*2.5. Genotyping*

Prior to DNA extraction, individual tardigrades from the three species were preliminarily identified in vivo using light microscopy (LM). Genomic DNA was extracted using a Chelex®® 100 resin (Bio-Rad, Hercules, CA, USA) extraction method [42] with modification in order to obtain voucher specimens, i.e., tardigrade exoskeletons [43]. After DNA extraction we performed morphological analysis following the protocol of Kaczmarek et al. [43]. Then, all exoskeletons were deposited in the collection of the Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University in Pozna ´n.

In total, four molecular markers were amplified: one mitochondrial gene, i.e., COI the cytochrome oxidase subunit I; three nuclear markers, i.e., 18S rRNA—the small ribosome subunit and 28S rRNA—the large ribosome subunit as well as ITS-2—the internal transcribed spacer-2. The polymerase chain reaction (PCR) amplification was performed according to Kaczmarek et al. [44]. The sequences of primers applied to amplify molecular markers are listed in Table 1. All PCR reactions were conducted in a Biometra TProfessional thermocycler. Prior to the sequencing, the PCR products were treated with the FastAP Alkaline Phosphatase and thermosensitive Exonuclease I (Fermentas, Thermo Scientific, Waltham, MA, USA) according to the manufacturer's guidelines. Sanger DNA sequencing in both directions was performed by Macrogen (Amsterdam, The Netherlands).


**Table 1.** Primers used for PCR amplification of four DNA molecular markers of *Bryodelphax mareki* sp. nov., *Macrobiotus birendrai* sp. nov. and *Mesobiotus skorackii*.
