High taxonomic diversity and miniaturization in benthic communities under persistent natural CO2 disturbances.

Metabarcoding techniques have revolutionized ecological research in recent years, facilitating the differentiation of cryptic species and revealing previously hidden diversity. In the current scenario of climate change and ocean acidification, biodiversity loss is one of the main threats to marine ecosystems. Here, we explored the effects of ocean acidification on marine benthic communities using DNA metabarcoding to assess the diversity of algae and metazoans. Specifically, we examined the natural pH gradient generated by the Fuencaliente CO2 vent system, located near La Palma Island (Canary Islands). High-resolution COI metabarcoding analyses revealed high levels of taxonomic diversity in an acidified natural area for the first time. This high number of species arises from the detection of small and cryptic species that were previously undetectable by other techniques. Such species are apparently tolerant to the acidification levels expected in future oceans. Hence and following our results, future subtropical communities are expected to keep high biodiversity values under an acidification scenario, although they will tend toward overall miniaturization due to the dominance of small algal and invertebrate species, leading to changes in ecosystem functions.

Resource competition drives an invasion-replacement event among shrew species on an island.

Invasive mammals are responsible for the majority of native species extinctions on islands. While most of these extinction events will be due to novel interactions between species (e.g. exotic predators and naive prey), it is more unusual to find incidences where a newly invasive species causes the decline/extinction of a native species on an island when they normally coexist elsewhere in their overlapping mainland ranges. We investigated if resource competition between two insectivorous small mammals was playing a significant role in the rapid replacement of the native pygmy shrew Sorex minutus in the presence of the recently invading greater white-toothed shrew Crocidura russula on the island of Ireland. We used DNA metabarcoding of gut contents from >300 individuals of both species to determine each species' diet and measured the body size (weight and length) during different stages of the invasion in Ireland (before, during and after the species come into contact with one another) and on a French island where both species have long coexisted (acting as a natural 'control' site). Dietary composition, niche width and overlap and body size were compared in these different stages. The body size of the invasive C. russula and composition of its diet changes between when it first invades an area and after it becomes established. During the initial stages of the invasion, individual shrews are larger and consume larger sized invertebrate prey species. During later stages of the invasion, C. russula switches to consuming smaller prey taxa that are more essential for the native species. As a result, the level of interspecific dietary overlap increases from between 11% and 14% when they first come into contact with each other to between 39% and 46% after the invasion. Here we show that an invasive species can quickly alter its dietary niche in a new environment, ultimately causing the replacement of a native species. In addition, the invasive shrew could also be potentially exhausting local resources of larger invertebrate species. These subsequent changes in terrestrial invertebrate communities could have severe impacts further downstream on ecosystem functioning and services.

DNA metabarcoding reveals the importance of gelatinous zooplankton in the diet of Pandalus borealis, a keystone species in the Arctic.

Information about the dietary composition of a species is crucial to understanding their position and role in the food web. Increasingly, molecular approaches such as DNA metabarcoding are used in studying trophic relationships, not least because they may alleviate problems such as low taxonomic resolution or underestimation of digestible taxa in the diet. Here, we used DNA metabarcoding with universal primers for cytochrome c oxidase I (COI) to study the diet composition of the northern shrimp (Pandalus borealis), an Arctic keystone species with large socio-economic importance. Across locations, jellyfish and chaetognaths were the most important components in the diet of P. borealis, jointly accounting for 40%-60% of the total read abundance. This dietary importance of gelatinous zooplankton contrasts sharply with published results based on stomach content analysis. At the same time, diet composition differed between fjord and shelf locations, pointing to different food webs supporting P. borealis in these two systems. Our study underlines the potential of molecular approaches to provide new insights into the diet of marine invertebrates that are difficult to obtain with traditional methods, and calls for a revision of the role of gelatinous zooplankton in the diet of the key Arctic species P. borealis, and in extension, Arctic food webs.

To denoise or to cluster, that is not the question: optimizing pipelines for COI metabarcoding and metaphylogeography.

