within the PCW, in contrast to sites located at the eastern and western portion of the country (Fig   252 4) . Data on assignment and diversity of MOTUS across study sites are available in the 253 supplementary material (S1 Table) . (Table 1 ; Fig 2) , and a large portion of these lineages appear to be unique to 272 each site (Fig 3) . In addition, our rarefaction/extrapolation approach showed that this diversity is 273 still undersampled across sites both within the PCW and along the Isthmus of Panama (Fig 4) .
These findings confirm that the diversity of freshwater macroinvertebrates in Neotropical 275 environments is largely understudied [10,34,68], and could be much higher than previously 276 thought. In addition, the fact that only a small number of specimens from these taxa matched 277 available sequences in public databases further highlights the potential for biodiversity discovery 278 in Neotropical freshwater environments. This seems particularly relevant for taxa such as 279 Hydropsychidae, Gerridae, Chironomidae, Leptophlebiidae, Libellulidae and Notonectidae, 280 which showed high lineage/haplotype diversity across sites (Table 1 ; Fig 2) . Some of these taxa 281 also showed high haplotype diversity in a previous molecular study across Panama [26] , and are 13 283 [6,10]. They also span several functional feeding groups such as filterers (Hydropsychidae), 284 scrapers (Chironomidae) and predators (Notonectidae), which play a crucial role in freshwater 