Diversification of the six main lineages
The phyletic relationships obtained in this contribution are overall in line with the first comprehensive phylogeny of the genusMilnesium (Morek & Michalczyk, 2020; see also Fig. 2 herein). Three out of the five main clades obtained in the previous paper were also recovered in the analysis presented herein (A, B and D), and only the two smallest clades (E and C in Morek & Michalczyk, 2020, consisting therein of one and two species, respectively) changed their composition and slightly changed their position in the currently presented tree. These changes are not surprising given the inclusion of a large portion of new data in the current analysis (Fig. 2) and the shallow divergence of the deep nodes.
The short branches and weakly supported nodes in the basal part of the tree may suggest ancient and rapid diversification within the genus. On the other hand, such a pattern could be an artefact resulting from fragmentary sampling. Although both taxonomic and geographic sampling in the present study are widest to date, the considerable number of species that were sequenced for the first time (see also below for details) suggests that new phyletic lineages could be out there, but have not been detected yet. Apart from new species, DNA sequences for the remaining three apochelan genera are likely to shed some strong light on the evolution of the genus and entire family. Moreover, the relatively long branch from the Apochela-Parachela split to the first diversification observed in the ingroup (187–270 My long; see Fig. 2) suggests that there probably were ancient lineages that did not persist to the present time. If this is indeed the case, it is possible that the genus diversified also earlier or later than the diversification recovered in our analysis. However, assuming that our phylogenetic tree accurately reflects the evolutionary history of the genus, the question what may have caused the rapid divergence calls for a hypothesis.
There are two mutually non-exclusive possible explanations for such a scenario: the breakdown of Gondwana and the switch to carnivory of the common ancestor of extant Milnesium species. The first hypothesis stems from the coincidence of the divergence and Gondwana breakdown (see Fig. 2). Under this scenario, the force behind the diversification was geographic isolation. The apparent low dispersal abilities of apochelans, as demonstrated in the present study, seem to support this hypothesis. On the other hand, the sister order Parachela started to diversify 83 My earlier and, if continental drift was supposed to be responsible for the diversification of limnoterrestrial tardigrades in general, we should expect to see roughly the same temporal pattern in both apochelans and parachelans. Moreover, it needs to be stressed that the Gondwana breakdown was a long process that did not result in an immediate geographic isolation and our molecular clock analysis is burdened with considerable error, meaning that the time correlation between geology and phylogeny observed in the present study may be accidental.
Whereas the Gondwana split hypothesis concerns an external, abiotic factor, the carnivory hypothesis concerns a physioecological property ofMilnesium itself. All known species in the genus are strictly carnivorous and are not able to reproduce on a herbivorous diet (Bryndová, Stec, Schill, Michalczyk, & Devetter, 2020). At the same time, all limnoterrestrial heterotardigrades and many eutardigrades are bacteri-, herbi- and/or fungivorous, only with macrobiotids being mostly omnivorous. Apart from tardigrades, there are no predators within this body size range dwelling in habitats occupied by apochelans that would hunt for the same prey (i.e. mainly nematodes, rotifers and non-apochelan tardigrades; Bryndová et al. 2020). In other words, the ecological niche of a microscopic predator in moss and lichen habitats is occupied exclusively by apochelans. Moreover, taking into consideration that global biodiversity grows over geological time (Crame, 2001), it is even more likely than the ecological niche of a microscopic predator was unoccupied at the time of the diversification of the genus Milnesium uncovered in our analysis (ca. 160 My ago or even earlier). Assuming that Milnesium was carnivorous at the time of the observed lineage diversification, which is the most parsimonious scenario, our analysis also suggests that the genus is older than other tardigrades that feed on micrometazoans, such as the omnivorous Paramacrobiotus Guidetti, Schill, Bertolani, Dandekar, & Wolf, 2009 (162 My vs 138 My, respectively), further lending credence to this hypothesis. On the other hand, this hypothesis requires a bold assumption that carnivory evolved within a relatively short period of time and relatively late in the natural history of the apochelan lineage (if it appeared earlier, then the divergence should have occurred earlier too). Moreover, the other three apochelan genera (which are probably also carnivorous) must have diverged at a similar time as the ‘basal’ lineages of Milnesium . Given that no molecular data for Bergtrollus Dastych, 2011, LimmeniusHorning, Schuster & Grigarick, 1978 and Milnesioides Claxton, 1999 are available, this hypothesis cannot be currently tested. Similarly, the Gondwana breakdown hypothesis remains a speculation and may require fossil evidence to be verified, meaning that it is likely to remain untested for a long time.