BACKGROUND: The recent blooming of metabarcoding applications to biodiversity studies comes with some relevant methodological debates. One such issue concerns the treatment of reads by denoising or by clustering methods, which have been wrongly presented as alternatives. It has also been suggested that denoised sequence variants should replace clusters as the basic unit of metabarcoding analyses, missing the fact that sequence clusters are a proxy for species-level entities, the basic unit in biodiversity studies. We argue here that methods developed and tested for ribosomal markers have been uncritically applied to highly variable markers such as cytochrome oxidase I (COI) without conceptual or operational (e.g., parameter setting) adjustment. COI has a naturally high intraspecies variability that should be assessed and reported, as it is a source of highly valuable information. We contend that denoising and clustering are not alternatives. Rather, they are complementary and both should be used together in COI metabarcoding pipelines. RESULTS: Using a COI dataset from benthic marine communities, we compared two denoising procedures (based on the UNOISE3 and the DADA2 algorithms), set suitable parameters for denoising and clustering, and applied these steps in different orders. Our results indicated that the UNOISE3 algorithm preserved a higher intra-cluster variability. We introduce the program DnoisE to implement the UNOISE3 algorithm taking into account the natural variability (measured as entropy) of each codon position in protein-coding genes.  This correction increased the number of sequences retained by 88%. The order of the steps (denoising and clustering) had little influence on the final outcome. CONCLUSIONS: We highlight the need for combining denoising and clustering, with adequate choice of stringency parameters, in COI metabarcoding. We present a program that uses the coding properties of this marker to improve the denoising step. We recommend researchers to report their results in terms of both denoised sequences (a proxy for haplotypes) and clusters formed (a proxy for species), and to avoid collapsing the sequences of the latter into a single representative. This will allow studies at the cluster (ideally equating species-level diversity) and at the intra-cluster level, and will ease additivity and comparability between studies.

Marine biomonitoring with eDNA: Can metabarcoding of water samples cut it as a tool for surveying benthic communities?

In the marine realm, biomonitoring using environmental DNA (eDNA) of benthic communities requires destructive direct sampling or the setting-up of settlement structures. Comparatively much less effort is required to sample the water column, which can be accessed remotely. In this study we assess the feasibility of obtaining information from the eukaryotic benthic communities by sampling the adjacent water layer. We studied two different rocky-substrate benthic communities with a technique based on quadrat sampling. We also took replicate water samples at four distances (0, 0.5, 1.5, and 20 m) from the benthic habitat. Using broad range primers to amplify a ca. 313 bp fragment of the cytochrome oxidase subunit I gene, we obtained a total of 3,543 molecular operational taxonomic units (MOTUs). The structure obtained in the two environments was markedly different, with Metazoa, Archaeplastida and Stramenopiles being the most diverse groups in benthic samples, and Hacrobia, Metazoa and Alveolata in the water. Only 265 MOTUs (7.5%) were shared between benthos and water samples and, of these, 180 (5.1%) were identified as benthic taxa that left their DNA in the water. Most of them were found immediately adjacent to the benthos, and their number decreased as we moved apart from the benthic habitat. It was concluded that water eDNA, even in the close vicinity of the benthos, was a poor proxy for the analysis of benthic structure, and that direct sampling methods are required for monitoring these complex communities via metabarcoding.

Trade-offs between reducing complex terminology and producing accurate interpretations from environmental DNA: Comment on "Environmental DNA: What's behind the term?" by Pawlowski et al., (2020).

In a recent paper, "Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring," Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived.

Enjoying the warming Mediterranean: Transcriptomic responses to temperature changes of a thermophilous keystone species in benthic communities.

Information about the genomic processes underlying responses to temperature changes is still limited in non-model marine invertebrates. In this sense, transcriptomic analyses can help to identify genes potentially related to thermal responses. We here investigated, via RNA-seq, whole-transcriptomic responses to increased and decreased temperatures in a thermophilous keystone sea urchin, Arbacia lixula, whose populations are increasing in the Mediterranean. This species is a key driver of benthic communities' structure due to its grazing activity. We found a strong response to experimentally induced cold temperature (7°C), with 1,181 differentially expressed transcripts relative to the control condition (13°C), compared to only 179 in the warm (22°C) treatment. A total of 84 (cold treatment) and three (warm treatment) gene ontology terms were linked to the differentially expressed transcripts. At 7°C the expression of genes encoding different heat shock proteins (HSPs) was upregulated, together with apoptotic suppressor genes (e.g., Bcl2), genes involved in the infection response and/or pathogen-recognition (e.g., echinoidin) and ATP-associated genes, while protein biosynthesis and DNA replication pathways were downregulated. At 22°C neither HSPs induction nor activation of the previously mentioned pathways were detected, with the exception of some apoptotic-related activities that were upregulated. Our results suggest a strong transcriptional response associated with low temperatures, and support the idea of low water temperature being a major limitation for A. lixula expansion across deep Mediterranean and northern Atlantic waters.

