Somatic muscolature of Tardigrada: phylogenetic signal and metameric patterns

Although studies describing molecular-based phylogenies within tardigrades are now frequently being published, this is not the case for studies combining molecular and morphological characters. Tardigrade phylogeny is still based, from a morphological point of view, almost exclusively on chitinous structures and little attention has been given to detecting and using novel morphological data. Consequently, we analysed the musculature of seven tardigrade species belonging to the main phyletic lines by confocal laser scanning microscopy and compared these morphological results with new molecular analyses (18S+28S rRNA genes). Finally, we analysed all the data with a total evidence approach. A consilience in the phylogenetic relationships among orders and superfamilies of tardigrades was obtained among the evolutionary trees obtained from morphological, molecular and total evidence approaches. Comparative analysis on the musculature allowed the identification of serial homologies and repeated metameric patterns along the longitudinal animal body axis. A phenomenon of mosaic evolution was detected in musculature anatomy, as dorsal musculature was found to be highly modified with respect to the other body muscle groups, probably related to the evolution of dorsal cuticular plates. An understanding of tardigrade musculature anatomy will give fundamental information to understand the evolution of segmental pattern within Panarthropoda

Comparative analyses of the cuticular and muscular structures of the buccal-pharyngeal apparatus of tardigrades

In spite of the great importance of the tardigrade buccal pharyngeal apparatus in taxonomic and phylogenetic studies, it received little attention as regards its evolution and operating mechanism. To understand the relationships between form and function of the structures acting in the functioning of buccal-pharyngeal apparatus (i.e. cuticular structures, muscular fibers, pharynx), and to increase our knowledge on this apparatus, a comparative analyses using different investigation techniques was performed. The buccal-pharyngeal apparatuses of three species have been studied, Echiniscus trisetosus, Milnesium tardigradum e Paramacrobiotus richtersi, as representative of the two classes and of three orders of tardigrades. The cuticular structures of the buccal-pharyngeal apparatus have been analyzed form a morphological (light microscopy, scanning electron microscopy \u2013 SEM, and confocal laser scanner microscopy -CLSM) and chemical (energy dispersive X-ray spectroscopy) point of views. The musculature associated to the sclerified structures of the buccal-pharyngeal apparatus has been analyzed by CLSM to identify the muscular fibers and their relationships with the sclerified structures. The differences in the general anatomy of the buccal-pharyngeal apparatus among the three species were high, even though homologous structures were recognizable. The higher differences among species were found in the organization of the muscular system responsible of the stylet movements. The detailed analyses of the buccal-pharyngeal apparatus allowed a new interpretation of the organization of the stylet systems, and to understand the muscular system related to the feeding. The chemical analyses showed that the piercing stylets were formed by calcium, in form of CaCO3. Heterotardigrada were differentiated from Eutardigrada for the presence of high concentration CaCO3 encrustations in the buccal tube. Within Eutardigrada, Apochela differs from Parachela since they are characterized by the absence of CaCO3 in the buccal tube

Strutture cuticolari e muscolatura dell\u2019apparato bucco-faringeo dei tardigradi

L\u2019apparato bucco-faringeo dei tardigradi \ue8 composto da parti cuticolari sclerificate (compresi due stiletti perforanti ritenuti una delle apomorfie del phylum) e muscolatura. Con questo studio si \ue8 voluto sia comprendere al meglio il meccanismo di funzionamento di questo apparato che individuare nuovi caratteri per gli studi di tassonomia e filogenesi. L\u2019apparato bucco-faringeo di Echniscus trisetosus, Milnesium tardigradum e Paramacrobiotus richtersi (appartenenti a due classi e tre ordini del phylum) \ue8 stato studiato morfologicamente (microscopia ottica, microscopia elettronica a scansione) e chimicamente (spettroscopia a raggi X). E\u2019 stata inoltre analizzata l\u2019anatomia della muscolatura associata all\u2019apparato mediante microscopia confocale a scansione laser. Nelle tre specie le differenze nell\u2019anatomia dell\u2019apparato bucco-faringeo risultano notevoli , cos\uec come quelle relative all\u2019organizzazione dei fasci muscolari ad esso associati. L\u2019analisi chimica ha rivelato che gli stiletti sono costituiti da CaCO3 e che le due classi di tardigradi si differenziano per la presenza/assenza di CaCO3 sul tubo boccale. L\u2019analisi dettagliata delle strutture cuticolari dell\u2019apparato bucco-faringeo e della sua muscolatura ha consentito una nuova interpretazione dell\u2019organizzazione e del funzionamento del sistema di protrazione e retrazione degli stiletti. Sebbene nelle strutture sclerificate dei tre ordini siano riconoscibili diverse omologie, queste sono pi\uf9 difficilmente riscontrabili nella muscolatura

Dating tardigrade evolution and early terrestrialization events.

