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

079º24’20.4’’ W), located 35 Km east of Frijoles, is intermediately disturbed and is surrounded by secondary forest with dense riparian vegetation. At each site, we haphazardly collected aquatic macroinvertebrates using standard kick-netting from the two dominant habitats types (riffles and pools). The sampling effort was approximately two hours at each site. All samples were sorted in the field and immediately preserved in 95% ethanol. Sampling permit was obtained from the Autoridad Nacional del Ambiente de Panama´ (Permit No. SC/A-44-12). In the laboratory, specimens were morphologically identified to the lowest possible taxo- nomic level (i.e., family or genus) using taxonomic keys for Neotropical macroinvertebrates [6,8,9,39]. However, given the low accuracy of morphological identification, and the fact that less than 50% of the individuals were successfully identified to species level using our barcod- ing data (see results), we focused our analyses and discussion on lineage rather than morpho- logical diversity. Representative specimens have been deposited in the invertebrate collection at Coleccio´n Zoolo´gica Dr. Eustorgio Me´ndez (CoZEM) at Instituto Conmemorativo Gorgas de Estudio de la Salud in Panama City (Voucher numbers: B001 –TR020). DNA sequencing Tissue samples were obtained from the hind leg or part of the body of each specimen, and total DNA was extracted by using the DNeasy Blood & Tissue kit (Qiagen, CA, USA), according to

Fig 1. Sampling sites of macroinvertebrates in the Panama Canal Watershed. Names on the inset map indicate the sites previously sampled by Mu´rria et al. [25]. https://doi.org/10.1371/journal.pone.0231683.g001 PLOS ONE Hidden biodiversity in Neotropical streams PLOS ONE | https://doi.org/10.1371/journal.pone.0231683 August 7, 2020 3 / 13