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Microbial community and diversity in the rhizosphere is strongly influenced by biotic and/or abiotic factors, like root exudates, nutrient availability, edaphon and climate. Here we report on the microbial diversity within the rhizosphere of Larix decidua , a dominant tree species in the Alps, as compared with the microbiome within the surrounding soil. We describe how increased light intensity influenced the rhizobiome and put emphasize on methane cycling microorganisms. Microbial taxa were classified into 26 bacterial, 4 archaeal and 6 fungal phyla revealing significant differences between bulk and rhizosphere soils. The dominant prokaryotic phyla were Proteobacteria, Acidobacteria, Actinobacteria (both, rhizosphere and bulk soil) and Bacteroidetes (rhizosphere soil only) and dominant fungal phyla in both fractions included Ascomycota and Basidiomycota. The rhizosphere community was indicated by Suillus sp., plant growth-promoting bacteria and Candidatus Saccharibacteria. Predicted genes in membrane transport and carbohydrate metabolism were significantly more abundant in rhizosphere soils while genes connected with energy metabolisms and cell motility increased in bulk soils. Dominant methanotrophic microorganisms were Upland Soil Cluster (USC) α methanotrophs, Methylogaea spp. and Methylosinus spp., while most methanogens belonged to Methanomassiliicoccales. The overall abundance of methanotrophs distinctly increased in the rhizosphere but to a very different species-specific extent. The increased light intensity only led to minor changes in the rhizobiome, nevertheless a couple of indicator species (e.g. Pseudomonas sp.) for intensified light conditions were established.

Metataxonomic studies of ecosystem microbiotas require the simultaneous processing of samples with contrasting physical and biochemical traits. However, there are no published studies of comparisons of different DNA extraction kits to characterize the microbiotas of the main components of terrestrial ecosystems. Here, and to our knowledge for the first time, five DNA extraction kits were used to investigate the composition and diversity of the microbiota of a subset of samples typically studied in terrestrial ecosystems such as bulk soil, rhizosphere soil, invertebrate taxa and mammalian feces. DNA extraction kit was associated with changes in the relative abundance of hundreds of ASVs, in the same samples, resulting in significant differences in alpha and beta diversity estimates of their microbiotas. Importantly, the impact of DNA extraction kit on sample diversity varies according to sample type, with mammalian feces and soil samples showing the most and least consistent diversity estimates across DNA extraction kits, respectively. We show that the MACHEREY–NAGEL NucleoSpin® Soil kit was associated with the highest alpha diversity estimates, providing the highest contribution to the overall sample diversity, as indicated by comparisons with computationally assembled reference communities, and is recommended to be used for any large-scale microbiota study of terrestrial ecosystems.

Background Metabarcoding targeting the internal transcribed spacer (ITS) region is commonly used to characterize fungal communities of various environments. Given their size and complexity, raw ITS sequences are necessarily processed and quality-filtered with bioinformatic pipelines. However, such pipelines are not yet standardized, especially for fungal communities, and those available may produce contrasting results. While some pipelines cluster sequences based on a specified percentage of base pair similarity into operational taxonomic units (OTUs), others utilize denoising techniques to infer amplicon sequencing variants (ASVs). While ASVs are now considered a more accurate representation of taxonomic diversity for prokaryote communities based on 16S rRNA amplicon sequencing, the applicability of this method for fungal ITS sequences is still debated. Results Here we compared the performance of two commonly used pipelines DADA2 (inferring ASVs) and mothur (clustering OTUs) on fungal metabarcoding sequences originating from two different environmental sample types (fresh bovine feces and pasture soil). At a 99% OTU similarity threshold, mothur consistently identified a higher fungal richness compared to DADA2. In addition, mothur generated homogenous relative abundances across multiple technical replicates ( n  = 18), while DADA2 results for the same replicates were highly heterogeneous. Conclusions Our study highlights a potential pipeline-associated bias in fungal metabarcoding data analysis of environmental samples. Based on the homogeneity of relative abundances across replicates and the capacity to detect OTUs/ASVs, we suggest using OTU clustering with a similarity of 97% as the most appropriate option for processing fungal metabarcoding data.

