Key words: barcoding, diagnostic key, development tracking, in vitro culture, M. berladnicorum, M. variefidum sp. nov. AbstractIn this paper we describe a new apochelan species, Milnesium variefidum sp. nov. from Scotland and provide novel morphological and molecular data for Milnesium berladnicorum Ciobanu et al., 2014. The new species differs from the most similar M. berladnicorum by the presence of developmental dimorphism in claw configuration, absent or weakly developed cuticular bars under claws I-III, a different arrangement of cuticular pseudoplates, and by differences in the sequences of three nuclear DNA fragments: 18S rRNA (p-distance: 0.6%), 28S rRNA (2.0%), ITS-2 (9.3%), and on mitochondrial gene COI (12.4%). Although ontogenetic claw configuration change was suspected to occur in some Milnesium species, we are the first to document it through the combined use of traditional, molecular and experimental methodologies. We discuss the implications of the observed phenomenon for the taxonomy of the genus and propose a new diagnostic key to all Milnesium species described up to the end of 2015. We also review other traits used for species differentiation in the genus and offer recommendations to improve the quality of future descriptions as well as suggest a need for integrative redescriptions of the known species. Finally, we propose to suppress M. dujiangensis and M. tardigradum trispinosum and suggest that M. alpigenum and M. quadrifidum are valid species that require thorough redescriptions.

A laboratory strain identified as “Hypsibius dujardini” is one of the best studied tardigrade strains: it is widely used as a model organism in a variety of research projects, ranging from developmental and evolutionary biology through physiology and anatomy to astrobiology. Hypsibius dujardini, originally described from the Île-de-France by Doyère in the first half of the 19th century, is now the nominal species for the superfamily Hypsibioidea. The species was traditionally considered cosmopolitan despite the fact that insufficient, old and sometimes contradictory descriptions and records prevented adequate delineations of similar Hypsibius species. As a consequence, H. dujardini appeared to occur globally, from Norway to Samoa. In this paper, we provide the first integrated taxonomic redescription of H. dujardini. In addition to classic imaging by light microscopy and a comprehensive morphometric dataset, we present scanning electron photomicrographs, and DNA sequences for three nuclear markers (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial marker (COI) that are characterised by various mutation rates. The results of our study reveal that a commercially available strain that is maintained in many laboratories throughout the world, and assumed to represent H. dujardini sensu stricto, represents, in fact, a new species: H. exemplaris sp. nov. Redescribing the nominal taxon for Hypsibiidae, we also redefine the family and amend the definitions of the subfamily Hypsibiinae and the genus Hypsibius. Moreover, we transfer H. arcticus (Murray, 1907) and Hypsibius conifer Mihelčič, 1938 to the genus Ramazzottius since the species exhibit claws and eggs of the Ramazzottius type. Finally, we designate H. fuhrmanni as subjectively invalid because the extremely poor description precludes identifying neotype material.

Despite advances in molecular systematics, the taxonomy of tardigrades still depends largely on morphological and morphometric traits. The fact that the variability of any biological trait is determined by the interaction between genetics and environment prompts a very fundamental question: is it possible for tardigrades of the same genotype, but originating from various habitats that differ in environmental conditions, to have phenotypes so different that they would be erroneously classified as different species taxa by means of classical taxonomy? Here, we present the results of a broad and fully controlled laboratory experiment in which we investigated the phenotypic plasticity of a number of traits that are traditionally considered to be taxonomically important. In order to achieve this, we have cultured six tardigrade species belonging to four eutardigrade families (Milnesiidae, Hypsibiidae, Isohypsibiidae, and Macrobiotidae) under five experimental regimes, reflecting key environmental factors that are likely to vary in natural habitats (i.e. temperature and food availability). We then measured a number of key taxonomic traits and compared their dimensions between the treatments. Over two years of experimentation we have obtained more than 28 000 morphometric measurements for over 2300 individuals. Such an extensive data set allowed us to test some of the fundamental assumptions of classic tardigrade taxonomy. We found that in the five parachelan species analysed, the great majority of both absolute and relative traits differed significantly between the treatments, whereas there were no significant differences in the apochelan species. Overall, tardigrades grew largest under the low-temperature treatment, whereas the smallest specimens were observed under high-temperature and low-food regimes. However, the prevalent statistical significance resulted mainly from the considerable statistical power of our analyses, and not from effect sizes, which varied mostly between low and moderate. In other words, the differences, although consistent, were minor in terms of taxonomical significance, and probably would not be considered by classic taxonomists as sufficient to designate animals from different treatments as separate taxa.

