High diversity in species, reproductive modes and distribution within the Paramacrobiotus richtersi complex (Eutardigrada, Macrobiotidae).

For many years, Paramacrobiotus richtersi was reported to consist of populations with different chromosome numbers and reproductive modes. To clarify the relationships among different populations, the type locality of the species (Clare Island, Ireland) and several Italian localities were sampled. Populations were investigated with an integrated approach, using morphological (LM, CLSM, SEM), morphometric, karyological, and molecular (18S rRNA, cox1 genes) data. Paramacrobiotus richtersi was redescribed and a neotype designed from the Irish bisexual population. Animals of all populations had very similar qualitative and quantitative characters, apart from the absence of males and the presence of triploidy in some of them, whereas some differences were recorded in the egg shell. All populations examined had the same 18S haplotype, while 21 haplotypes were found in the cox1 gene. In four cases, those qualitative characters were correlated with clear molecular (cox1) differences (genetic distance 14.6-21.8%). The integrative approach, which considered the morphological differences in the eggs, the reproductive biology and the wide genetic distances among putative species, led to the description of four new species (Paramacrobiotus arduus sp. n., Paramacrobiotus celsus sp. n., Paramacrobiotus depressus sp. n., Paramacrobiotus spatialis sp. n.) and two Unconfirmed Candidate Species (UCS) within the P. richtersi complex. Paramacrobiotus fairbanksi, the only ascertained parthenogenetic, triploid species, was redescribed and showed a wide distribution (Italy, Spain, Poland, Alaska), while the amphimictic species showed limited distributions. The difference in distribution between apomictic and amphimictic populations can be explained by the difference in the dispersal potentials associated with these two types of reproduction.

Distribution of Calcium and Chitin in the Tardigrade Feeding Apparatus in Relation to its Function and Morphology.

The cuticular portion of the tardigrade feeding apparatus is a complex structure that can be schematically divided into four parts: a buccal ring, a buccal tube, a stylet system (formed by two piercing stylets, each within a stylet coat, and two stylet supports), and the lining of a myoepithelial sucking pharynx. To better understand the function and evolution of the feeding apparatus, the morpho-functional traits and chemical composition of the structures forming the feeding apparatuses of eight different species of tardigrades were analyzed. These eight species are representative of almost all main phylogenetic lineages of the phylum. The calcium and chitin in the feeding apparatus were examined by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, energy dispersive X-ray spectroscopy, and Raman microspectroscopy (Raman). In all species, the feeding apparatus had been subjected to biomineralization due to CaCO3 encrustations organized in the crystalline form of aragonite. Aragonite and chitin are present in different concentrations in the feeding apparatus according to the structures and species considered. Generally, where the structures are rigid there is more aragonite than chitin, and vice versa. The buccal tube and piercing stylets are rich in calcium, with the piercing stylets apparently composed exclusively of aragonite. In eutardigrades, chitin is in higher concentration in the structures subject to higher mechanical stresses, such as the crests of the buccal crown and the condyles of the stylet furca.

Aquatic tardigrades in the Great Smoky Mountains National Park, North Carolina and Tennessee, U.S.A., with the description of a new species of Thulinius (Tardigrada, Isohypsibiidae).

As part of the All Taxa Biodiversity Inventory (http://www.dlia.org), an extensive survey of tardigrades has been conducted in the Great Smoky Mountains National Park (GSMNP) in Tennessee and North Carolina, U.S.A., by Bartels and Nelson. Freshwater tardigrades include three species in the aquatic genus Thulinius (Eutardigrada, Isohypsibiidae). A new species, Thulinius romanoi, described from stream sediment, is distinguished from all other congeners by having a sculptured cuticle. In addition, the presence of Thulinius augusti (Murray, 1907) was verified by combined morphological and molecular analysis, and nine specimens of a third species, Thulinius cf. saltursus, were also found. Thulinius augusti is a new record for the United States. Thulinius saltursus (Schuster, Toftner & Grigarick, 1978) was previously recorded in California and Ohio, but our specimens vary slightly in morphology. The list of tardigrades from streams in the GSMNP was updated to a total of 44 species, 22 of which were predominantly or exclusively aquatic.

Phylogeny of Eutardigrada: new molecular data and their morphological support lead to the identification of new evolutionary lineages.