Lower atmospheric pressure affects biologically relevant physical parameters such as gas partial pressure and concentration, leading to increased water vapor diffusivity and greater soil water content loss through evapotranspiration. This might impact plant photosynthetic activity, resource allocation, water relations, and growth. However, the direct impact of low air pressure on plant physiology is largely unknown. This study examined the effects of low air pressure, alone and combined with two water inputs, on different functional traits of three plant species transplanted from montane grasslands at 1,500 m a.s.l. during the first four weeks of their early phenological stage: Trifolium pratense , Hieracium pilosella , and B rachypodium rupestre . Using the terraXcube Ecotron facility which can simulate different climatic conditions, we isolated the effect of air pressure from those of other, related environmental factors (temperature, humidity, and solar radiation) by simulating three different elevations with corresponding air pressures: 1,500 m a.s.l. (85 kPa, control scenario ), 2,500 m a.s.l. (75 kPa), and 4,000 m a.s.l. (62 kPa) and we used two different water regimes to observe the combined effect of low air pressure and the impact of varying water inputs on plants. In T . pratense and H . pilosella , we observed an increase in stomatal conductance but a reduction in aboveground biomass at the lowest pressure compared to the control scenario after four weeks of incubation. Contrastingly, B . rupestre showed an interactive effect of air pressure and water treatment on chlorophyll and biomass nitrogen content, which were reduced under higher soil water conditions at 85kPa. This study serves as an initial step in isolating the specific impact of air pressure on plant physiology, demonstrating the potential of the facility for future research. The mixed response patterns across species highlight that atmospheric pressure could be a driving factor to consider when assessing plant responses along elevational gradient.

Metataxonomy has become the standard for characterizing the diversity and composition of microbial communities associated with multicellular organisms and their environment. Currently available protocols for metataxonomy assume a uniform DNA extraction, amplification and sequencing efficiency for all sample types and taxa. It has been suggested that the addition of a mock community (MC) to biological samples before the DNA extraction step could aid identification of technical biases during processing and support direct comparisons of microbiota composition, but the impact of MC on diversity estimates of samples is unknown. Here, large and small aliquots of pulverized bovine fecal samples were extracted with no, low or high doses of MC, characterized using standard Illumina technology for metataxonomics, and analysed with custom bioinformatic pipelines. We demonstrated that sample diversity estimates were distorted only if MC dose was high compared to sample mass (i.e. when MC > 10% of sample reads). We also showed that MC was an informative in situ positive control, permitting an estimation of the sample 16S copy number, and detecting sample outliers. We tested this approach on a range of sample types from a terrestrial ecosystem, including rhizosphere soil, whole invertebrates, and wild vertebrate fecal samples, and discuss possible clinical applications.

The mountain hare ( Lepus timidus ) is an arctic-alpine species with relictual populations in the Italian Alps, typically occurring at elevations above 2000 m a.s.l. This species is threatened by habitat loss and fragmentation, and declining snow cover due to climate warming. Moreover, as treelines shift upward, the European brown hare ( L. europaeus ) is expanding its distribution into areas previously dominated by the mountain hare, potentially leading to resource competition, and loss of local adaptation through hybridization and inter-specific gene flow. In particular, the consequences of sympatry on diversity and composition of prokaryote and fungal communities of the gut microbiota, which are critical to individual health, are currently unknown. Here, we compared the gut microbiota of these two hare species in an area of overlap in the central Alps by analysing fresh faecal pellets collected from Val Mazia/Matschertal, Italy along an elevational gradient (1000 to 2500 m a.s.l.). For the first time, we describe the prokaryote diversity and composition of L. timidus , and the fungal gut communities (mycobiota) of both Lepus species. Species identity was confirmed for 95 samples via mtDNA barcoding, while gut microbiota richness and composition were investigated using amplicon sequencing, targeting the V3-V4 region of the prokaryote 16S rRNA gene and fungal ITS2 regions. Distinct prokaryote and fungal communities were observed for each species, even where their distributions overlap, indicating differences in their functional diversity. Interestingly, for both Lepus species, elevation influenced fungal but not prokaryote diversity. Therefore, sympatry appears to have had minimal impact on gut microbiota composition of either species thus far. Given the expected upward range shift of L. europaeus under climate warming and its continued restocking for hunting, our findings provide an important baseline for assessing the health and adaptability of L. timidus as well as the effectiveness of conservation efforts aimed at protecting this species. However, expanding this research to other areas of sympatry will be essential to understand if gut microbiota composition is indicative of L. timidus conservation status across its range.