Intraspecific variability of tardigrades is a poorly investigated field. The great majority of tardigrade species were described using single populations, sometimes even single individuals. Moreover, intraspecific variability between multiple, integratively verified populations has never been described for any tardigrade species. In this study, we analyzed morphological, morphometric, and genetic variability of nine European populations of Milnesium tardigradum, one of the first described tardigrade species that until recently was thought to be cosmopolitan. We found that the nine populations of M. tardigradum exhibited low to moderate diversity in the nuclear ITS‐2 marker (0.2%–4.0%, 1.1% on average) and low to high divergence in the mitochondrial COI fragment (0.2%–11.4%, 4.4% on average). Despite the considerable variability in COI, all M. tardigradum populations exhibited mostly uniform morphology and morphometry. Moreover, we analyzed ontogenetic variability in claw and lamellae configuration under scanning electron microscopy, which resulted in the description of novel taxonomically important traits for M. tardigradum. The phylogenetic analysis unambiguously showed that all GenBank sequences predating the redescription of M. tardigradum and labeled as “M. tardigradum” or “M. cf. tardigradum” represent other species. Thus, the neotype population from Germany and the additional eight European populations analyzed in this study are the only confirmed records of M. tardigradum to date. Finally, in order to clarify ontogenetic nomenclature, we propose distinct names for the first and second immature instars in the order Apochela (hatchling and juvenile, respectively) and concise definitions of time and direction of ontogenetic changes in claw configuration (i.e., early/late and positive/negative change).

Tardigrade taxonomy is based largely on classical methodology, with morphological and morphometric traits being most often the sole basis for species delineation and identification. However, despite over 1200 described tardigrade species, so far there have been no studies estimating the sample size that would allow confident interpretation of dimensions of taxonomically important characters. We defined such optimal sample size as a minimal number of structures that need to be measured in order to result in a mean and a range that are not significantly different from the global (population) values. We estimated the optimal sample size by employing a randomized sampling approach to an extensive data set of more than 28 000 morphometric measurements for over 2300 individuals of six tardigrade species representing four major families (Milnesiidae, Hypsibiidae, Isohypsibiidae and Macrobiotidae) of the two eutardigrade orders, Apochela and Parachela. We found that the optimal sample size for the accurate estimation of trait means varied between 6 and 40, with 19 measurements being the overall average. In the case of trait ranges, sample size was much higher (26-413; 130 on average). Given that the range can be covered by measuring the smallest and largest specimens, we suggest that for accurate mean estimation at least 20 measurements ought to be taken, although 30 should be aimed at. We discuss our findings in light of the current practice in tardigrade taxonomy and provide suggestions that could improve the quality of tardigrade species descriptions.

The genus Echiniscus C.A.S. Schultze, 1840, one of the earliest established and speciose tardigrade genera, has been hypothesized to be polyphyletic. Moreover, the presence or absence of trunk appendages, the type of cuticular sculpturing and body colour have been argued to hold taxonomic significance at the genus level in Echiniscus‐line taxa. Here, by combining morphological and genetic analyses, we demonstrate that the so‐called “arctomys group”, i.e. Echiniscus spp. lacking trunk appendages, comprises numerous separate evolutionary lineages within the family Echiniscidae. As a result, we erect five new echiniscid genera: Barbariagen. nov., presumably of Neotropical (Gondwanan) origin, previously classified as the Echiniscus bigranulatus group; the pantropical and subtropical Kristenseniscusgen. nov. (the tessellatus group), characterized by a peculiar subdivision of dorsal plates; Claxtoniagen. nov. (the wendti group), with large and evident endocuticular pillars in the form of polygons; Nebularmisgen. nov. (the reticulatus group), with an elusive dorsal sculpturing; and Viridiscusgen. nov. (the viridis group), with body colour ranging from light green through brownish to even almost black. Additionally, we briefly address appendaged Echiniscus s.s. and divide the genus into several groups based on dorsal plate sculpturing and suggest that these could also represent separate supraspecific entities.

Given the constantly growing number of described tardigrade species and the scarcity of taxonomically meaningful morphological characters that these microinvertebrates exhibit, in many cases morphometric traits are the primary or even the only means of classic species differentiation, especially between closely related taxa. However, the validity of comparisons is contingent on the assumption that the mounting does not significantly affect the dimensions of morphometric traits, meaning that specimens mounted on different microscope slides may be compared with confidence. Despite the importance of the accuracy of morphometric measurements in tardigrade taxonomy and a long-standing supposition that the pressure exerted by the coverslip on a tardigrade specimen mounted on a microscope slide may deform some of the taxonomically important morphometric traits, the effect of mounting on specimen quality has not yet been addressed conclusively. In this paper we experimentally tested nine mounting methods using a lab-bred clonal strain of Milnesium cf. alpigenum Ehrenberg, 1853. Among all known tardigrades, species of the genus Milnesium Doy ere, 1840 exhibit the widest buccal tubes, thus they provide the most sensitive gauge of deformation caused by coverslip pressure. Moreover, using equal-age clonal animals reared under uniform lab conditions allowed us to minimize background noise in the data. Our study showed that among the nine mounting methods tested, thermal inactivation of animals at 60°C for 30 min followed by briefly pressing the coverslip with an entomological pin produced specimens fixed optimally for morphometry and imaging (i.e. maximally stretched and levelled but not deformed).

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