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Molecular Applied to Invertebrate Evolution. Systematics on Tardigrada

Final Report Summary - MAIE. ST (Molecular Applied to Invertebrate Evolution. Systematics on Tardigrada)

The main objective of the proposal presented was to progress knowledge on the evolution of Tardigrada, using all the information we would be able to generate: morphological, molecular, biological, etc. Once we would have a reasonably stable phylogeny, we would study the evolution of different characteristics in tardigrades e.g. reproductive modes, their distribution through different habitats, claws and buccopharyngeal apparatus morphology, substances from cryptobiosis and cryptobiosis as a process itself, etc. These results would be useful both in invertebrate and within Tardigrada evolution, and in biomedicine (within the evolutionary biomedicine framework). One of the main objectives was to include more taxa and genes (mainly those used in other invertebrate analyses, so our results could be used in other invertebrate evolution studies) in phylogenetic analyses. Besides the above, we have tried to study and analyse different morphological characters (examining Tardigrada collection from the Museum of Zoology at the University of Copenhagen), to complete previous information, so that complementary information will be available for further analyses. Finally, we would perform phylogenetic comparative analyses to study the evolution of certain interesting characters within tardigrades.
Molecularly, we have advanced in protocols from its more basic steps to primers useful for tardigrades, and we have settled the bases of general DNA protocols for tardigrades in accordance with other Tardigrada group working on molecular, Prof. R. Bertolani´s team. Moreover, we have increased greatly in taxa and genes (majority of 28S RNA information available at present days) for Eutardigrada analyses comparing with what was published either in journals or in GenBank. A total of 153 sequences have been obtained (DNA extraction, DNA amplifications of specific fragments, cleaning those amplifications and sequencing them), 93 from class Eutardigrada and 60 from class Heterotardigrada, belonging to 45 species (27 Eutardigrada and 18 Heterotardigrada) and 88 specimens (40 were eutardigrades and 48 heterotardigrades). For each specimen I have sequenced a 700 bp fragment of the 18S RNA and a 1500 bp fragment of the 28S RNA. In the analyses about Eutardigrada genera, 15 out of 27 species sequenced have not been sequenced before: Doryphoribius macrodon (Greenland), D. zyxiglobus (Australia), Hypsibius dujardini (Greenland), Ramazzottius cataphractus (Greenland), Diphascon prorsirostre (Denmark), Dactylobiotus ambiguus and D. octavi (Greenland), Murrayon pullari (Greenland), Adorybiotus granulatus (Greenland), M. echinogenitus (Faroe Island), M. grandis (Greenland), M. harsmworthi (Faroe Island), M. islandicus (Greenland), M. liviae (Chile), and Milnesium eurystomum (Spain). Species sequenced before for 18S RNA but for which we have included geographical variability are: Ramazzottius oberhaeuseri (from Sweden and Greenland), Milnesium tardigradum (Greenland, Disko Island and Norway), and Isohypsibius prosostomus (Egypt and Denmark). For majority of Eutardigrada sequences I have sequenced 28S RNA information not available before. There are 11 out of 18 Heterotardigrada species not sequenced before, and are: Bryochoerus intermedius (Greenland), Bryodelphax aaseae n. sp. (Easter Island, Chile), Proechiniscus hanneae (Disko Island, Greenland), Pseudechiniscus novaezelindae (Australia), P. suillus (Norway), Echiniscus bigranulatus (Chile), E. blumi (Chile and Greenland), E. merokensis suecica (Greenland), E. oihonnae (Normay), E. spiniger (Sweden), and E. wendti (Greenland). New, universal and specifically designed, primers for both nuclear genetic markers (18S RNA and 28S RNA) have been tried successfully. Almost all 28S RNA information I have created is new in GenBank data base. Sequences have been check and are now ready in a matrix for phylogenetic analyses.
Morphological analyses of specimens from the Prof. R. M. Kristensen collection and identification of individuals for subsequent DNA extraction have required the use of an optical microscope. I have had the opportunity to study species from three families from class Eutardigrada I have not observed before (Calohypsibius ornatus, from family Calohypsibiidae; Bertolanius from Amphibolidae; and Eohypsibius and Microhypsibius from Microhypsibiidae), analysing diverse morphological structures, making digital photographies of them, and recording them in a data base. Besides, I have studied many genera from Eutardigrada and Heterotardigrada I had not observed before: Pseudobiotus, Richtersius and Xerobiotus from class Eutardigrada, Echiniscoides, Halobiotus, Briodelphax, Bryochoerus, Proechiniscus, and Testechiniscus from class Heterotardigrada, and several species from genera Echiniscus and Pseudechiniscus (Echiniscidae, Echiniscoidea, Heterotardigrada). Besides, a study of tardigrades from Morocco belonging to new material, donated by Drs. J. Hortal Muñóz and Ana Santos from the Imperial College of London (U.K.) have required to fixed in Carnoy and mount in Faure liquid for optical microscope preparations: 108 heterotardigrades, 356 eutardigrades and 58 eggs, now deposited in the invertebrate collection of the Museum of Zoology at the University of Copenhagen. These specimens have been observed and identified in a first round, but a second round for all of them and more photos will be recorded. Revisions of Morocco material from Prof. R. M. Kristensen collection and comparisons with new material are final steps to this task.