An extensive study of the phylogeny of Eutardigrada, the largest class of Tardigrada, has been performed analyzing one hundred and forty sequences (eighty of which newly obtained) representative of one hundred and twenty-nine specimens belonging to all families (except Necopinatidae) of this class. The molecular (18S and 28S rRNA) results were compared with new and previous morphological data, allowing us to find new phylogenetic relationships, to identify new phylogenetic lineages, to erect new taxa for some lineages, and to find several morphological synapomorphies supporting the identified clusters. The class Eutardigrada has been confirmed and, within it, the orders Apochela and Parachela, the superfamilies Macrobiotoidea, Hypsibioidea, Isohypsibioidea, and Eohypsibioidea, and all the families and subfamilies considered, although with emended diagnoses in several cases. In addition, new taxa have been erected: the new subfamily Pilatobiinae (Hypsibiidae) with the new genus Pilatobius, as well as an upgrading of Diphascon and Adropion to genus level, previously considered subgenera of Diphascon. Our results demonstrate that while molecular analysis is an important tool for understanding phylogeny, an integrative and comparative approach using both molecular and morphological data is necessary to better elucidate evolutionary relationships.

Considerations on the taxonomy of the Phylum Tardigrada.

An analysis of the taxonomy of the Tardigrada is offered, based on the latest checklist version. A total of 1167 species from 113 genera were counted, but marine species are misrepresented on account of being understudied. Moreover, many poor descriptions and synonyms remain in this constantly growing list. We advocate more accurate future taxonomic work, aiming for an official list of species that better represents true values of biodiversity.

Integrative taxonomy allows the identification of synonymous species and the erection of a new genus of Echiniscidae (Tardigrada, Heterotardigrada).

The taxonomy of tardigrades is challenging as these animals demonstrate a limited number of useful morphological characters, therefore several species descriptions are supported by only minor differences. For example, Echiniscus oihonnae and Echiniscus multispinosus are separated exclusively by the absence or presence of dorsal spines at position Bd. Doubts were raised on the validity of these two species, which were often sampled together. Using an integrative approach, based on genetic and morphological investigations, we studied two new Portuguese populations, and compared these with archived collections. We have determined that the two species must be considered synonymous with Echiniscus oihonnae the senior synonym. Our study showed generally low genetic distances of cox1 gene (with a maximum of 4.1%), with specimens displaying both morphologies sharing the same haplotype, and revealed character Bd to be variable. Addition-ally, a more detailed morphological and phylogenetic study based on the 18S gene uncovered a new evolutionary line within the Echiniscidae, which justified the erection of Diploechiniscus gen. nov. The new genus is in a sister group relationship with Echiniscus and is, for the moment, composed of a single species.

Survival of freezing by hydrated tardigrades inhabiting terrestrial and freshwater habitats.

The seasonality and unpredictability of environmental conditions at high altitudes and latitudes govern the life cycle patterns of organisms, giving rise to stresses that cause death or development of specific adaptations. Ice formation is a major variable affecting the survival of both freshwater fauna and fauna inhabiting lichens, mosses and leaf litter. Tardigrades occupy a wide range of niches in marine, freshwater and terrestrial environments. The highest number of species is found in terrestrial habitats thanks to their ability to enter anhydrobiosis and cryobiosis. The cryobiotic ability of tardigrade species from polar regions is well known. Consequently, we focused our research on the ability to survive freezing in the active hydrated state using seven tardigrade species differing in phylogenetic position and collected at various altitudes and from different habitats in a temperate area. Specimens were cooled at different cooling rates (from 0.31° C min(-1) to 3.26° C min(-1)). Even though the final survival and the time required by animals to recover to active life were both inversely related to the cooling rate, highly significant interspecific differences were found. Species survival ability ranged from excellent to none. Species living in xeric habitats withstood freezing better than those living in hygrophilous habitats, while true limnic species did not exhibit any cryobiotic ability. The ability to withstand freezing seems linked to the anhydrobiotic ability. The differences in cryptobiotic performance among tardigrade species seem more influenced by selective pressures linked to local adaptation to habitat characteristics than by phylogenetic relationships.

Tardigrade Resistance to Space Effects: first results of experiments on the LIFE-TARSE mission on FOTON-M3 (September 2007).

