Calcium Oxalate Crystals, the Plant 'Gemstones': Insights into Their Synthesis and Physiological Implications in Plants.

From simple algal forms to the most advanced angiosperms, calcium oxalate (CaOx) crystals (CRs) occur in the majority of taxonomic groups of photosynthetic organisms. Various studies have demonstrated that this biomineralization is not a simple or random event but a genetically regulated coordination between calcium uptake, oxalate (OX) synthesis and, sometimes, environmental stresses. Certainly, the occurrence of CaOx CRs is old; however, questions related to their genesis, biosynthesis, significance and genetics exhibit robust evolution. Moreover, their speculated roles in bulk calcium regulation, heavy metal/OX detoxification, light reflectance and photosynthesis, and protection against grazing and herbivory, besides other characteristics, are gaining much interest. Thus, it is imperative to understand their synthesis and regulation in relation to the ascribed key functions to reconstruct future perspectives in harnessing their potential to achieve nutritious and pest-resistant crops amid anticipated global climatic perturbations. This review critically addresses the basic and evolving concepts of the origin (and recycling), synthesis, significance, regulation and fate vis-à-vis various functional aspects of CaOx CRs in plants (and soil). Overall, insights and conceptual future directions present them as potential biominerals to address future climate-driven issues.

Species composition of root-associated mycobiome of ruderal invasive Anthemis cotula L. varies with elevation in Kashmir Himalaya.

Investigating the microbial communities associated with invasive plant species can provide insights into how these species establish and thrive in new environments. Here, we explored the fungal species associated with the roots of the invasive species Anthemis cotula L. at 12 sites with varying elevations in the Kashmir Himalaya. Illumina MiSeq platform was used to identify the species composition, diversity, and guild structure of these root-associated fungi. The study found a total of 706 fungal operational taxonomic units (OTUs) belonging to 8 phyla, 20 classes, 53 orders, 109 families, and 160 genera associated with roots of A. cotula, with the most common genus being Funneliformis. Arbuscular mycorrhizal fungi (AMF) constituted the largest guild at higher elevations. The study also revealed that out of the 12 OTUs comprising the core mycobiome, 4 OTUs constituted the stable component while the remaining 8 OTUs comprised the dynamic component. While α-diversity did not vary across sites, significant variation was noted in ß-diversity. The study confirmed the facilitative role of the microbiome through a greenhouse trial in which a significant effect of soil microbiome on height, shoot biomass, root biomass, number of flower heads, and internal CO2 concentration of the host plant was observed. The study indicates that diverse fungal mutualists get associated with this invasive alien species even in nutrient-rich ruderal habitats and may be contributing to its spread into higher elevations. This study highlights the importance of understanding the role of root-associated fungi in invasion dynamics and the potential use of mycobiome management strategies to control invasive species.

Risk analysis of fast spreading species in a Kashmir Himalayan National Park (Dachigam) for better monitoring and management.

Huge economic costs and ecological impacts of invasive alien species (IAS) in the protected areas (PAs) worldwide make their timely prediction and potential risk assessment of central importance for effective management. While the preborder weed risk assessment framework has been extensively evaluated and implemented, the postborder species risk assessment framework has not been subjected to the same degree of scrutiny. Here we used a rather more realistic modified version of the Australian Weed Risk framework (AWRM) for Dachigam National Park (DNP) in Kashmir Himalaya against 84 plant species, including 55 alien species and 29 fast spreading native species, for risk analysis. We found two very high-risk species, three high-risk species, 10 medium-risk species, 29 low-risk species, and 40 negligible-risk species in the DNP. The containment scores accordingly ranged from 14.4 to 293.5 comprising of 27 species that can be contained with very high feasibility, 23 species with high feasibility, 14 species with medium feasibility, and 12 species which cannot be contained easily thereby having low feasibility of containment (FOC) score. However, eight species which have a negligible FOC score are difficult to contain within their infestation sites. Our results demonstrate the merit of the AWRM with a caution that the necessary region-specific modifications may help in its better implementation. Overall, these results provide quite a promising tool in the hands of protected area managers to timely and effectively deal with the problem of plant invasions.

Site and Organ-Specific Culture-Dependent Endophytic Diversity of Crocus sativus L. (Saffron) in Kashmir Himalaya, India.

