Abstract
DNA barcoding is an essential tool in modern biodiversity sciences. Despite considerable work to barcode the tree of life, many groups, including insects, remain partially or totally unreferenced, preventing barcoding from reaching its full potential. Aquatic insects, especially the three orders Ephemeroptera, Plecoptera, and Trichoptera (EPT), are key freshwater quality indicators worldwide. Among them, Plecoptera (stoneflies), which are among the most sensitive aquatic insects to habitat modification, play a central role in river monitoring surveys. Here, we present an update of the Plecoptera reference database for (meta)barcoding in Switzerland, now covering all 118 species known from this country. Fresh specimens, mostly from rare or localized species, were collected, and 151 new CO1 barcodes were generated. These were merged with the 422 previously published sequences, resulting in a dataset of 573 barcoded specimens. Our CO1 dataset was delimited in 115 CO1 clusters based on a priori morphological identifications, of which 17% are newly reported for Switzerland, and 4% are newly reported globally. Among the 115 CO1 clusters, 85% showed complete congruence with morphology. Distance-based analysis indicated local barcoding gaps in 97% of the CO1 clusters. This study significantly improves the Swiss reference database for stoneflies, enhancing future species identification accuracy and biodiversity monitoring. Additionally, this work reveals cryptic diversity and incongruence between morphology and barcodes, both presenting valuable opportunities for future integrative taxonomic studies. Voucher specimens, DNA extractions and reference barcodes are available for future developments, including metabarcoding and environmental DNA surveys.