*2.6. Comparative Genetic Analysis*

Obtained mtDNA and nrDNA sequences were quality checked and consensus sequences were created for individual tardigrades in BioEdit v. 7.2.5 [49]. All COI sequences were translated into amino acid sequences to check against pseudogenes using the EMBOSS-TRANSEQ application [50,51]. The translation was performed with the invertebrate mitochondrial codon table. To verify the homology of the amplified DNA region, Basic Local Alignment Search Tool [52] searches at the National Centre for Biotechnology Information NCBI were applied.

All obtained sequences were deposited in GenBank and the accession numbers are listed in Table 2.

**Table 2.** The GenBank accession numbers of obtained molecular markers of three tardigrade species.


For molecular comparisons, all sequences of the mtDNA and nrDNA fragments of the genera *Bryodelphax*, *Macrobiotus* and *Mesobiotus* were downloaded from the GenBank and trimmed to the same length in BioEdit v. 7.2.5. The COI sequences could be unambiguously aligned without inserting gaps. In turn, the sequences of nrDNA were aligned using ClustalW Multiple Alignment tool [53] implemented in BioEdit v. 7.2.5. with default settings. Uncorrected pairwise distances were calculated using MEGA X [54].

#### **3. Results**