The Tardigrade Resistance to Space Effects (TARSE) project, part of the mission LIFE on FOTON-M3, analyzed the effects of the space environment on desiccated and active tardigrades. Four experiments were conducted in which the eutardigrade Macrobiotus richtersi was used as a model species. Desiccated (in leaf litter or on paper) and hydrated tardigrades (fed or starved) were flown on FOTON-M3 for 12 days in September 2007, which, for the first time, allowed for a comparison of the effects of the space environment on desiccated and on active animals. In this paper, we report the experimental design of the TARSE project and data on tardigrade survival. In addition, data on survival, genomic DNA integrity, Hsp70 and Hsp90 expressions, antioxidant enzyme contents and activities, and life history traits were compared between hydrated starved tardigrades flown in space and those maintained on Earth as a control. Microgravity and radiation had no effect on survival or DNA integrity of active tardigrades. Hsp expressions between the animals in space and the control animals on Earth were similar. Spaceflight induced an increase of glutathione content and its related enzymatic activities. Catalase and superoxide dismutase decreased with spaceflight, and thiobarbituric acid reactive substances did not change. During the flight mission, tardigrades molted, and females laid eggs. Several eggs hatched, and the newborns exhibited normal morphology and behavior.

DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae).

Morphological and molecular studies on a tardigrade species have been carried out to verify the possibility of using a DNA barcoding approach for species identification in this phylum. Macrobiotus macrocalix Bertolani & Rebecchi, 1993 was chosen as the test species since it belongs to a group of species in which the taxonomy is quite problematic. Animals and eggs belonging to three Italian and one Swedish populations have been investigated. Both morphological and molecular analyses show that all the populations belong to the same species. The low genetic distances recorded among the studied populations (0.3-1.0%) and the high genetic distance (15.9-16.3%) between these populations and a closely related species confirm the possibility of identifying a specimen of this species by its cytochrome oxidase subunit I sequence. Data from other authors support our results indicating that DNA barcoding can be applied to tardigrades. With our protocols, we have obtained voucher specimens that enable us to show a correspondence between morphology and molecular data.

Diapause in tardigrades: a study of factors involved in encystment.

Stressful environmental conditions limit survival, growth and reproduction, or these conditions induce resting stages indicated as dormancy. Tardigrades represent one of the few animal phyla able to perform both forms of dormancy: quiescence and diapause. Different forms of cryptobiosis (quiescence) are widespread and well studied, while little attention has been devoted to the adaptive meaning of encystment (diapause). Our goal was to determine the environmental factors and token stimuli involved in the encystment process of tardigrades. The eutardigrade Amphibolus volubilis, a species able to produce two types of cyst (type 1 and type 2), was considered. Laboratory experiments and long-term studies on cyst dynamics of a natural population were conducted. Laboratory experiments demonstrated that active tardigrades collected in April produced mainly type 2 cysts, whereas animals collected in November produced mainly type 1 cysts, indicating that the different responses are functions of the physiological state at the time they were collected. The dynamics of the two types of cyst show opposite seasonal trends: type 2 cysts are present only during the warm season and type 1 cysts are present during the cold season. Temperature represents the environmental factor involved in induction, maintenance and termination of the cyst. We also obtained evidence that A. volubilis is able to perform both diapause and cryptobiosis, even overlapping the two phenomena. The induction phase of tardigrade encystment can be compared to the induction phase of insect diapause, also indicating an involvement of endogenous factors in tardigrade encystment. As in insect diapause, tardigrade encystment can be considered a diapausing state controlled by exogenous and endogenous stimuli.

Banding techniques on tardigrade chromosomes: the karyotype of Macrobiotus richtersi (Eutardigrada, Macrobiotidae).

This work represents the first attempt to define tardigrade chromosomes using banding techniques. Macrobiotus richtersi, a eutardigrade morphospecies with amphimictic diploid and thelytokous triploid cytotypes, was used as a model. Prime consideration was given to oocyte chromosomes because they are larger than those of spermatocytes and of mitotic chromosomes. With Giemsa staining, the chromatids of the 6 bivalents of the diploid cytotypes and those of the 17-18 univalents of the triploid cytotypes were very similar to each other and appeared rod- or flame-shaped. In the amphimictic strain, a chiasma was generally present in each bivalent at diplotene, whereas there were no chiasmata in the oocyte prophase chromosomes of the triploid strain. Both in diploid and triploid cytotypes, C-banding and fluorescence showed a heterochromatic centromeric band on the telomere of each chromosome oriented towards the spindle pole, indicating that all of them were acrocentric. Silver staining showed the presence of a NOR in only a pair of chromosomes, close to the centromeric C-banded site. NOR was particularly evident in the oocyte prophases. Other silver positive regions, corresponding to the kinetochore, were located on all other chromosomes on the telomeres towards the spindle pole.