2.1 Taxon sampling and data collection
Specimens were collected between 2014 and 2017 from 34 localities, in a diverse array of habitats including small streams and ponds to lakes, rivers and bays in Argentina, Brazil, Chile and Dominican Republic. The main emphasis was on adult sampling, collected with a sweep net near aquatic systems. A 20 cm diameter D-frame kick net (mesh size 250 μm) was also used to collect immature stages at some localities. All sampled adults were preserved in 75%-85% ethanol and larvae in 96-100% ethanol and stored at 4°C in the dark prior to the extraction. Specimens were identified using the classification proposed by Townes (1945), Bidawid & Fittkau (1995), Bidawid-Kafka (1996), Shimabakuro et al. (2019), Pinho & Silva (2020), and eventual examination of type material. Voucher specimens are deposited in the Museum of Comparative Zoology (MCZ) at Harvard University and in the National Institute of Amazonian Research (INPA).
In addition to data generated for this publication, we also searched for public COI barcodes in the Barcode of Life Data Systems (BOLD,www.boldsystems.org) belonging to the genus Polypedilum that were longer than 300 base pairs and without stop codons. Searches were performed on 25 January 2022 in BOLD. In total, 9,540 COI barcodes were included in our dataset, of which 149 barcodes of 54 identified species were not previously used in any molecular analysis. A reduced data set, containing 1,492 sequences, was generated based on the manual deletion of the highly similar sequences based on an UPGMA tree. Duplicate sequences occurring at different sampling localities were retained in our dataset. The detailed specimen records and sequence information, including trace files, are available in BOLD through the dataset ‘DS-RPPPOL - Reduced personal and publicly available records of Polypedilum (Diptera: Chironomidae)” with DOI: https://doi.org/XXXX.