In response to climate change, plants in mountain regions are shifting their distribution ranges upward, exposing them to novel abiotic conditions such as reduced atmospheric pressure. While these changes are likely to affect plant physiology, their impact on plant-associated microorganisms in the rhizosphere has not yet been investigated. In this study, we used the terraXcube Ecotron facility to experimentally discriminate air pressure from other elevation-related factors like humidity and temperature, and to assess its influence on the rhizosphere microbiota of three plant species: a grass ( Brachipodium rupestre ), a forb ( Hieracium pilosella ), and a legume ( Trifolium pratense ). Plants were grown under controlled environmental conditions at four simulated elevations (260, 1500, 2500, and 4000 m a.s.l.), corresponding to pressure levels of 98, 85, 75, and 62 kPa, respectively. Microbial biomass and activity were significantly influenced by air pressure, but in a plant-specific manner. In addition, air pressure also led to notable and plant-specific shifts in the community composition of prokaryotes and, to a lesser extent, fungi. Redundancy analysis identified air pressure as a central predictor of these rhizosphere community shifts. Notably, no correlations were detected between microbial community composition and morphological and physiological plant traits, suggesting that air pressure should directly affect microorganisms, independently of plant-mediated effects. This study demonstrates that even under constant temperature and humidity, air pressure alone can restructure rhizosphere microbial communities, highlighting a critical yet often overlooked driver of plant–microbe dynamics during uphill range shifts. Whether such alterations in the rhizosphere microbiota ultimately enhance or impair soil chemistry, plant health, and ecosystem functioning remains an important question for future research.

High mountain areas above the alpine zone are, despite the low-temperature conditions, inhabited by evolutionary and functionally differing organism groups. We compared the abundance and species richness of vascular plants, oribatid mites, springtails, spiders, and beetles, as well as bacterial and methanogenic archaeal prokaryotes (only abundance), at 100 m vertical intervals from 2,700-3,400 m in the Central Alps. We hypothesized that the less mobile microarthropods and microorganisms are more determined by and respond in similar ways to soil properties as do vascular plants. In contrast, we expected the more mobile surface-dwelling groups to forage also in places devoid of vegetation and thus to show patterns that deviate from that of vascular plants. Surprisingly, the observed patterns were diametrically opposed to our expectations: soil-living oribatid mites and springtails showed high individual numbers at high elevations, even where vascular plants barely occurred. Springtails also showed a rather constant species richness throughout the entire gradient. In contrast, patterns of surface-dwelling organisms and of archaeal prokaryotes did not differ significantly from vascular plants, because of either comparable climate sensitivity or their dependency on vegetated habitats. This study may serve as a baseline to estimate the risks of biodiversity losses in response to climate change across different biotic ecosystem components and to explore the potential and limitations of vascular plants as proxy for other organism groups that are far more challenging to monitor.

Five differently developed soils aged 6, 35, 80, 150, and >5000 years with the same bedrock and the same (current) climate conditions were chosen to assess abiotic and enzymatic properties as well as methanogenic and methanotrophic activities. Most abiotic properties (dry weight, pH, soil organic matter, and ammonium content), enzyme activities (dehydrogenase [DH] activity, ammonification [AM] rate, dimethylsulfoxide reduction), and potential methane oxidation (PoMO) per gram of dry weight (DW) increased with soil age. In contrast, potential methane production (PoMP) as well as the nitrate content per gram of DW and most enzymatic properties per gram of soil organic matter (SOM) did not increase with soil age but reached its maximum in the middle-aged soils (80–150 years). Our results show that (i) microbial activity does not consequently increase with SOM content/soil age; (ii) methane production can be measured in undeveloped soils, whereas methane oxidation is more restricted to fully developed soils; and (iii) certain soil modifications (change in water content, ammonium addition) could influence potential methane production/oxidation. When considering the concurrent release of raw soil because of the melting of perpetual ice, these data could help to better understand and assess the consequences of global change.

ABSTRACT Due to the activity of methane-oxidizing bacteria, forest soils are usually net sinks for the greenhouse gas methane (CH4). Despite several hints that CH4 balances might be influenced by vegetation, there are only few investigations dealing with this connection. Therefore, we studied this soil–plant–microbe interaction by using mesocosm experiments with forest soil and Larix decidua, a common coniferous tree species within the Alps. Gas measurements showed that the presence of L. decidua significantly reduced CH4 oxidation of the forest soil by ∼10% (−0.95 µmol m−2 h−1 for soil vs −0.85 µmol m−2 h−1 for soil plus L. decidua) leading to an increased net CH4 balance. Increased light intensity was used to intensify the influence of the plant on the soil's CH4 balance. The increase in light intensity strengthened the effect of the plant and led to a greater reduction of CH4 oxidation. Besides, we examined the impact of L. decidua and light on the abundance of methanogens and methanotrophs in the rhizosphere as compared with bulk soil. The abundance of both methane-oxidizing bacteria and methanogenic archaea was significantly increased in the rhizosphere compared with bulk soil but no significant response of methanogens and methanotrophs upon light exposure was established. L. decidua affects the abundance of methanogenic and methanotrophic microorganisms and reduces the methane sink capacity of forest soils, which can be additionally reduced by increasing light.