DNA metabarcoding unveils multiscale trophic variation in a widespread coastal opportunist.

A thorough understanding of ecological networks relies on comprehensive information on trophic relationships among species. Since unpicking the diet of many organisms is unattainable using traditional morphology-based approaches, the application of high-throughput sequencing methods represents a rapid and powerful way forward. Here, we assessed the application of DNA metabarcoding with nearly universal primers for the mitochondrial marker cytochrome c oxidase I in defining the trophic ecology of adult brown shrimp, Crangon crangon, in six European estuaries. The exact trophic role of this abundant and widespread coastal benthic species is somewhat controversial, while information on geographical variation remains scant. Results revealed a highly opportunistic behaviour. Shrimp stomach contents contained hundreds of taxa (>1,000 molecular operational taxonomic units), of which 291 were identified as distinct species, belonging to 35 phyla. Only twenty ascertained species had a mean relative abundance of more than 0.5%. Predominant species included other abundant coastal and estuarine taxa, including the shore crab Carcinus maenas and the amphipod Corophium volutator. Jacobs' selectivity index estimates based on DNA extracted from both shrimp stomachs and sediment samples were used to assess the shrimp's trophic niche indicating a generalist diet, dominated by crustaceans, polychaetes and fish. Spatial variation in diet composition, at regional and local scales, confirmed the highly flexible nature of this trophic opportunist. Furthermore, the detection of a prevalent, possibly endoparasitic fungus (Purpureocillium lilacinum) in the shrimp's stomach demonstrates the wide range of questions that can be addressed using metabarcoding, towards a more robust reconstruction of ecological networks.

Spatio-temporal patterns of genetic variation in Arbacia lixula, a thermophilous sea urchin in expansion in the Mediterranean.

The genetic structure of 13 populations of the amphiatlantic sea urchin Arbacia lixula, as well as temporal genetic changes in three of these localities, were assessed using ten hypervariable microsatellite loci. This thermophilous sea urchin is an important engineer species triggering the formation of barren grounds through its grazing activity. Its abundance seems to be increasing in most parts of the Mediterranean, probably favoured by warming conditions. Significant genetic differentiation was found both spatially and temporally. The main break corresponded to the separation of western Atlantic populations from those in eastern Atlantic and the Mediterranean Sea. A less marked, but significant differentiation was also found between Macaronesia (eastern Atlantic) and the Mediterranean. In the latter area, a signal of differentiation between the transitional area (Alboran Sea) and the rest of the Mediterranean was detected. However, no genetic structure is found within the Mediterranean (excluding Alboran) across the Siculo-Tunisian Strait, resulting from either enough gene flow to homogenize distance areas or/and a recent evolutionary history marked by demographic expansion in this basin. Genetic temporal variation at the Alboran Sea is as important as spatial variation, suggesting that temporal changes in hydrological features can affect the genetic composition of the populations. A picture of genetic homogeneity in the Mediterranean emerges, implying that the potential expansion of this keystone species will not be limited by intraspecific genetic features and/or potential impact of postulated barriers to gene flow in the region.

Environmental DNA metabarcoding as an effective and rapid tool for fish monitoring in canals.

We focus on a case study along an English canal comparing environmental DNA (eDNA) metabarcoding with two types of electrofishing techniques (wade-and-reach and boom-boat). In addition to corroborating data obtained by electrofishing, eDNA provided a wider snapshot of fish assemblages. Given the semi-lotic nature of canals, we encourage the use of eDNA as a fast and cost-effective tool to detect and monitor whole fish communities.

A new sampling device for metabarcoding surveillance of port communities and detection of non-indigenous species.

