Deceptive conservatism of claws : distinct phyletic lineages concealed within Isohypsibioidea (Eutardigrada) revealed by molecular and morphological evidence

Isohypsibioidea are most likely the most basally branching evolutionary lineage of eutardigrades. Despite being second largest eutardigrade order, phylogenetic relationships and systematics within this group remain largely unresolved. Broad taxon sampling, especially within one of the most speciose tardigrade genera, Isohypsibius Thulin, 1928, and application of both comparative morphological methods (light contrast and scanning electron microscopy imaging of external morphology and buccal apparatuses) and phylogenetic framework (18S + 28S rRNA sequences) resulted in the most comprehensive study devoted to this order so far. Two new families are erected from the currently recognised family Isohypsibiidae: Doryphoribiidae fam. nov., comprising all aquatic isohypsibioids and some terrestrial isohypsibioid taxa equipped with the ventral lamina; and Halobiotidae fam. nov., secondarily marine eutardigrades with unique adaptations to sea environment. We also split Isohypsibius into four genera to accommodate phylogenetic, morphological and ecological variation within the genus: terrestrial Isohypsibius s.s. (Isohypsibiidae), with smooth or sculptured cuticle but without gibbosities; terrestrial Dianea gen. nov. (Isohypsibiidae), with small and pointy gibbosities; terrestrial Ursulinius gen. nov. (Isohypsibiidae), with large and rounded gibbosities; and aquatic Grevenius gen. nov. (Doryphoribiidae fam. nov.), typically with rough cuticle and claws with branches of very similar heigths. Claw morphology is reviewed and, for the first time, shown to encompass a number of morphotypes that correlate with clades recovered in the molecular analysis. The anatomy of pharynx and cuticle are also shown to be of high value in distinguishing supraspecific taxa in Isohypsibioidea. Taxonomy of all isohypsibioid families and genera is discussed, with special emphasis on the newly erected entities. Finally, a dychotomous diagnostic key to all currently recognised isohypsibioid families and genera is provided

Experimental taxonomy exposes ontogenetic variability and elucidates the taxonomic value of claw configuration in Milnesium Doyère, 1840 (Tardigrada : Eutardigrada : Apochela)

In 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

An integrative redescription of Hypsibius dujardini (Doyère, 1840), the nominal taxon for Hypsibioidea (Tardigrada : Eutardigrada)

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 19^{th} 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

Redescription of Milnesium alpigenum Ehrenberg, 1853 (Tardigrada: Apochela) and a description of Milnesium inceptum sp. nov., a tardigrade laboratory model

Intra- and interspecific variability, being at the very core of alpha taxonomy, has been a long-standing topic of debate among tardigrade taxonomists. Early studies tended to assume that tardigrades exhibit wide intraspecific variation. However, with more careful morphological studies, especially those incorporating molecular tools that allow for an independent verification of species identifications based on phenotypic traits, we now recognise that ranges of tardigrade intraspecific variability are narrower, and that differences between species may be more subtle than previously assumed. The taxonomic history of the genus Milnesium, and more specifically that of the nominal species, M. tardigradum described by Doyère in 1840, is a good illustration of the evolution of views on intraspecific variability in tardigrades. The assumption of wide intraspecific variability in claw morphology led Marcus (1928) to synonymise two species with different claw configurations, M. alpigenum and M. quadrifidum, with M. tardigradum. Currently claw configuration is recognised as one of the key diagnostic traits in the genus Milnesium, and the two species suppressed by Marcus have recently been suggested to be valid. In this study, we clarify the taxonomic status of M. alpigenum, a species that for nearly a century was considered invalid. We redescribe M. alpigenum, using a population collected from the locus typicus, by the means of integrative taxonomy, i.e. including light microscopy, scanning electron microscopy, ontogenetic observations, and genetic barcoding. Moreover, the redescription of M. alpigenum allowed us to verify the uncertain taxonomic status of two popular laboratory models that were originally considered to be M. tardigradum; though one was recently reidentified as M. cf. alpigenum. Our analysis showed that both laboratory strains, despite being morphologically and morphometrically nearly identical to M. alpigenum, in fact represent a new species, M. inceptum sp. nov. The two species, being disnguishable only by statistical morphometry and/or DNA sequences, are the first example of pseudocryptic species in tardigrades

Morfometria rodzaju Milnesium DOYÈRE, 1840 (Tardigrada: Eutardigrada: Milnesiidae) – podejście eksperymentalne

