BackgroundProtura is a group of tiny, primarily wingless hexapods living in soil habitats. Presently about 800 valid species are known. Diagnostic characters are very inconspicuous and difficult to recognize. Therefore taxonomic work constitutes an extraordinary challenge which requires special skills and experience. Aim of the present pilot project was to examine if DNA barcoding can be a useful additional approach for delimiting and determining proturan species.Methodology and Principal FindingsThe study was performed on 103 proturan specimens, collected primarily in Austria, with additional samples from China and Japan. The animals were examined with two markers, the DNA barcoding region of the mitochondrial COI gene and a fragment of the nuclear 28S rDNA (Divergent Domain 2 and 3). Due to the minuteness of Protura a modified non-destructive DNA-extraction method was used which enables subsequent species determination. Both markers separated the examined proturans into highly congruent well supported clusters. Species determination was performed without knowledge of the results of the molecular analyses. The investigated specimens comprise a total of 16 species belonging to 8 genera. Remarkably, morphological determination in all species exactly mirrors molecular clusters. The investigation revealed unusually huge genetic COI distances among the investigated proturans, both maximal intraspecific distances (0–21.3%), as well as maximal congeneric interspecifical distances (up to 44.7%).ConclusionsThe study clearly demonstrates that the tricky morphological taxonomy in Protura has a solid biological background and that accurate species delimitation is possible using both markers, COI and 28S rDNA. The fact that both molecular and morphological analyses can be performed on the same individual will be of great importance for the description of new species and offers a valuable new tool for biological and ecological studies, in which proturans have generally remained undetermined at species level.

1. Successful restoration of semi-natural grasslands on grasslands previously subject to intensive management needs to overcome manifold barriers. These include high soil fertility, the dominance of a few fast-growing plant species, degraded soil faunal communities and missing propagules of the targeted above-and belowground flora and fauna. A combination of removing the topsoil and introducing propagules of target plants has become one of the major tools for nature conservation agencies and practitioners to reduce soil fertility and restore former species-rich grasslands in various European countries.2. Using topsoil removal as a restoration measure has provoked an ongoing debate between supporting nature conservation and rejecting soil protection agencies. Although it favours species-rich plant communities, it strongly disturbs soil communities and affects physical and chemical soil properties and processes.Currently, there is a lack of long-term data to assess how restored grassland ecosystems develop and recover after topsoil removal. Here, we used two well-established bioindicators, soil nematodes and plants, to quantify restoration success of topsoil removal in comparison with alternative restoration measures and target communities 22 years after intervention.3. The nematode community composition indicated reduced nutrient availability in the restored systems, as was aimed at by topsoil removal. Nevertheless, after this 22-year period following topsoil removal, nematode composition and structure revealed successful recovery. 4. Plant communities benefitted from the reduction of soil nutrients after topsoil removal as indicated by higher numbers of plant species and higher Shannon diversity. Furthermore, topsoil removal strongly promoted the re-establishment of plant species of the target plant community. Synthesis and applications.Overall, our study demonstrates how a massive intervention by topsoil removal proved successful in converting intensively managed into species-rich grasslands. This contrasts with the mild intervention of repeated mowing and removing of the harvested plant material. We show that, in the long run, potential negative effects of topsoil removal on the soil fauna can be | 1783

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It is generally assumed that restoring biodiversity will enhance diversity and ecosystem functioning. However, to date, it has rarely been evaluated whether and how restoration efforts manage to rebuild biodiversity and multiple ecosystem functions (ecosystem multifunctionality) simultaneously. Here, we quantified how three restoration methods of increasing intervention intensity (harvest only < topsoil removal < topsoil removal + propagule addition) affected grassland ecosystem multifunctionality 22 yr after the restoration event. We compared restored with intensively managed and targeted seminatural grasslands based on 13 biotic and abiotic, above‐ and belowground properties. We found that all three restoration methods improved ecosystem multifunctionality compared to intensively managed grasslands and developed toward the targeted seminatural grasslands. However, whereas higher levels of intervention intensity reached ecosystem multifunctionality of targeted seminatural grasslands after 22 yr, lower intervention missed this target. Moreover, we found that topsoil removal with and without seed addition accelerated the recovery of biotic and aboveground properties, and we found no negative long‐term effects on abiotic or belowground properties despite removing the top layer of the soil. We also evaluated which ecosystem properties were the best indicators for restoration success in terms of accuracy and cost efficiency. Overall, we demonstrated that low‐cost measures explained relatively more variation of ecosystem multifunctionality compared to high‐cost measures. Plant species richness was the most accurate individual property in describing ecosystem multifunctionality, as it accounted for 54% of ecosystem multifunctionality at only 4% of the costs of our comprehensive multifunctionality approach. Plant species richness is the property that typically is used in restoration monitoring by conservation agencies. Vegetation structure, soil carbon storage and water‐holding capacity together explained 70% of ecosystem multifunctionality at only twice the costs (8%) of plant species richness, which is, in our opinion, worth considering in future restoration monitoring projects. Hence, our findings provide a guideline for land managers how they could obtain an accurate estimate of aboveground‐belowground ecosystem multifunctionality and restoration success in a highly cost‐efficient way.

Seminatural grasslands are important biodiversity hotspots, but they are increasingly degraded by intensive agriculture. Grassland restoration is considered to be promising in halting the ongoing loss of biodiversity, but this evaluation is mostly based on plant communities. Insect herbivores contribute substantially to grassland biodiversity and to the provisioning of a variety of ecosystem functions. However, it is unclear how they respond to different measures that are commonly used to restore seminatural grasslands from intensively used agricultural land. We studied the long‐term success of different restoration techniques, which were originally targeted at reestablishing seminatural grassland plant communities, for herbivorous insect communities on taxonomic as well as functional level. Therefore, we sampled insect communities 22 yr after the establishment of restoration measures. These measures ranged from harvest and removal of biomass to removal of the topsoil layer and subsequent seeding of plant propagules. We found that insect communities in restored grasslands had higher taxonomic and functional diversity compared to intensively managed agricultural grasslands and were more similar in composition to target grasslands. Restoration measures including topsoil removal proved to be more effective, in particular in restoring species characterized by functional traits susceptible to intensive agriculture (e.g., large‐bodied species). Our study shows that long‐term success in the restoration of herbivorous insect communities of seminatural grasslands can be achieved by different restoration measures and that more invasive approaches that involve the removal of the topsoil layer are more effective. We attribute these restoration successes to accompanying changes in the plant community, resulting in bottom‐up control of the herbivore community. Our results are of critical importance for management decisions aiming to restore multi‐trophic communities, their functional composition and consequently the proliferation of ecosystem functions.