**4. Discussion**

Out of 10 provinces and three territories of Canada, limno-terrestrial tardigrades have been reported in eight provinces and two territories. Up to now, no tardigrades have been reported from Northwest Territories, Nova Scotia nor Prince Edward Island. The highest number of tardigrade species were recorded from Nunavut (70) and the lowest Manitoba (only one). Moreover, 18 species were recorded from Alberta, 55 from British Columbia, 33 from New Brunswick, 29 from Newfoundland and Labrador, 12 from Ontario, 13 from Quebec, 3 from Saskatchewan and 5 from Yukon [13,14]. Only one species of the genus *Bryodelphax,* i.e., *Bry. parvulus* has been recorded from British Columbia and Nunavut. In case of the genus *Macrobiotus*, four species i.e., *Mac. echinogenitus* Richters [81], *Mac. hufelandi*, *Mac. occidentalis* Murray [82] and *Mac. virgatus* Murray [82] were recorded from Alberta, British Columbia, New Brunswick, Newfoundland and Labrador, Nunavut, Ontario and Quebec. Among these, only *Mac. hufelandi* belongs to the *hufelandi* group. Three species of the genus *Mesobiotus*, i.e., *Meb. harmsworthi* (Murray [83]), *Meb. montanus* (Murray [82]) and *Meb. pilatoi* (Binda and Rebecchi [84]) were recorded from Alberta, British Columbia, New Brunswick, Newfoundland and Labrador, Nunavut, Ontario and Quebec [14]. What is more intriguing, some species reported from Canada in the past are now considered as group of species or species with problematic taxonomical status [14].

Summarizing, from Canada only 121 tardigrade species and subspecies are known. Taking into consideration the area of the country (ca. 10 million km2) and its diversity such as habitats and ecosystem, it is obvious that this number is highly underestimated. For comparison, USA, with the similar country area, has more than 220 species reported [14]. This contrast is even more spectacular while comparing Canadian tardigrade fauna with the number of tardigrade species from much smaller areas like e.g., Costa Rica (ca. 51,000 km2 and 63 species known), Finland (ca. 340,000 km<sup>2</sup> and 68 species known), Italy (ca. 300,000 km2 and 234 species known) or Poland (ca. 312,000 km<sup>2</sup> and 111 species known) [68,85–89]. The number of tardigrade species from Canada is expected to be much higher than reported up to date, especially that in the present study, in one analyzed sample, we found two species new for science and one new record for the country.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/d13080394/s1, SM1—Estimates of evolutionary divergence between 28S rRNA sequences of *Bryodelphax mareki* sp. nov based on p-distances, SM2—Estimates of evolutionary divergence between COI sequences of *Macrobiotus birendrai* sp. nov based on p-distances, SM3—Estimates of evolutionary divergence between 18S rRNA sequences of *Macrobiotus birendrai* sp. nov based on p-distances, SM4—Estimates of evolutionary divergence between 28S rRNA sequences of *Macrobiotus birendrai* sp. nov based on p-distances, SM5—Estimates of evolutionary divergence between ITS-2 sequences of *Macrobiotus birendrai* sp. nov based on p-distances, SM6—Estimates of evolutionary divergence between COI sequences of *Mesobiotus skorackii* based on p-distances, SM7— Estimates of evolutionary divergence between 28S rRNA sequences of *Mesobiotus skorackii* based on p-distances [13,15–17,23,30,38,39,41,60,67,70,73–80,90–103].

**Author Contributions:** Conceptualization, M.R. and Ł.K.; data curation, P.K.; formal analysis, P.K., M.R., M.M., M.G. and Ł.K.; investigation, P.K. and Ł.K.; methodology, M.R., M.M. and Ł.K.; supervision, Ł.K.; validation, P.K., M.R., M.M. and Ł.K.; visualization, M.R. and M.G.; writing—original draft, P.K., M.M. and Ł.K.; writing—review and editing, P.K., M.R., M.M., M.G. and Ł.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are presented in the article and in SM which are part of article.

**Acknowledgments:** Milena Roszkowska and Pushpalata Kayastha are scholarship holders of Passport to the future—Interdisciplinary doctoral studies at the Faculty of Biology, Adam Mickiewicz University, Pozna ´n POWR.03.02.00-00-I006/17. The work of Monika Mioduchowska was supported

by grant no. 2017/27/B/NZ8/01056 from the National Science Centre, Poland. Studies have been partially conducted in the framework of activities of BARg (Biodiversity and Astrobiology Research group).

**Conflicts of Interest:** The authors declare no conflict of interest.
