LAUSR

Simple Summary Polypedilum is a genus of aquatic non-biting midges in the family Chironomidae. This genus is widely distributed in neotropical rivers from lowlands to Andean highlands. Nevertheless, making species identification based on morphology is quite complex, even more so in the Neotropics, since systematic studies of this group are scarce. DNA barcoding can help to overcome this problem using a short DNA sequence as a barcode for species delimitation. A fragment of the mitochondrial gene cytochrome c oxidase I (CO1) has been successfully employed as a barcode in the genus Polypedilum. In this study, our aim was to understand the effect of environmental characteristics on Polypedilum diversity and distribution. We examined the CO1 sequence of 68 Polypedilum specimens from rivers with different environmental conditions located in an important biogeographic area of Ecuador. We identified five morphotypes and seven putative species which revealed high genetic variability among them. Polypedilum distribution seems to be affected mainly by two environmental factors, dissolved oxygen, and temperature. Our study is the first evidence of richness within the genus in Ecuador, highlighting the importance of developing taxonomic studies along with ecological assessments to further describe and identify new species. Abstract Chironomids show a wide distribution and can occupy several habitats due to their high adaptive capacity in different freshwater environments. The genus Polypedilum is found along a wide elevational and environmental gradient in the neotropics, and its genetic variability could help to elucidate factors determining its distribution and tolerance to the environmental changes of different species or populations. This study examines the genetic variability of Polypedilum in an important biogeographic area that acts as a geographical barrier of biodiversity at the border of the Choco and Tumbes biomes. We identified five Polypedilum morphotypes using classic taxonomic methods. We examined 68 Polypedilum individuals from eight sampling sites in El Oro Province, Ecuador, analyzing the putative molecular species using the cytochrome c oxidase subunit 1 (CO1) mitochondrial gene fragment. Then, we calculated molecular diversity indices, Haplotype diversity (Hd), and θs and θπ estimators. Seven Polypedilum OTUs were determined from which a high molecular diversity was registered. A CCA was conducted to understand the population composition in relation to environmental characteristics. Results indicated that dissolved oxygen and temperature are the main environmental factors affecting Polypedilum distribution across elevational gradients and between basins.