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

Introduction Aquatic macroinvertebrates are a fundamental component of freshwater environments. They mediate important processes such as food web dynamics, energy flow, and nutrient cycling, and therefore play a central role in sustaining the biodiversity and functioning of freshwater ecosystems [1–3]. However, the diversity of Neotropical freshwater macroinvertebrates remains poorly described, and even less is known about the processes that drive their diversity, and the scale at which they operate [4]. For instance, despite considerable efforts by local tax- onomists (e.g., [5–8], most of the published literature use genus and family as a standard taxo- nomic unit for Neotropical macroinvertebrates (e.g., [9–14]. This is partially due to the complexity of these communities, which are often composed of multiple life-stages existing at the interface between the terrestrial and aquatic environment [15,16]. Another limitation is the low efficiency of traditional morphological methods, which are generally time-consuming, and highly variable in the quality of identification across taxa and experts. This “taxonomic void” has important consequences for our general understanding of biodi- versity patterns and processes, both in Neotropical environments and globally. For example, species diversity is generally expected to increase at lower latitudes [17,18], but no consensus has been reached for macroinvertebrates, given the current lack of taxonomic knowledge [19– 22]. Within the Neotropics, our current understanding of the drivers of species diversity in

benthic macroinvertebrates is also limited [23–25]. Similar to other freshwater taxa (e.g., [26,27], spatial isolation is likely a major factor driving diversification in macroinvertebrates, but few studies have tested this expectation [25,28,29]. In particular, Mu´rria et al. [25] found a high frequency of unique haplotypes associated with the geographical distance across watersheds in Panama. While confirmatory, these findings are not surprising, given the large geographic distance among the watersheds included in Mu´r- ria et al. [25]. However, patterns of haplotype (or lineage) diversity at smaller scales (e.g., among streams within watersheds), where dispersal and gene flow might be less restricted, have received less attention. We use DNA-barcoding to assess patterns of lineage diversity (and the diversity of unique haplotypes) in freshwater macroinvertebrates in four streams within the Panama Canal Watershed (PCW). In addition, we compiled available barcoding data [25] to contrast macroinvertebrate diversity at a broader spatial scale, among eight streams along the Isthmus of Panama. Assessing the patterns and drivers of macroinvertebrate diversity at different scales is par- ticularly relevant, given the increasing rate of environmental degradation in Neotropical regions [30–33]. This includes alterations such as introduction of alien species [34,35], the development of megaprojects [36], habitat degradation, water pollution, and climate change [30,33,37,38]. As a consequence, a large portion of this biodiversity risks being lost before discovery.

Material and methods Study sites and sample processing Samples were collected from four streams within the PCW (Frijolito, Frijoles, Trinidad, and Indio) between April and May of 2013 (Fig 1). Frijolito (09º08’57.9’’ N, 79º43’53.2’’ W) and Frijoles (09º09’08.2’’ N, 79º44’05.3’’ W) are typical Neotropical streams separated by approxi- mately 300 m and located inside Soberanı´a National Park. These streams are surrounded by dense secondary forest and present low levels of disturbance. Rı´o Trinidad (8º58’28.50’’ N, 79º57’23.9’’ W) is located approximately 30 km West Rı´o Frijoles in an agricultural landscape dominated by pasture, but it has abundant riparian vegetation. Rı´o Indio (09º12’04.1’’ N, PLOS ONE Hidden biodiversity in Neotropical streams PLOS ONE | https://doi.org/10.1371/journal.pone.0231683 August 7, 2020 2 / 13 De Leo´n. Additional funding was provided by Secretarı´a Nacional de Ciencia, Tecnologı´a e Innovacio´n (SENACYT; Grants No. ITE12-002 and FID16-116 to L. F. De Leo´n), Sistema Nacional de Investigacio´n (SNI; to C. Aguilar), and the University of Massachusetts Boston (to Luis F. De Leo´n). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.