Metabarcoding techniques are revolutionizing studies of marine biodiversity. They can be used for monitoring non-indigenous species (NIS) in ports and harbors. However, they are often biased by inconsistent sampling methods and incomplete reference databases. Logistic constraints in ports prompt the development of simple, easy-to-deploy samplers. We tested a new device called polyamide mesh for ports organismal monitoring (POMPOM) with a high surface-to-volume ratio. POMPOMS were deployed inside a fishing and recreational port in the Mediterranean alongside conventional settlement plates. We also compiled a curated database with cytochrome oxidase (COI) sequences of Mediterranean NIS. COI metabarcoding of the communities settled in the POMPOMs captured a similar biodiversity than settlement plates, with shared molecular operational units (MOTUs) representing ca. 99% of reads. 38 NIS were detected in the port accounting for ca. 26% of reads. POMPOMs were easy to deploy and handle and provide an efficient method for NIS surveillance.

Taxonomic accuracy and complementarity between bulk and eDNA metabarcoding provides an alternative to morphology for biological assessment of freshwater macroinvertebrates.

Determining biological status of freshwater ecosystems is critical for ensuring ecosystem health and maintaining associated services to such ecosystems. Freshwater macroinvertebrates respond predictably to environmental disturbances and are widely used in biomonitoring programs. However, many freshwater species are difficult to capture and sort from debris or substrate and morphological identification is challenging, especially larval stages, damaged specimens, or hyperdiverse groups such as Diptera. The advent of high throughput sequencing technologies has enhanced DNA barcoding tools to automatise species identification for whole communities, as metabarcoding is increasingly used to monitor biodiversity. However, recent comparisons have revealed little congruence between morphological and molecular-based identifications. Using broad range universal primers for DNA barcode marker cox1, we compare community composition captured between morphological and molecular-based approaches from different sources - tissue-based (bulk benthic and bulk drift samples) and environmental DNA (eDNA, filtered water) metabarcoding - for samples collected along a gradient of anthropogenic disturbances. For comparability, metabarcoding taxonomic assignments were filtered by taxa included in the standardised national biological metric IBMWP. At the family level, bulk benthic metabarcoding showed the highest congruence with morphology, and the most abundant taxa were captured by all techniques. Richness captured by morphology and bulk benthic metabarcoding decreased along the gradient, whereas richness recorded by eDNA remained constant and increased downstream when sequencing bulk drift. Estimates of biological metrics were higher using molecular than morphological identification. At species level, diversity captured by bulk benthic samples were higher than the other techniques. Importantly, bulk benthic and eDNA metabarcoding captured different and complementary portions of the community - benthic versus water column, respectively - and their combined use is recommended. While bulk benthic metabarcoding can likely replace morphology using similar benthic biological indices, water eDNA will require new metrics because this technique sequences a different portion of the community.

Chromosome-level genome assembly and annotation of the black sea urchin Arbacia lixula (Linnaeus, 1758).

The black sea urchin (Arbacia lixula) is a keystone species inhabiting the coastal shallow waters of the Mediterranean Sea, which is a key driver of littoral communities' structure. Here, we present the first genome assembly and annotation of this species, standing as the first Arbacioida genome, including both nuclear and mitochondrial genomes. To obtain a chromosome-level assembly, we used a combination of PacBio high fidelity (HiFi) reads and chromatin capture reads (Omni-C). In addition, we generated a high-quality nuclear annotation of both coding and non-coding genes, by using published RNA-Seq data from several individuals of A. lixula and gene models from closely related species. The nuclear genome assembly has a total span of 607.91 Mb, being consistent with its experimentally estimated genome size. The assembly contains 22 chromosome-scale scaffolds (96.52% of the total length), which coincides with its known karyotype. A total of 72,767 transcripts were predicted from the nuclear genome, 24,171 coding, and 48,596 non-coding that included lncRNA, snoRNA, and tRNAs. The circularized mitochondrial genome had 15,740 bp comprising 13 protein-coding genes, 2 rRNA, and 22 tRNA. This reference genome will enhance ongoing A. lixula studies and benefit the wider sea urchin scientific community.

Exceptional population genomic homogeneity in the black brittle star Ophiocomina nigra (Ophiuroidea, Echinodermata) along the Atlantic-Mediterranean coast.

