## 2 Materials and Methods
### DNA Sequencing and Phylogenetic Analysis  
DNA was extracted from seven specimens (isolates 1 BAN, 2 BAN, 3 BAN, 4 BAA, 5 BAN, 6 BAA, 7 BAN, Poland locality). Genomic DNA was isolated without modifying the protocol using the Bio-Trace DNA Purification Kit (EURx, Gdansk, Poland). To elute the purified DNA, we applied 50 uL of an Elution Buffer onto the silica membrane. Two genes were amplified, mitochondrial COI and nuclear EF1\(\alpha\). To amplify a fragment of the mitochondrial cytochrome c oxidase I (COI) gene, the primer pair LCO-1490 and HCO1850 [21] was used. For the amplification of the Elongation factor 1-alpha, alpha (EF1\(\alpha\)) primers EF2 [22] and EF3 [23] were used. Polymerase chain reaction (PCR) amplification for all DNA fragments was performed in a final volume of 20 uL containing 30 ng of DNA, 10 uL of 2\(\times\) Phanta Master Mix (Vazyme Biotech, Nanjing, China), 0.4 uL of 20 uM of each primer, in a Mastercycler ep system (Eppendorf, Germany). The cycling profile for the PCR was: 95 \({}^{\circ}\)C for 3 min, 35 cycles of 95 \({}^{\circ}\)C for 15 s, Tm of oligos for 15 s, 72 \({}^{\circ}\)C for 1 min and a final extension period of 72 \({}^{\circ}\)C for 5 min. In order to assess the quality of the amplification, the PCR products were electrophoresed in 1% agarose gel for 45 min at 85 V with a DNA molecular weight marker (Mass Ruler Low Range DNA Ladder, Thermo-Scientific, Waltham, MA, USA). The PCR products were purified using Clean-Up Concentrator (A & A Biotechnology, Gdansk, Poland). Samples were sequenced in both directions using the same primers as for the PCR reactions, except EF1\(\alpha\) products where two or more internal primers, selected from: EF4 (5\({}^{\prime}\)-GAACCACCATACAGCGAAAG) or EF4a (5\({}^{\prime}\)-GAACCACCGTACAGTGAAAG), EF5 (5\({}^{\prime}\)-TTGTAACTGTGACTTC), EF7 (5\({}^{\prime}\)-ATTGGAGGTTATGGAAACAGT) and EF8 (5\({}^{\prime}\)-GGGACTGTCCAATACCTCC) [24] were used combined with a BigDye Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA) in the chain termination reaction method [25]. The sequencing reaction was carried out with the PCR product at a total volume of 20 uL, containing 2 uL of BigDye Terminator Reaction Ready Mix v. 3.1, 2 uL 5\(\times\)sequencing buffer, 3.2 mol/uL of the primer solution, and 6 uL of the purified PCR product. The cycle-sequencing profile was 3 min at 94 \({}^{\circ}\)C followed by 30 cycles of 10 s at 96 \({}^{\circ}\)C, 5 s at 50 \({}^{\circ}\)C, and 2 min at 60 \({}^{\circ}\)C.  
Sequencing products were precipitated using CleanDTR (CleanNa, Waddinxveen, The Netherlands), and were separated on an ABI PRISM 3130 xl DNA Sequencer (Applied Biosystems, Waltham, MA, USA). Raw chromatograms were evaluated and corrected in Geneious v10. 2.6 ([[https://www.geneious.com](https://www.geneious.com)]([https://www.geneious.com](https://www.geneious.com)), accessed on 15 February 2023) [26]. In order to identify the nunts [27; 28], the mitochondrial COI and EF1 sequences were translated into amino acid sequences with Geneious v R10.2.6 using the respectively standard invertebrate mitochondrial genetic code and standard code. All of the nucleotide sequences were verified using BLAST searches of NCBI ([[http://blast.ncbi.nlm.nih.gov/](http://blast.ncbi.nlm.nih.gov/)]([http://blast.ncbi.nlm.nih.gov/](http://blast.ncbi.nlm.nih.gov/)) Blast.cgi, accessed on 20 February 2023). Protein coding data were aligned using the MAFFT versions 7.263 [29] plugin within Geneious v R10.2.6 and introns were removed from the EF1 \(\alpha\) alignment. All sequences used in the phylogenetic analysis are presented in Table 1.