The tempo and mode of cryptobiosis evolution within tardigrades are still unknown, but it is clear that comprehending this process is the key to better understand evolutionary history and ecology of this phylum, and the process of animal terrestrialization, given the ubiquitous continental distribution of tardigrades. Our study was mainly aimed at timing tardigrade radiation and key events in tardigrade evolution. Given the existence of two lineages of continental tardigrades, i.e. Eutardigrada and Echiniscidae, we decided to implement a relaxed molecular clock based approach to attempt to derive a minimal time interval for tardigrade terrestrialization. Data from new and Genbank partial 18S and 28S rDNA sequences of 41 specimens, belonging to 31 species from 26 genera, representing all known tardigrade orders and superfamilies, were acquired. Phylogenetic inference was achieved using Bayesian mixture models that have allowed the most accurate estimates of evolutionary rates. The following molecular clock analyses were then based on 3 tardigrade calibration points derived from fossil records and improved by the addition of 5 new calibrations points pertaining to Arthropoda, Priapulida and Kinoryncha, emerged from paleontological studies. To clarify the evolutionary history of cryptobiosis, and to evaluate whether its origin might have played a role in the process of tardigrade terrestrialization, a maximum-likelihood based ancestral character state reconstruction was used. Molecular phylogeny analyses yielded a robustly supported and well-resolved evolutionary tree for all considered tardigrade taxa. Our results suggest the origin of the tardigrade stem group in the Ediacaran age (~620 Million years ago -Mya), with the two major extant tardigrade lineages (Eutardigrada and Heterotardigrada) split quite recently ~443 Mya. Ancestral character state reconstruction indicated a probability of ~30% for cryptobiosis to have been present in the last common ancestor of Heterotardigrada and Eutardigrada. Hence, it is most likely that this adaptive trait evolved twice independently within this phylum. Cryptobiosis most likely emerged in the time interval between 443 and 359 Mya, in the stem Eutardigrada lineage that existed for ~84 My. This is in accordance with available fossil evidence suggesting terrestrial ecosystems first flourished in the Silurian. The second independent origin (within Heterotardigrada) was within the Echiniscoidea clade and can be dated to an interval included between 238 and 141 Mya (Middle Triassic to early Cretaceous). In both cases, existence of a link between cryptobiosis and terrestrialization was clear. All the eutardigrade superfamilies originated within a short-time span of ~257-200 Mya, suggesting an early Mesozoic eutardigrade radiation. Moreover, within each parachelan superfamily a great mid Jurassic to Early Cretaceous process of diversification was recorded

Comparative analysis and phylogenetic implications of tardigrade musculature architectures.