The endophytic diversity of a host plant has been shown to vary across various spatio-temporal scales and between different organs of a plant, but no such study has been carried out on the economically important plant Crocus sativus (saffron). To fill this knowledge gap, the present study was undertaken to document the diversity of culturable bacterial, actinomycete and fungal endophytes at multiple sites from vegetative and reproductive organs of C. sativus. A total of 1170 culturable endophytic isolates were recovered from 6480 tissue segments of C. sativus collected from six different study sites in Pampore region of Kashmir valley in India. These isolates were identified using 16S and ITS (internal transcribed spacer) rDNA barcode sequence analysis and were classified into 84 operational taxonomic units (OTUs), including 52 bacterial OTUs, 7 actinomycete OTUs and 25 fungal OTUs. The phylogenetic analysis of sequences separated them into four phyla, namely, Firmicutes (46%), Ascomycota (30%), Proteobacteria (16%) and Actinobacteria (8%). Significant differences were observed in the diversity of endophytic assemblages across various study sites and different plant organs (P ≤ 0.001). Species richness was highest at the Baroosa site and lowest at the Chandhar site while the Shannon index was highest at the Baroosa site and lowest at the Letpur site. Among organs, species richness was highest in stigma and lowest in leaf. Similarly, Shannon index was highest for root and lowest for leaf. Further, 15 culturable endophytic OTUs showed organ specificity. The present study is the first comprehensive report that not only brings out differences in the diversity of endophytes associated with different organs and at different sites but also highlights the complexity of host-endophyte interaction at multiple scales.

Morel mushroom, Morchella from Kashmir Himalaya: a potential source of therapeutically useful bioactives that possess free radical scavenging, anti-inflammatory, and arthritic edema-inhibiting activities.

Morel mushrooms, Morchella species are highly nutritious and excellently edible wild mushrooms abundantly growing in Kashmir Himalayas. The free radical scavenging, anti-inflammatory, and arthritis edema-inhibiting properties of bioactive extract of Morchella elata (EAE) were evaluated. EAE inhibited 53.2% formalin-induced paw edema at a dose of 500 mg/kg b.wt and 75.0% croton oil-induced skin inflammation at a dose of 50 mg topical application. EAE exhibited 51.8% COX inhibiting activity at a concentration of 100 µg/ml when assayed using LPS-stimulated RAW 264.7 cells exposed to the extract. NF-kB inhibiting activity of EAE was assayed using Lentix-293T P65 Ds Red stable cell line. High-throughput fluorescent imaging and flow cytometry showed profound ability of EAE to inhibit NF-kB activity. HPTLC analysis revealed that EAE is composed of several chemical components. The mushroom is a source of therapeutically useful functional food that can provide relief in arthritis.

Decline in the suitable habitat of dominant Abies species in response to climate change in the Hindu Kush Himalayan region: insights from species distribution modelling.

Reliable predictions of future distribution ranges of ecologically important species in response to climate change are required for developing effective management strategies. Here we used an ensemble modelling approach to predict the distribution of three important species of Abies namely, Abies pindrow, Abies spectabilis and Abies densa in the Hindu Kush Himalayan region under the current and two shared socioeconomic pathways (SSP245 and SSP585) and time periods of 2050 and 2090s. A correlative ensemble model using presence/absence data of the three Abies species and 22 environmental variables, including 19 bioclimatic variables and 3 topographic variables, from known distributions was built to predict the potential current and future distribution of these species. The individual models used to build the final ensemble performed well and provided reliable results for both the current and future distribution of all three species. For A. pindrow, precipitation of the driest month (Bio14) was the most important environmental variable with 83.3% contribution to model output while temperature seasonality (Bio4) and annual mean diurnal range (Bio2) were the most important variables for A. spectabilis and A. densa with 48.4% and 46.1% contribution to final model output, respectively. Under current climatic conditions, the ensemble models projected a total suitable habitat of about 433,003 km2, 790,837 km2 and 676,918 km2 for A. pindrow, A. spectabilis and A. densa, respectively, which is approximately 10.36%, 18.91% and 16.91% of the total area of Hindu Kush Himalayan region. Projections of habitat suitability under future climate scenarios for all the shared socioeconomic pathways showed a reduction in potentially suitable habitats with a maximum overall loss of approximately 14% of the total suitable area of A. pindrow under SSP 8.5 by 2090. A decline in total suitable habitat is predicted to be 9.6% in A. spectabilis by 2090 under the SSP585 scenario while in A. densa 6.67% loss in the suitable area is expected by 2050 under the SSP585 scenario. Furthermore, there is no elevational change predicted in the case of A. pindrow while A. spectabilis is expected to show an upward shift by about 29 m per decade and A. densa is showing a downward shift at a rate of 11 m per decade. The results are interesting, and intriguing given the occurrence of these species across the Hindu Kush Himalayan region. Thus, our study underscores the need for consideration of unexpected responses of species to climate change and formulation of strategies for better forest management and conservation of important conifer species, such as A. pindrow, A. spectabilis and A. densa.

Risk assessment and management framework for rapidly spreading species in a Kashmir Himalayan Ramsar site.

