DNA barcoding reference libraries of Italian Plecoptera: a gap analysis

Plecoptera is a small order of aquatic insects that is considered one of the most endangered groups of insects due to increasingly altered freshwater ecosystems. Plecoptera nymphs can be challenging to identify at the species level because diagnostic characters for most species are lacking, while adults can be difficult to collect routinely given their short life span. Species identification by DNA barcoding is increasingly used as an alternative to morphological identification, but gaps and inaccuracies in reference databases needed for taxonomic assignment can undermine the utility of barcoding in real case studies. Here we aim to: i) quantify the number of Italian species of Plecoptera with barcodes from specimens collected worldwide and from Italy; ii) perform a regional assessment of DNA barcoding coverage; iii) calculate the intraspecific distance among available sequences to evaluate the potential presence of errors and cryptic species. As reference databases, we used both a non-curated database (BOLD) and a curated database (MIDORI2) to test the effect of sequence selection on the availability of reference sequences. We found that 67.6% and 51.8% of the Italian Plecoptera species were represented in BOLD and MIDORI2. Most of the available sequences were obtained from specimens collected outside Italy, with only 21.8% and 13.5% of the Italian species having sequences from specimens collected in Italy. Endemisms were poorly represented, and intraspecific distances for some species were high, which suggest cryptic diversity or erroneous assignments. Our results support the growing need to increase international cooperation among barcode initiatives and to promote the integration between molecular biologists and zoologists to exploit the full potential of DNA barcoding to protect biodiversity.