Abstract
Psyllids (Hemiptera: Psylloidea) are plant sap-sucking insects whose identification is often difficult for non-experts. Despite the rapid development of DNA barcoding techniques and their widespread use, only a limited number of sequences of psyllids are available in the public databases, and those that are available are often misidentified. Here, we provide 80 sequences of two mitochondrial genes, cytochrome c oxidase I (COI) and cytochrome b (Cytb), for 25 species of Aphalaridae, mainly from Bulgaria. The DNA barcodes for 15 of these species are published for the first time. In cases where standard primers failed to amplify the target gene fragment, we designed new primers that can be used in future studies. The distance-based thresholds for the analysed species were between 0.0015 and 0.3415 for COI and 0.0771 and 0.4721 for Cytb, indicating that the Cytb gene has a higher interspecific divergence, compared to COI, and therefore allows for more accurate species identification. The species delimitation based on DNA barcodes is largely consistent with the differences resulting from morphological and host plant data, demonstrating that the use of DNA barcodes is suitable for successful identification of most aphalarid species studied. The phylogenetic reconstruction based on maximum likelihood and Bayesian inference analyses, while showing similar results at high taxonomic levels to previously published phylogenies, provides additional information on the placement of aphalarids at the species level. The following five species represent new records for Bulgaria: Agonoscena targionii, Aphalara affinis, Colposcenia aliena, Co. bidentata, and Craspedolepta malachitica. Craspedolepta conspersa is reported for the first time from the Czech Republic, while Agonoscena cisti is reported for the first time from Albania.