In view of huge ecological impacts and exorbitantly high economic costs of biological invasions, the risk assessment for timely prediction of potential invaders and their effective management assumes central importance, yet having been little addressed. Hence, we did the risk analysis of 39 plant species, including both alien and fast-spreading native species, in Hokera wetland, an important Ramsar site in Kashmir Himalaya, using the post-border Australian Weed Risk Management (AWRM) framework. Based on the AWRM scores, we listed these species into different categories, such as alert, destroy infestation, contain spread, manage weed, manage sites and monitor, with management implications. Out of the eight decisions created for Hokera wetland, alien Alternanthera philoxeroides was identified as 'alert species', while Typha angustifolia, Typha latifolia, Phragmites australis, Sparganium ramosum and Myriophyllum aquaticum were placed under the 'manage weed' category of the management priorities. To check the predictability and reliability of the AWRM scheme, we developed the receiver operating characteristic (ROC) curve that yielded a positive diagonal value of above 0.5, with 88.6% and 83.1% area under the curve for comparative weed risk (CWR) score and the feasibility of coordinated control (FOC) score, respectively. The outcomes of the ROC analysis were compared with the results of the WRM evaluation of other regions across the globe. Our results indicate that the risk assessment using the AWRM model is quite efficient at discriminating and flagging the most troublesome plant species and offsetting their impacts on native biodiversity and ecosystem functioning in wetland ecosystems. Given the growing threat of biological invasions in the protected areas, we recommend an integrated and strategic approach, well informed by the data on the species biology and ecology, in the form of the AWRM management system to effectively deal with the alarmingly spreading species.

Next Generation High Throughput Sequencing to Assess Microbial Communities: An Application Based on Water Quality.

Traditional techniques to identify different contaminants (biological or chemical) in the waters are slow, laborious, and can require specialized expertise. Hence, the rapid determination of water quality using more sensitive and reliable metagenomic based approaches attains special importance. Metagenomics deals with the study of genetic material that is recovered from microbial communities present in environmental samples. In traditional techniques cultivation-based methodologies were used to describe the diversity of microorganisms in environmental samples. It has failed to function as a robust marker because of limited taxonomic and phylogenetic implications. In this backdrop, high-throughput DNA sequencing approaches have proven very powerful in microbial source tracking because of investigating the full variety of genome-based analysis such as microbial genetic diversity and population structure played by them. Next generation sequencing technologies can reveal a greater proportion of microbial communities that have not been reported earlier by traditional techniques. The present review highlights the shift from traditional techniques for the basic study of community composition to next-generation sequencing (NGS) platforms and their potential applications to the biomonitoring of water quality in relation to human health.

Differential Bioaccumulation of Select Heavy Metals from Wastewater by Lemna minor.

The capacity of Lemna minor to remediate toxic heavy metals from wastewater is reasonably well documented. In view of the pivotal role of this species in the environmental clean-up, here we evaluated the bioaccumulation potential of L. minor for cadmium (Cd), lead (Pb), and nickel (Ni) through a controlled experiment. L. minor tolerated the metals Cd, Ni, and Pb up to 0.5, 5, and 8 mg/L, respectively, and beyond these concentrations the toxicity symptoms appeared. Bio-concentration factor varied at different concentrations of heavy metals tested. Overall, L. minor showed good phytoremediation potential for all the three tested heavy metals (Cd, Ni, and Pb), though in relative terms it was more effective in extracting Ni and Cd, as compared to Pb, both in single and mixed concentrations. In view of the growing pollution in Kashmir Himalayan aquatic habitats the phytoremediation by invasive species such as L. minor promises to be one of the best choices than other native plants for cleaning up of polluted soils/water because of its fast growth rate, high abundance, easy handling, and wide distribution in Kashmir Himalayan aquatic ecosystems.

Chalcone synthases (CHSs): the symbolic type III polyketide synthases.

MAIN CONCLUSION: Present review provides a thorough insight on some significant aspects of CHSs over a period of about past three decades with a better outlook for future studies toward comprehending the structural and mechanistic intricacy of this symbolic enzyme. Polyketide synthases (PKSs) form a large family of iteratively acting multifunctional proteins that are involved in the biosynthesis of spectrum of natural products. They exhibit remarkable versatility in the structural configuration and functional organization with an incredible ability to generate different classes of compounds other than the characteristic secondary metabolite constituents. Architecturally, chalcone synthase (CHS) is considered to be the simplest representative of Type III PKSs. The enzyme is pivotal for phenylpropanoid biosynthesis and is also well known for catalyzing the initial step of the flavonoid/isoflavonoid pathway. Being the first Type III enzyme to be discovered, CHS has been subjected to ample investigations which, to a greater extent, have tried to understand its structural complexity and promiscuous functional behavior. In this context, we vehemently tried to collect the fragmented information entirely focussed on this symbolic enzyme from about past three-four decades. The aim of this review is to selectively summarize data on some of the fundamental aspects of CHSs viz, its history and distribution, localization, structure and analogs in non-plant hosts, promoter analyses, and role in defense, with an emphasis on mechanistic studies in different species and vis-à-vis mutation-led changes, and evolutionary significance which has been discussed in detail. The present review gives an insight with a better perspective for the scientific community for future studies devoted towards delimiting the mechanistic and structural basis of polyketide biosynthetic machinery vis-à-vis CHS.