Most knowledge on tardigrade musculature architecture dates back to the end of XIX century, and the beginning of XX century. It has been only in the last five years that a great deal of new information on tardigrade musculature system has become available, mainly thanks to the use of rhodamine-phalloidin staining of F-actin in combination with three-dimensional microscopical techniques such as confocal laser scanning microscopy (CLSM). In spite of all these information, only few and fragmentary evolutionary considerations on tardigrade musculature system have been done. This is probably due to the relatively low number of analyzed taxa, and to the difficulty in the comparisons of data that often have been obtained with different degree of accuracy, and are presented using different terminologies. In this study we increased the number of analyzed species, by studying CLSM the musculature architectures of 7 species representative of most tardigrade higher taxa: the heterotardigrades Batillipes bullacaudatus (Arthrotardigrada), and Echiniscus testudo (Echiniscoidea), and the eutardigrades Paramacrobiotus richtersi, Dactylobiotus parthenogeneticus (Parachela, Macrobiotoidea), Bertolanius volubilis (Parachela, Eohypsibioidea), Acutuncus antarcticus (Parachela, Hypsibioidea) and Milnesium tardigradum (Apochela, Milnesiidae). We were able to define all the muscular fibers associated with the body movement. The number of fibers and their organization changed among taxa, with heterotardigrades being the least complex. Muscular fibers have been schematically organized into three systems: dorsal, lateral, and ventral. The ventral system was the most conservative, showing a clear metameric pattern and only few differences among taxa, while the lateral system was the most derived and so precise homologies were not always well defined. Using these new morphological information and literature data on Halobiotus crispae (Parachela, Isohypsibioidea), it was possible to analyse the phylogenetic signal of the musculature system. Two matrixes were constructed: a morphological matrix of 94 characters based on musculature data, and a matrix for a total evidence analysis combining the previous data with molecular data (18S, 28S). Both matrixes have been analyzed in Bayesian and parsimony frameworks. The phylogenetic trees, obtained by both analyses using both matrixes, differ only for the position of Eohypsibioidea. Heterotardigrade taxa were the sister group of all Eutardigrada, within this last class, Apochela and Parachela were sister taxa; among parachelan superfamilies the Isohypsibioidea was the most basal, the Macrobiotoidea the most derived, while the Eohypsibioidea changed position according to the analyses. Our data demonstrated that musculature architecture can be used for phylogenetic purposes, but it is only applicable at higher taxonomic levels. Indeed, the musculature of Paramacrobiotus and Dactylobiotus, although belonging to different families of Macrobiotoidea, showed the same muscular organization

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

Seventy-nine specimens belonging to six of the nine current eutardigrade families have been considered in a wide and extensive study of the phylogeny of the largest class of Tardigrada (Eutardigrada). The molecular (18S rDNA) and morphological data partly support previous results. In particular, the applied integrative approach allowed us to find morphological synapomorphies, supporting the clusters here identified by molecular data and the previous taxa erected only on molecular basis. The class Eutardigrada has been confirmed and, within it, the orders Apochela and Parachela, the superfamilies Macrobiotoidea, Hypsibioidea and Isohypsibioidea, and all the families and subfamilies considered, even though in several cases with an emended diagnosis. In addition, new taxa have been erected: the superfamily Eohypsibioidea, the new families Hexapodibiidae and Isohypsibiidae, the new subfamily Pilatobiidae (Hypsibiidae) with the new genus Pilatobius, in addition to an upgrading to genus level of Diphascon and Adropion, to date considered subgenera of Diphascon. Our results demonstrate that while molecular analysis is an important tool for understanding phylogeny, an integrative approach using molecular and morphological data is necessary to fully elucidate evolutionary relationships

Two tardigrade species on board of the STS-134 space flight

The TARDIKISS project (Tardigrades in Space) investigated the physiological and molecular effects of space stresses on alive desiccated tardigrades, as representative of multicellular organisms. It has been a part of the BIOKIS (Biokon in Space), a set of multidisciplinary experiments of DAMA (DArk MAtter) mission on board of STS-134 space flight, the last of the shuttle Endeavour, in the frame of a joint between ASI and Italian Air Force. In TARDIKISS, experimentally desiccated (anhydrobiotic) specimens of Paramacrobiotus richtersi and Ramazzottius oberhaeuseri have been used. Both species have very good anhydrobiotic ability, but differ for several biological and ecological characters. The experiment units of TARDIKISS were hosted into the Biokon, a standard transportable container designed and manufactured by Kayser Italia. In May 2011, the Biokon containing the TARDIKISS experiment unit was integrated on Middeck Locker of ISS and had flown for 16 days at Low Earth Orbit. Two sample sets were used as controls: the former (Laboratory Control) was maintained in Modena laboratory for the duration of the flight, and the latter one (Temperature Control) was a post-flight control in which samples were exposed to the temperature profile experienced by tardigrades the days immediately before, during, and just after the flight mission. For both species, the flight animals did not show differences in survival with regards of Laboratory control and Temperature control animals. Only in R. oberhaeuseri differences have been recorded between Flight and Temperature Control samples. Live specimens of P. richtersi from Flight samples, Laboratory control and Temperature control samples have been reared in lab. Flight females laid eggs with normal shape; several eggs have been able to hatch, and newborns exhibited normal morphology, behaviour and capability to reproduce. Moreover, a comparative analyses of the antioxidant metabolism between Flight samples and Temperature control samples has been done. No differences have been evidenced between the two groups, with the exception of the reductase activity: significant differences in reductase activity between Flight and Temperature control were evidenced (p<0.05) in R. oberhaeuseri. These first results lead us to deduce that during the DAMA mission, microgravity and cosmic radiations did not significantly affect survival of flown tardigrades, confirming that tardigrades represent a useful animal tool for space research

