Pinpointing the microbiota of tardigrades: What is really there?

Microbiota are considered significant in the biology of tardigrades, yet their diversity and distribution remain largely unexplored. This is partly due to the methodological challenges associated with studying the microbiota of small organisms that inhabit microbe-rich environments. In our study, we characterized the microbiota of 31 species of cultured tardigrades using 16S rRNA amplicon sequencing. We employed various sample preparation strategies and multiple types of controls and estimated the number of microbes in samples using synthetic DNA spike-ins. We also reanalysed data from previous tardigrade microbiome studies. Our findings suggest that the microbial communities of cultured tardigrades are predominantly composed of bacterial genotypes originating from food, medium, or reagents. Despite numerous experiments, we found it challenging to identify strains that were enriched in certain tardigrades, which would have indicated likely symbiotic associations. Putative tardigrade-associated microbes rarely constituted more than 20% of the datasets, although some matched symbionts identified in other studies. We also uncovered serious contamination issues in previous tardigrade microbiome studies, casting doubt on some of their conclusions. We concluded that tardigrades are not universally dependent on specialized microbes. Our work underscores the need for rigorous safeguards in studies of the microbiota of microscopic organisms and serves as a cautionary tale for studies involving samples with low microbiome abundance.

"Everything is not everywhere": Time-calibrated phylogeography of the genus Milnesium (Tardigrada).

There is ample evidence that macroscopic animals form geographic clusters termed as zoogeographic realms, whereas distributions of species of microscopic animals are still poorly understood. The common view has been that micrometazoans, thanks to their putatively excellent dispersal abilities, are subject to the "Everything is everywhere but environment selects" hypothesis (EiE). One of such groups, <1 mm in length, are limnoterrestrial water bears (Tardigrada), which can additionally enter cryptobiosis that should further enhance their potential for long distance dispersion (e.g., by wind). However, an increasing number of studies, including the most recent phylogeny of the eutardigrade genus Milnesium, seem to question the general applicability of the EiE hypothesis to tardigrade species. Nevertheless, all Milnesium phylogenies published to date were based on a limited number of populations, which are likely to falsely suggest limited geographic ranges. Thus, in order to test the EiE hypothesis more confidently, we considerably enlarged the Milnesium data set both taxonomically and geographically, and analysed it in tandem with climate type and reproductive mode. Additionally, we time-calibrated our phylogeny to align it with major geological events. Our results show that, although cases of long distance dispersal are present, they seem to be rare and mostly ancient. Overall, Milnesium species are restricted to single zoogeographic realms, which suggests that these tardigrades have limited dispersal abilities. Finally, our results also suggest that the breakdown of Gondwana may have influenced the evolutionary history of Milnesium. In conclusion, phylogenetic relationships within the genus seem to be determined mainly by paleogeography.

What If Multiple Claw Configurations Are Present in A Sample? A Case Study with the Description of Milnesium pseudotardigradum sp. nov. (Tardigrada) with Unique Developmental Variability.

The tardigrade fauna of Iceland has been a subject of studies since mid XX century. So far, only a single species of the genus Milnesium has been reported from the island, M. tardigradum, which at the time was assumed to have a cosmopolitan distribution. However, the record comes from before the redescription of M. tardigradum, thus the validity of the Icelandic report is questionable. Some species of Milnesium are characterised by developmental variability, which is most pronounced in the morphology of secondary branches of claws, exhibited by shifts in the number of spurs. In this contribution, we present a case study in which multiple claw configurations (CC) were found in a single lichen sample from Iceland, indicating the presence of more than one species and/or ontogenetic variability. To elucidate this puzzle, we utilised a range of integrative tools, including detailed morphology, morphometry, barcoding and development tracking. We present the workflow, which enabled us to collect the data for each species/morphotype. In result, we revealed the presence of three species, two characterised by ontogenetic CC change (M. variefidum and a new species) and one with a stable CC (a new species). Here, we describe one of these new species, M. pseudotardigradum, which is extremely similar to M. tardigradum, but can be phenotypically differentiated by a unique, double CC change pattern.