Specimens of Trichocera (Saltrichocera) maculipennis Meigen, 1818: female [indicated as Polar in the diagrams], Arctowski Polish Station, data, collected from sewage system as described in  (IBB PAS, coll. B. Matuszczak); female [CH]: Switzerland, cave, 2. Galerie Sieben Hengste-Hohgant, alt. 1486 m, 26.12.1986–29.12. 1987 (MNHN, now ISEZ; coll. A. Hof); female: Poland, cave [PL] Pod Sokolą, 19.02.2018 (ISEA; coll. J. Zalewska). Trichocera (Saltrichocera) regelationis (L.), 1758, female, Poland, Ojców National Park, Wąwóz [gorge] Skałbania, 11.04. 1999 (ISEA, coll. A. Palaczyk); Trichocera (Saltrichocera) nordica Krzemińska & Gorzka, 2014 : female, Finland, Oulanka Research Station, 5.09.2011; Trichocera (Saltrichocera) parva Meigen, 1804: female, Finland, Oulanka Research Station, 10.09.2011; Trichocera (Trichocera) major Meigen, 1818: female, Poland, Ojców National Park, Wąwóz [gorge] Korytania, 19.11.1992 (coll. E. Krzemińska). All specimens are housed in ISEA if not otherwise stated.
IBB—Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
ISEA—Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków. Poland.
MNHN—Museé national d’histoire naturelle, Neuchâtel, Switzerland.
DNA isolation from specimens
DNA isolation from insects was carried out as described by Gilbert et al.  with minor modifications as described below. The whole specimens were transferred into 1.5 ml microcentrifuge tubes and washed twice with 1 ml TE buffer (10 mM Tris–HCl, 1 mM EDTA pH 8.0) to remove residual ethanol from the sample, then 500 µl of lysis buffer (3 mM CaCl2, 2% (w/v) sodium dodecyl sulphate (SDS), 40 mM dithiotreitol (DTT), 50 µg/ml proteinase K, 100 mM Tris buffer pH 8 and 100 mM NaCl; final concentrations) were added. The homogenates were incubated overnight at 55 °C. Specimens were removed from the buffer, placed in 1 ml 95% (v/v) ethanol and replaced in their respective collections. The lysates were then extracted 3 times with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1; v/v/v) until the interface was clear. Nucleic acids were precipitated by addition of 0.7 volume of isopropanol. Three micro litres of glycogen (20 mg/ml) was added during the precipitation step to improve DNA yields. Samples were incubated at room temperature for 20 min and centrifuged using a MiniSpin Plus centrifuge (Eppendorf AG, Hamburg, Germany) at 14,000×g for 15 min. The supernatant was then removed and the DNA pellet washed twice in 500 µl room temperature 80% (v/v) ethanol, allowed to air-dry at 37 °C, and resuspended in 50 µl low-TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA). After isolation DNA quantity was measured using Qubit 3.0 fluorometer (Thermo Scientific, Waltham, USA) and High Sensitivity DNA quantification kit (Thermo Scientific, Waltham, USA). DNA concentration was normalized to final concentration of 5 ng/µl. All reagents used in the purification step were molecular biology grade and were purchased from Sigma.
PCR amplification and sequencing of mitochondrial barcodes
The standard cytochrome oxidase (COI) fragment was amplified using the primer pair described by Folmer et al. :
LCO1490: 5′- GGTCAACAAATCATAAAGATATTGG-3
HCO2198: 5′- TAAACTTCAGGGTGACCAAAAAATCA-3'
16S mtrRNA fragment was amplified with following primer pair:
LR-N-13398: 5′-CGCCTGTTTAACAAAAACAT -3’
described by Simon et al. .
PCR products were amplified using KAPA Robust PCR kit (Roche, Basel, Switzerland). PCR reactions were carried out in 20 µl final volume consisting of: 4 µl of KAPA 2G A buffer, 0.4 µl of 10 mM dNTPs, 1U of KAPA Robust polymerase (5 U/µl), 0.5 µl of each primer (10 µM), 11.45 µl of PCR-grade water and 2 µl of DNA template (10 ng). Amplification reaction conditions for both sets of barcoding primers were as follows: 3 min of initial denaturation at 95 °C, followed by 38 cycles of 30 s at 95 °C, 20 s at 50 °C, 30 s at 72 °C, and final extension period of 2 min at 72 °C. The amplified products were visualized through agarose gel electrophoresis (1.5%, wt/v) and ethidium bromide staining. The amplicons were purified using EPPiC Fast kit (A&A Biotechnology, Gdańsk, Poland) and directly sequenced with the same primers used for PCR amplification. Sanger sequencing was done using BigDye Terminator v3.1 chemistry and ABI3730xl genetic analyzer (Thermo Scientific, Waltham, USA).
Sequence data were analyzed using FinchTV ver. 1.4.0 (Geospiza, Akron, USA). Consensus sequences were obtained with Seqman Pro ver. 9.1 software (DNAStar, Madison, USA).
Molecular phylogenetic analysis by maximum likelihood method
Phylogenetic tree of mitochondrial 16S mtr RNA and and COI gene fragments
The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model . Evolutionary analyses were conducted in MEGA7 .
Nucleotide sequence submission and GenBank accession numbers
Accession numbers for each novel nucleotide sequence of COI and 16S mtrRNA genes of the Trichocera species are given in Table 1.