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.

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 acute toxicity of ammonia on Thulinius ruffoi (Bertolani, 1981), a eutardigrade isolated from a small waste water treatment plant (WWTP) in Poland, was estimated. Our results show that no active individuals survived a 24 h exposure to solutions equal to or higher than 125 mg/L of total ammonia nitrogen (NH3–N + NH4+–N), which, under the conditions in our experiment, was equivalent to 1.17 mg/L of un-ionised ammonia (NH3). The LC50 concentration of total ammonia nitrogen was equal to 52 mg/L (or 0.65 mg/L un-ionised ammonia). Given that the norms for the concentration of ammonia in treated waters leaving WWTPs are usually several times lower than the LC50 for T. ruffoi, this species does not seem to be a good bioindicator candidate for WWTPs. In this paper we also note that various ecotoxicological studies use different methodological approaches and we suggest that a more uniform methodology may aid interspecific comparisons of LC50 values.

The maintenance of males and outcrossing is widespread, despite considerable costs of males. By enabling recombination between distinct genotypes, outcrossing may be advantageous during adaptation to novel environments and if so, it should be selected for under environmental challenge. However, a given environmental change may influence fitness of male, female, and hermaphrodite or asexual individuals differently, and hence the relationship between reproductive system and dynamics of adaptation to novel conditions may not be driven solely by the level of outcrossing and recombination. This has important implications for studies investigating the evolution of reproductive modes in the context of environmental changes, and for the extent to which their findings can be generalized. Here, we use Caenorhabditis elegans—a free-living nematode species in which hermaphrodites (capable of selfing but not cross-fertilizing each other) coexist with males (capable of fertilizing hermaphrodites)—to investigate the response of wild type as well as obligatorily outcrossing and obligatorily selfing lines to stressfully increased ambient temperature. We found that thermal stress affects fitness of outcrossers much more drastically than that of selfers. This shows that apart from the potential for recombination, the selective pressures imposed by the same environmental change can differ between populations expressing different reproductive systems and affect their adaptive potential.

Radical shifts in reproductive systems result in radical changes in selective pressures acting on reproductive traits. Nematode Caenorhabditis elegans constitutes one of rare model systems where such shifts can be experimentally induced, providing an opportunity for studying the evolution of reproductive phenotypes in real time. Evolutionary history of predominantly selfing reproduction in has led to degeneration of traits involved outcrossing, making it inefficient. Here, we introduced obligatory outcrossing into isogenic lines of C. elegans and allowed replicate populations to evolve under the new reproductive system. We predicted that they should evolve higher outcrossing efficiency, leading to increased fitness relative to unevolved ancestors. To test this prediction, we assayed fitness of both ancestral and evolved outcrossing populations. To control for the potentially confounding effect of adaptation to laboratory conditions, we also assayed populations with wild-type (selfing) reproductive system. In five experimental blocks, we measured competitive fitness of 12 evolved populations (6 outcrossing, 6 selfing) after ca. 95 generations of evolution, along with their respective ancestors. On average, we found that fitness increased by 0.72 SD (± 0.3 CI) in outcrossing and by 0.52 (± 0.35 CI) in selfing populations, suggesting further adaptation to laboratory conditions in both types. Contrary to predictions, fitness increase was not significantly higher in outcrossing populations, suggesting no detectable adaptation to the changed reproductive system. Importantly, the results for individual populations varied strongly between experimental blocks, in some cases even differing in effect direction. This emphasises the importance of experimental replication in avoiding reporting false findings.