Final Report Summary - DSM.GBPP.LT (Dispersal and Speciation in Micrometazoans. Geographic Barriers, Phylogeography and Phylogeny in Limnoterrestrial Tardigrades) The project proposed for the ERG (annex I) was focused on a main objective: to study tardigrade speciation processes, their dispersal and effect of geographic barriers. Lack of information about tardigrades at all levels has forced us to advance in several multidisciplinary fields from more basic to more complex questions. To achieve the final objective, we firstly had to fill morphological (biodiversity) information gaps since majority of areas studied were not explored before searching for tardigrades. A second step was generating molecular data of specimens among different geographic scales (e.g. between slopes of several mountain systems, among mountains, or within and among samples) based on previous species check list with their distributions produced. Genetic structure of those more widespread species (found previously in the biodiversity study) have to be analysed respect environmental, as well as historical and geographical (phylogeography) variables. Finally, the effect of geographic barriers (mountains) as well as efficiency of dispersal and levels of gene flow among tardigrade units (demes, populations and species) would be determined at different geographic scales, so we could understand how dispersal and genetic isolation (and differentiation) are acting on tardigrades.We have designed and carried out the collecting field work, sampling the three mountain systems proposed: 'Sistema Central', 'Montes de Toledo' and 'Serrania de Cuenca'. There, we have collected 94 terrestrial samples distributed in seven sampling points within each mountain, and five samples per sampling point (i.e. moss and lichen on rocks and tree trunks and leaf litters) that later were stored dried within paper envelops. In addition, we explored the neglected for tardigrades freshwater habitats, collecting 24 aquatic samples that were stored frozen from rivers located in the studied area. One half of each sample was studied under a stereoscope after its moisturising, and tardigrades isolated for their preparation in permanent slides. A total of 8871 specimens (1466 from class Heterotardigrada, 7405 from class Eutardigrada, and 170 eggs) have been isolated and fixed in Carnoy liquid (a fixative for tardigrade storing) before preparing them for optical microscope slides. Approximately 1000 slides have been needed to mount those 8871 tardigrades in Faure liquid as permanent preparations for optical microscope that will be deposited in the Invertebrates collection at the National Museum of Natural History (CSIC) once identified. A total of 1041 individuals have been identified, showing high diversity so far: 28 species belonging to 13 genera from the two classes (Heterotardigrada and Eutardigrada); which include four out of the five superfamilies, and five out of the nine families in Eutardigrada. A data base with 2582 photographies has been created.Milnesium tardigradum was one of the species selected for studying its genetic structure with the advantage that Milnesiums are recognisable even using a stereoscope. A total of 135 Milnesium specimens from 32 terrestrial samples (where Milnesium individuals were observed during their isolation for morphological purposes) were isolated for molecular studies, identified with the optical microscope and isolated individually in sterile tubes for their DNA extraction. A data base with a total of 3209 photographies of relevant structures has been created, which are considered hologenophores (photographies as morphological evidences that link those sequences and morphologies of the exactly individual that was extracted; at least part of this morphological evidences will be deposited in Morphobank, an internet data base with photographies linked to molecular studies). Additional 1754 tardigrades (1698 eutardigrades, 56 heterotardigrades and 25 eggs) found in those 32 samples were stored frozen for future molecular studies, once morphological identifications were finished. From those 135 Milnesium individuals, 13 were used for a study in collabouration with the team of tardigradologists from Modena University (Italy) about Milnesium genetic variability worldwide (18 localities worldwide), including three out of the 32 Spanish samples studied. For the Milnesium study worldwide, we have sequenced 305 sequences (fragments of 18S and 28S rRNA, COI -cytochrome oxidase I- and ITS2 -internal transcribed spacer 2-). For the project focused at the regional scale (Iberian mountain systems), we have sequenced same genetic markers mentioned above of 125 Milnesium specimens (a total of 479 sequences) from 21 localities in the three mountain systems. Due to results obtained with the Milnesium project worldwide, and conflict found between genotype and phenotype, we decided to collect another Milnesium, M. eurystomum (found in Spain during N. Guil's PhD), collecting during this expedition two individuals that were isolated and sequenced. Some Spanish specimens were cloned because of their variability within individual (the so-called intragenomic variation) for the ITS2 sequence (50 individuals were cloned, ca. 200 clones sequenced, tuning up all protocols for tardigrades).Biodiversity found so far is high, and expected diversity will be higher since the remaining samples to be identified belong to unexplored areas for tardigrades. Probably, we will find new record for species and genera not found before, and probably, families, genera and species not sequenced before. We anticipate to find high diversity locally, with important effect to environmental variables measured in macro-scale, but also determinant influence of novel variables such as slope of a mountain or new leaf litter types (such as cork litter). In this sense, we will publish at least two papers: one with the taxonomic check list of species found and their distributions and population variability, and another with the biodiversity patterns. A great advance will be done and a more compete knowledge about Iberian tardigrades could be provided for the scientific community. A new, more complete and geographically wider, biodiversity study will be performed, which will confirm or reject previous patterns found. Moreover, a deeper exploration about the use and patterns found with dynamics of trophic groups and their diversity could be explored. We have edited the first Tardigrada webpage in Spanish (see http://www.tardigrada.es online), which fills a gap since the existence only of webpages on Tardigrada in English. This media also serves as meeting point to resolve questions about tardigrades at all academic level plus general public.Milnesium worldwide project has revealed phylogenetically close related haplotypes with no morphological differentiation in localities separated by thousands of kilometres (e.g. New Zealand and Slovakia), but also haplotypes genetically remote (up to 20 % of genetic differentiation for COI sequences), sharing the same moss or lichen neither with no morphological differentiation. Besides, distribution of genetic variability with no geographic barriers has questioned dispersal capabilities and isolation for tardigrades. Moreover, these phylogenetic patterns found in tardigrades without geographic or morphological relationships, are relatively common in unicellular organisms, which open a new evolutionary frame of discussion respect the relative effect of historical events and ecology in organisms' evolution. Regionally (Iberian mountain systems), Milnesium showed great part of the genetic variability found worldwide. Evolutionary hypotheses, such as the 'everything is everywhere, but environment selects', although simplistic (since confront historical events and ecological episodes, when they are in fact interrelated), could be partially supported by our results, in terms of haplotypes geographically widely distributed, and with high local diversity respect the global one. For the first time in tardigrades, we have found intragenomic variation for the ITS2 in several Spanish individuals, which suggests that events such as hybridisation, reticulated evolution, or duplication due to asexual reproduction may influence tardigrade evolution. We also have found a conflict between phenotype and genotype regarding the commonly used morphological characteristics for taxonomic differentiation among Milnesium species (e.g. several buccopharyngeal apparatus measurements and shape). Phylogenetically and statistically morphological speciation need to be discarded, which probably would question the way Milnesium species has been recently established and re-described. Our ability to collect two different Milnesium species (M. tardigradum and M. eurystomum) has allowed us to establish accurately morphological and molecular species differentiation based on morphological and molecular information. In spite of their tiny size, as animals, tardigrades share a common evolutionary history with the rest of multicellular organisms, but due to that tiny size tardigrades share biological characteristics relevant to dispersal and speciation (e.g. latency stages that provides survival in extreme conditions in both terrestrial and extraterrestrial environments, effective passive dispersal over unlimited distances with high survival rates, or similar population dynamics). As a consequence, a new spectrum of possible evolutionary questions will be opened, where the limits between uni- and multicellularity are not as accentuated and worldwide connection is possible. This means that we will have a broader understanding of how organisms evolve, with a more general perspective, at least in evolutionary scenarios tested.