Molecular clock dating
Molecular clock estimations of the first split within Milnesiumlineages present in our dataset is dated for the Jurassic, 162 million years (My) ago. Therefore, the analysis confirms the previously hypothesised ancient origin of the genus (Bertolani & Grimaldi, 2000; Guil & Giribet, 2012; Morek & Michalczyk, 2020), which may be even older than the first split observed in our analysis. Moreover, this suggest that the remaining genera within Milnesiidae Ramazzotti, 1962 should be even older, possibly >200 My old (late Triassic/early Jurassic or older). This, however, requires the assumption that all three are valid genera, and are not nested within the currently recognised Milnesium lineages. The age of split between the Apochela and the Parachela is dated for 432 Mya (Silurian) but has a very wide 95% HPD, (540 to 323 Mya; early Cambrian to Carboniferous). This is the first ever estimation of this divergence event between the two eutardigrade orders. However, given the scarce available fossil data for Milnesium (Bertolani & Grimaldi, 2000) and tardigrades in general (Guidetti et al., 2017), the uncertainty of the exact node ages is expected and needs to be verified and narrowed down by a more robust phylogenetic analysis, encompassing more genetic data for the numerous parachelan lineages.
The molecular clock analysis also allowed us to estimate mutation rates for all four utilised molecular markers (Table 3). As expected, 18S rRNA is characterised by the lowest rate whereas the COI fragment mutates the fastest. Importantly, our estimation of the 18S rRNA mutation rate (2.37×10-4 substitutions per site per million years; SSM) is lower than the previously calculated joint 18S and 28S rRNA rate for the entire Ecdysozoa (1.56×10-3 SSM; Rota-Stabelli, Daley, & Pisani, 2013) by an order of magnitude. The estimation of the 28S rRNA mutation rate indicated 1.05×10-3 SSM, which is also lower than the joined 18S and 28S rRNA for Ecdysozoa. The much lower mutation rates obtained in our analysis indicate that tardigrades are most likely characterised by slower pace of evolution, which is mirrored in the lower mutation rate. However, given that this is the first time that substitution rates have been calculated exclusively for tardigrades, they should be used with extreme caution, especially when applied to other groups of tardigrades. This is so because the substitution rates have been estimated in the genus Milnesium , which is characterised by a considerable morphological stasis compared to the parachelans, and thus is it possible that this lineage also exhibits a slower pace of evolution than other tardigrades. Therefore, analyses concerning taxa belonging to all major tardigrade lineages and a larger number of calibration points should be taken into consideration to assess the values of these rates for the entire phylum, or independently for each of the classes and/or orders.