The Atlantic-Mediterranean marine transition is characterised by strong oceanographic barriers and steep environmental gradients that generally result in connectivity breaks between populations from both basins and may lead to local adaptation. Here, we performed a population genomic study of the black brittle star, Ophiocomina nigra, covering most of its distribution range along the Atlantic-Mediterranean region. Interestingly, O. nigra is extremely variable in its coloration, with individuals ranging from black to yellow-orange, and different colour morphs inhabiting different depths and habitats. In this work, we used a fragment of the mitochondrial COI gene and 2,374 genome-wide ddRADseq-derived SNPs to explore: (a) whether the different colour morphs of O. nigra represent different evolutionary units; (b) the disruptive effects of major oceanographic fronts on its population structure; and (c) genomic signals of local adaptation to divergent environments. Our results revealed exceptional population homogeneity, barely affected by oceanographic fronts, with no signals of local adaptation nor genetic differentiation between colour morphs. This remarkable panmixia likely results from a long pelagic larval duration, a large effective population size and recent demographic expansions. Our study unveils an extraordinary phenotypic plasticity in O. nigra, opening further research questions on the ecological and molecular mechanisms underpinning coloration in Ophiuroidea.

Using eDNA to find Micrognathozoa.

Over the past decades the sampling of environmental DNA (eDNA) - encompassing the DNA of all organisms present in an environmental sample1 - has emerged as a technique for biodiversity monitoring and discovery in a diversity of environments. Avoiding the physical collection and identification of biota, this approach is praised for its independence of taxonomic expertise and has changed the way biologists study biodiversity. However, a common result in eDNA studies is the finding of unexpected taxa which are often removed by conservative bioinformatic filters or disregarded, since the authors are uncertain about the result and rarely have the interest, time, skills, and/or resources to return to the field and confirm with actual specimens2. Here, we report a case in which an eDNA discovery led to the physical localization of a member of the Micrognathozoa (Figure 1B) - a rare group of limnic micrometazoans, and the animal phylum to be discovered last3, which is the sister group to rotifers4,5. To this day, Micrognathozoa still comprises only a single named species from Greenland and a few additional disparate places.

DnoisE: distance denoising by entropy. An open-source parallelizable alternative for denoising sequence datasets.

DNA metabarcoding is broadly used in biodiversity studies encompassing a wide range of organisms. Erroneous amplicons, generated during amplification and sequencing procedures, constitute one of the major sources of concern for the interpretation of metabarcoding results. Several denoising programs have been implemented to detect and eliminate these errors. However, almost all denoising software currently available has been designed to process non-coding ribosomal sequences, most notably prokaryotic 16S rDNA. The growing number of metabarcoding studies using coding markers such as COI or RuBisCO demands a re-assessment and calibration of denoising algorithms. Here we present DnoisE, the first denoising program designed to detect erroneous reads and merge them with the correct ones using information from the natural variability (entropy) associated to each codon position in coding barcodes. We have developed an open-source software using a modified version of the UNOISE algorithm. DnoisE implements different merging procedures as options, and can incorporate codon entropy information either retrieved from the data or supplied by the user. In addition, the algorithm of DnoisE is parallelizable, greatly reducing runtimes on computer clusters. Our program also allows different input file formats, so it can be readily incorporated into existing metabarcoding pipelines.

Fine-scale differences in eukaryotic communities inside and outside salmon aquaculture cages revealed by eDNA metabarcoding.

Aquaculture impacts on marine benthic ecosystems are widely recognized and monitored. However, little is known about the community changes occurring in the water masses surrounding aquaculture sites. In the present study, we studied the eukaryotic communities inside and outside salmonid aquaculture cages through time to assess the community changes in the neighbouring waters of the farm. Water samples were taken biweekly over five months during the production phase from inside the cages and from nearby points located North and South of the salmon farm. Eukaryotic communities were analyzed by eDNA metabarcoding of the partial COI Leray-XT fragment. The results showed that eukaryotic communities inside the cages were significantly different from those in the outside environment, with communities inside the cages having higher diversity values and more indicator species associated with them. This is likely explained by the appearance of fouling species that colonize the artificial structures, but also by other species that are attracted to the cages by other means. Moreover, these effects were highly localized inside the cages, as the communities identified outside the cages, both North and South, had very similar eukaryotic composition at each point in time. Overall, the eukaryotic communities, both inside and outside the cages, showed similar temporal fluctuations through the summer months, with diversity peaks occurring at the end of July, beginning of September, and in the beginning of November, with the latter showing the highest Shannon diversity and richness values. Hence, our study suggests that seasonality, together with salmonid aquaculture, are the main drivers of eukaryotic community structure in surface waters surrounding the farm.