Tardigrades on board of the STS-134 space flight

The joint ASI-AM DAMA mission supported a set of scientific experiments executed in short-duration microgravity on board the Space Shuttle docked to the International Space Station. BIOKIS payload (by Kayser Italia) took advantage of the last STS-134 Endeavour mission lasted 16 days. It hosted a multidisciplinary set of experiments in the field of biology and dosimetry. Among them, the TARDIKISS project investigated the responses of live desiccated tardigrades, multicellular heterotrophic organisms, under space stresses. In particular, specimens of Paramacrobiotus richtersi and Ramazzottius oberhaeuseri have been used. The experimental flight units were hosted into the Biokon, a standard transportable container. In addition, a Laboratory Control sample was maintained in Modena laboratory for the duration of the flight, while a post-flight Temperature Control sample was exposed to the temperature profile experienced by tardigrades during the mission. For both species, the Flight animals did not show differences in survival with regard to Laboratory and Temperature control animals. Only in R. oberhaeuseri differences have been recorded between Flight and Temperature Control samples. Specimens of P. richtersi from Flight, Laboratory and Temperature control samples have been reared in lab. Flight females laid eggs with normal shape; several eggs have been able to hatch, and newborns exhibited, when adult, normal capability to reproduce. The comparative analysis of the antioxidant metabolism between Flight and Temperature control samples did not evidenced significantly differences between the two groups. These data confirm that tardigrades represent a useful animal tool for space research. The authors thanks the Italian Space agency, Italian Air Force and Kayser Italia S.r.l

MicroRNA and phylogenomics congruently resolve the phylogenetic relationships of the Tardigrada within Ecdysozoa.

MicroRNA and phylogenomics congruently resolve the phylogenetic relationships of the Tardigrada within Ecdysozo

TARDIKISS: tardigrades in the mission STS-134, the last of the shuttle Endeavour.

The TARDIKISS project (Tardigrades in Space) investigates the physiological and molecular effects of space stresses on alive desiccated multicellular organisms, the tardigrades. It is a part of the BIOKIS (Biokon in Space), a set of multidisciplinary experiments of DAMA (DArk MAtter) mission on board of STS-134 space flight, the last of the shuttle Endeavour, in the frame of a joint between ASI and Italian Air Force. In TARDIKISS, experimentally desiccated (anhydrobiotic) specimens of Paramacrobiotus richtersi and Ramazzottius oberhaeuseri have been used. Both species have very good anhydrobiotic ability, but differ for several biological and ecological characters. Paramacrobiotus richtersi is carnivorous, soil-dwelling and white in colour, while R. oberhaeuseri is lichen-dwelling, herbivorous, and characterized by a brown/red pigmentation. The experiment units of TARDIKISS were hosted into the Biokon, a standard transportable container designed and manufactured by Kayser Italia. In May 2011, the Biokon containing the TARDIKISS experiment unit was integrated on Middeck Locker of ISS and had flown for 16 days. As control, ground experiments replicating the temperature profile recorded within the Biokon during the flight are currently running. To this day, we have completed the analysis on tardigrade survival after the space flight and obtained preliminary data on life cycle parameters. Both tardigrade species showed a very high survival (98.33% for P. richtersi and 90.0% for R. oberhaeuseri) and no significant differences were scored between flight or Earth tardigrades. After the flight, specimens of P. richtersi were reared in lab. Females laid eggs with normal shape; several eggs hatched with a short hatching time and newborns exhibited normal morphology and behaviour. In the next step, genomic DNA quality and oxidative metabolism of flown tardigrades vs ground control tardigrades will be analysed