Specimens were collected within the project “Hymenopteran biodiversity of the Vashlovani National Park” by the team from the Invertebrate Research Center. Every 20 days, from the beginning of April to the end of August, we placed four colored traps (plates) of similar diameter in different colors (yellow, white, blue, and pink) at two locations (Fig. 1). Traps were located in two different habitats of Vashlovani NP: Mijniskure (41.111663, 46.649028) and at the central bungalows (41.159442, 46.567060).

Traps were set on the ground and filled with water (100–150 ml) and a drop of unscented detergent to break surface tension. After 24 hours, the material was collected with tweezers and preserved in vials with 96% ethanol for further study. The species were morphologically identified by the second author using the key by Schmid-Egger et al. (2017). Images of specimens were taken with an Optika Microscopes-ITALY CP-20 camera mounted on a Zeiss Stemi 508 stereomicroscope.

Four Polistes specimens belonging to three species from Georgia were subjected to DNA barcoding. Genomic DNA was extracted from the middle legs using the Oxgen Soft Tissue DNA Purification Kit (Oxgen, Mtskheta, Georgia) following the manufacturer’s protocol. Partial sequences of cytochrome c oxidase subunit I (COI) were amplified by polymerase chain reaction (PCR) using the primer pair LCO1490 and HCO2198 (Folmer et al. 1994). Thermal conditions included denaturing at 95 °C for 1 min, followed by a first cycle set (15 cycles): 94 °C for 30 sec, annealing at 55 °C for 1 min, and extension at 72 °C for 1:30 min. A second set of cycles was used as follows (25 cycles): 94 °C for 35 sec, 50 °C for 1 min, 72 °C for 1:30 min, and a final extension step at 72 °C for 10 min. PCR products were stored at 4 °C and visualized on 1% agarose gels. PCR products were purified with ExoSAP-IT (Thermo Fisher, Waltham, Massachusetts) according to the manufacturer’s protocols. Sequencing of the purified PCR products in both directions was conducted at BGI Tech Solutions (Hong Kong).

We generated further new reference sequences from Georgia (2 × P. cf. gallicus, P. cf. semenowi, P. associus, P. bucharensis), Bulgaria (P. nimpha), Armenia (P. mongolicus), and Iran (Polistes sp.). DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, but with an extended overnight incubation time at 56 °C (Thermomixer, with gentle shaking) for cell lysis. For elution, 100 µL of AE buffer was used. The extracted DNA was stored at −20 °C until use. Primers used for amplification and sequencing were LCO1490-JJ and HCO2198-JJ (Astrin and Stüben 2008). The amplified mitochondrial region is a part of the cytochrome c oxidase subunit I gene (COI) and corresponds to the standard barcode region of the animal kingdom (Hebert et al., 2003).

PCR reactions were carried out in a total volume of 25 µL containing 1.0 µL DNA, 2.0 µL of primers (5 pmol each), and 12.5 µL of 2× Multiplex PCR Plus Master mix (QIAGEN). The PCR protocol consisted of an initial DNA polymerase (HotStar Taq) activation step at 95 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, 90 s at 49 °C, and 90 s at 72 °C; the last cycle was followed by a final 10 min extension step at 72 °C. 3 µL of the PCR product were visualized on a 1% agarose gel. The PCR product was purified using 3 M sodium acetate and ethanol precipitation. Sanger sequencing was performed using the BigDye Terminator v. 3.1 Cycle Sequencing kit (Applied Biosystems, Thermo Fisher Scientific Inc.) with a total amount of 20 ng of purified PCR product. To obtain unequivocal sequences, both sense and antisense strands were sequenced.

The obtained sequences were trimmed, checked, and aligned using the MUSCLE algorithm implemented in Geneious Prime v. 2022.1.1 (http://www.geneious.com). The remaining DNA extracts were deposited in the scientific collection of the Institute of Entomology, Agricultural University of Georgia, Tbilisi, Georgia. Newly generated DNA barcodes had a length of 648 bp and were compared against the BOLD database to confirm morphological species identification (Ratnasingham and Hebert 2013).

Furthermore, we provided phylogenetic background by performing Bayesian phylogenetic analysis using our 12 newly generated and an additional 21 ingroup sequences of related species taken from BOLD (all newly generated sequences were submitted to NCBI GenBank (www.ncbi.nlm.nih.gov; under the accession numbers: PX502332–PX502343). We included all known species from Georgia for which publicly available sequences exist. A sequence of Vespa crabro Linnaeus, 1758, was used as the outgroup (GenBank Accession Number: ACUFI813). Analyses were performed with MrBayes v. 3.2.6 (Ronquist et al. 2012). We used the reversible jump model to provide high flexibility in the analyses and ran the analyses for 1,000,000 generations, sampling every 100 trees to generate a sample of 10,000 trees. Convergence was confirmed by average split frequencies below 0.05. The resulting consensus tree was summarized using FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).

We generated new sequences for the genus Polistes from Georgia and constructed a phylogenetic tree, including published sequences from the same species from other locations and related species to confirm identification (Fig. 3). In regard to the outgroup, all Polistes formed one cluster with high support. With the exception of one specimen of P. dominula (BCHYM12109) grouping with P. bucharensis and a specimen of P. associus grouping within P. nimpha, all species formed monophyletic groups. Polistes biglumis, with two specimens of P. cf. gallicus, as well as P. gallicus and P. mongolicus, were the basal taxa, but formed a polytomy with another group that included all other taxa. This group is further divided into P. foederatus, including our specimen from Georgia. Next, P. nimpha splits off, together with one specimen identified as P. associus. The last cluster splits into two groups: one of P. dominula and P. bucharensis (including two sequences from Georgia). However, one specimen identified as P. dominula groups at the base of the P. bucharensis branch, albeit with low support. The sister group to these two species consists of P. atrimandibularis, which is again basal to P. sulcifer and P. semenowi, which formed a cluster.

The two specimens breaking up species monophylies may be the result of misidentifications, as the respective close relatives are morphologically very similar. In all other cases, morphological and genetic assignments are in agreement.