Using DNA Metabarcoding to Characterize the Prey Spectrum of Two Co-Occurring Themisto Amphipods in the Rapidly Changing Atlantic-Arctic Gateway Fram Strait.

The two congeneric hyperiids Themisto libellula and T. abyssorum provide an important trophic link between lower and higher trophic levels in the rapidly changing Arctic marine ecosystem. These amphipods are characterized by distinct hydrographic affinities and are hence anticipated to be impacted differently by environmental changes, with major consequences for the Arctic food web. In this study, we applied DNA metabarcoding to the stomach contents of these Themisto species, to comprehensively reveal their prey spectra at an unprecedented-high-taxonomic-resolution and assess the regional variation in their diet across the Fram Strait. Both species feed on a wide variety of prey but their diet strongly differed in the investigated summer season, showing overlap for only a few prey taxa, such as calanoid copepods. The spatially structured prey field of T. libellula clearly differentiated it from T. abyssorum, of which the diet was mainly dominated by chaetognaths. Our approach also allowed the detection of previously overlooked prey in the diet of T. libellula, such as fish species and gelatinous zooplankton. We discuss the reasons for the differences in prey spectra and which consequences these may have in the light of ongoing environmental changes.

For all audiences: Incorporating immature stages into standardised spider inventories has a major impact on the assessment of biodiversity patterns.

Although arthropods are the largest component of animal diversity, they are traditionally underrepresented in biological inventories and monitoring programmes. However, no biodiversity assessment can be considered informative without including them. Arthropod immature stages are often discarded during sorting, despite frequently representing more than half of the collected individuals. To date, little effort has been devoted to characterising the impact of discarding nonadult specimens on our diversity estimates. Here, we used a metabarcoding approach to analyse spiders from oak forests in the Iberian Peninsula, to assess (1) the contribution of juvenile stages to local diversity estimates, and (2) their effect on the diversity patterns (compositional differences) across assemblages. We further investigated the ability of metabarcoding to inform on abundance. We obtained 363 and 331 species as adults and juveniles, respectively. Including the species represented only by juveniles increased the species richness of the whole sampling in 35% with respect to those identified from adults. Differences in composition between assemblages were greatly reduced when immature stages were considered, especially across latitudes, possibly due to phenological differences. Moreover, our results revealed that metabarcoding data are to a certain extent quantitative, but some sort of taxonomic conversion factor may be necessary to provide accurate informative estimates. Although our findings do not question the relevance of the information provided by adult-based inventories, they also reveal that juveniles provide a novel and relevant layer of knowledge that, especially in areas with marked seasonality, may influence our interpretations, providing more accurate information from standardised biological inventories.

High resolution ancient sedimentary DNA shows that alpine plant diversity is associated with human land use and climate change.

The European Alps are highly rich in species, but their future may be threatened by ongoing changes in human land use and climate. Here, we reconstructed vegetation, temperature, human impact and livestock over the past ~12,000 years from Lake Sulsseewli, based on sedimentary ancient plant and mammal DNA, pollen, spores, chironomids, and microcharcoal. We assembled a highly-complete local DNA reference library (PhyloAlps, 3923 plant taxa), and used this to obtain an exceptionally rich sedaDNA record of 366 plant taxa. Vegetation mainly responded to climate during the early Holocene, while human activity had an additional influence on vegetation from 6 ka onwards. Land-use shifted from episodic grazing during the Neolithic and Bronze Age to agropastoralism in the Middle Ages. Associated human deforestation allowed the coexistence of plant species typically found at different elevational belts, leading to levels of plant richness that characterise the current high diversity of this region. Our findings indicate a positive association between low intensity agropastoral activities and precipitation with the maintenance of the unique subalpine and alpine plant diversity of the European Alps.