Wraz ze wzrastającą liczbą gatunków należących do rodzaju Milnesium (DOYÈRE, 1840), z których część została opisana wyłącznie na podstawie cech morfometrycznych, standaryzacja morfometrii w tym rodzaju nigdy nie była tak istotna. Ponieważ pomiary tuby bukalnej są jednymi z kluczowych cech używanych do rozróżniania gatunków, oraz dlatego, że niektórzy autorzy sugerowali, że jej wymiary mogą zależeć od sposobu preparatyki, zdecydowałem się by zbadać tę kwestię eksperymentalnie. Użyłem klonalnego szczepu Milnesium cf. alpigenum, zarówno osobników juwenilnych (o długości tuby bukalnej ok. 25 μm) jak i dorosłych samic (z tubą bukalną o długości ok. 50 μm). Podzielono je na dziesięć zabiegów eksperymentalnych, wraz ze wzrastającym naciskiem na szkiełko nakrywkowe, oraz zabiegami polegającymi na temperaturowym uśmierceniu osobników (30 min w temperaturze 60 °C) i/lub naciskaniu na szkiełko nakrywkowe (aż osobnik będzie w pełni rozprostowany). W każdym zabiegu minimum 15 niesporczaków z obu stadiów zostało umieszczonych na preparatach. Po wyschnięciu preparatów zostały one polakierowane a tuby bukalne zostały zmierzone pod mikroskopem kontrastowo-fazowym. Drugim celem eksperymentu było ustalenie sposobu preparacji Milnesium cf. alpigenum, który pozwalałby na pełne rozciągnięcie osobnika, przy równoczesnym nie naruszeniu szerokości tuby bukalnej. By osiągnąć ten cel posłużono się metodami statystycznymi – testami ANOVA i post hoc Tukey’a.With a constantly increasing number of Milnesium (DOYÈRE, 1840) species and with many newly described species being established solely on the basis of morphometric traits, the accuracy and standardisation of morphometrics in the genus has never been so important. Given that the buccal tube dimensions are one of the key morphometric traits used for species differentiation and identification and because in the past some researchers have hypothesised that pressure exerted by the cover slip may affect the measurements of the tube, I decided to address this issue experimentally using a clonal laboratory strain of Milnesium cf. alpigenum (Ehrenberg, 1853). In this experiment, I have subjected both juveniles (with buccal tubes ca. 20 µm in length) and mature, fourth instar females (with ca. 40 µm long buccal tubes ) to ten experimental regimes of increasing pressure exerted by the cover slip and temperature killing animals (30 min exposure to 60 °C) and/or pressing (cover slip gently pressed until the specimen is stretched). In each of the regimes at least 15 individuals of each of the two instars were mounted. After the slides have dried, they were sealed with nail polish and then the buccal tubes were measured under a phase contrast light microscope. The second goal of the experiment was to identify the most optimal preparation technique that would allow to obtain fully stretched but at the same time intact Milnesium specimens mounted on microscope slides. To achieve this, we employed a statistical analyses – ANOVAs and post hoc Tukey’s tests

Novel integrative data for two Milnesium Doyère, 1840 (Tardigrada: Apochela) species from Central Asia

Tardigrada are a phylum of microscopic animals inhabiting a variety of ecosystems, both aquatic and terrestrial, being recognised for their remarkable abilities to withstand tough environmental conditions. The order Apochela groups exclusively carnivorous species, with the vast majority representing the genus Milnesium Doyère, 1840. Representatives of this genus are characterised by simplified morphology, therefore possessing an extremely limited set of taxonomically meaningful morphological traits. Nevertheless, the taxonomy of Milnesium is mostly based on classical data: observations and measurements in light microscopy with the majority of descriptions lacking integrative data, most importantly DNA barcodes, but also scanning electron microscopy photographs and developmental variability analysis. Hence, re-descriptions that include novel integrative data are urgently needed. In this contribution, we provide new taxonomic data for two species described from Central Asia, Milnesium almatyense (a single population) and Milnesium reductum Tumanov, 2006 (five populations): morphometrics, DNA barcodes, SEM observations and description of developmental variability. As a result, we amend the description of both species and reveal phylogenetic relationships of those species and other sequenced congeners. The integrative data confirm the validity of the two species and include them in the growing set of Milnesium species associated with DNA sequences

Reaching the monophyly : re-evaluation of the enigmatic species Tenuibiotus hyperonyx (Maucci, 1983) and the genus Tenuibiotus (Eutardigrada)

Revisions and redescriptions of taxa described in the past and that are now categorized as insufficiently diagnosed often play a crucial role in making further progress in modern taxonomy in many groups of organisms. Here we revised an enigmatic tardigrade species Tenuibiotus hyperonyx (Maucci, 1983) based on the newly discovered topotypic population from the Italian Alps. We performed an integrative analysis of morphological and genetic data in order to present an upgraded species description and elucidate its phylogenetic position. Our results enabled us to confidently place T.hyperonyx within the family Richtersiusidae, as a member of the genus Diaforobiotus. This change, together with a re-assessment of microphotographs of the Tenuibiotus willardi (Pilato, 1977) and Tenuibiotus bozhkae Pilato, Kiosya, Lisi, Inshina & Biserov, 2011 types, led to the discussion on species composition with narrative taxa amendments for the taxonomic parties involved in the proposed alteration