Rheum australe, an endangered high-value medicinal herb of North Western Himalayas: a review of its botany, ethnomedical uses, phytochemistry and pharmacology.

Rheum australe (Himalayan Rhubarb) is a multipurpose, endemic and endangered medicinal herb of North Western Himalayas. It finds extensive use as a medicinal herb since antiquity in different traditional systems of medicine to cure a wide range of ailments related to the circulatory, digestive, endocrine, respiratory and skeletal systems as well as to treat various infectious diseases. The remedying properties of this plant species are ascribed to a set of diverse bioactive secondary metabolite constituents, particularly anthraquinones (emodin, chrysophanol, physcion, aloe-emodin and rhein) and stilbenoids (piceatannol, resveratrol), besides dietary flavonoids known for their putative health benefits. Recent studies demonstrate the pharmacological efficacy of some of these metabolites and/or their derivatives as lead molecules for the treatment of various human diseases. Present review comprehensively covers the literature available on R. australe from 1980 to early 2018. The review provides up-to-date information available on its botany for easy identification of the plant, and origin and historical perspective detailing its trade and commerce. Distribution, therapeutic potential in relation to traditional uses and pharmacology, phytochemistry and general biosynthesis of major chemical constituents are also discussed. Additionally, efficient and reproducible in vitro propagation studies holding vital significance in preserving the natural germplasm of the plant and for its industrial exploitation have also been highlighted. The review presents a detailed perspective for future studies to conserve and sustainably make use of this endangered plant species at a commercial scale.

Low genetic diversity despite multiple introductions of the invasive plant species Impatiens glandulifera in Europe.

BACKGROUND: Invasive species can be a major threat to native biodiversity and the number of invasive plant species is increasing across the globe. Population genetic studies of invasive species can provide key insights into their invasion history and ensuing evolution, but also for their control. Here we genetically characterise populations of Impatiens glandulifera, an invasive plant in Europe that can have a major impact on native plant communities. We compared populations from the species' native range in Kashmir, India, to those in its invaded range, along a latitudinal gradient in Europe. For comparison, the results from 39 other studies of genetic diversity in invasive species were collated. RESULTS: Our results suggest that I. glandulifera was established in the wild in Europe at least twice, from an area outside of our Kashmir study area. Our results further revealed that the genetic diversity in invasive populations of I. glandulifera is unusually low compared to native populations, in particular when compared to other invasive species. Genetic drift rather than mutation seems to have played a role in differentiating populations in Europe. We find evidence of limitations to local gene flow after introduction to Europe, but somewhat less restrictions in the native range. I. glandulifera populations with significant inbreeding were only found in the species' native range and invasive species in general showed no increase in inbreeding upon leaving their native ranges. In Europe we detect cases of migration between distantly located populations. Human activities therefore seem to, at least partially, have facilitated not only introductions, but also further spread of I. glandulifera across Europe. CONCLUSIONS: Although multiple introductions will facilitate the retention of genetic diversity in invasive ranges, widespread invasive species can remain genetically relatively invariant also after multiple introductions. Phenotypic plasticity may therefore be an important component of the successful spread of Impatiens glandulifera across Europe.

Conyza canadensis suppresses plant diversity in its nonnative ranges but not at home: a transcontinental comparison.

The impact of invasive species across their native and nonnative ranges is poorly quantified and this impedes a complete understanding of biological invasions. We compared the impact of the native North American plant, Conyza canadensis, which is invasive to Eurasia, on species richness at home and in a number of introduced regions through well replicated transcontinental field studies, glasshouse experiments and individual-based models. Our results demonstrated mostly negative relationships between C. canadensis abundance and native species richness in nonnative ranges, but either positive or no relationships in its native North American range. In glasshouse experiments, the total biomass of Conyza was suppressed more by species from its native range than by species from regions where it is nonnative, but the effects of Conyza on other species did not show a consistent biogeographical pattern. Finally, individual-based models led to the exclusion of Conyza from North American scenarios but to high abundances in scenarios with species from the nonnative ranges of Conyza. We illustrate biogeographical differences in the impact of an invader across regional scales and suggest that inherent differences in one specific aspect of competitive ability, tolerance to the effects of other species, may play some role in these differences.

Uncovering the secret weapons of an invasive plant: The endophytic microbes of Anthemis cotula.

Understanding plant-microbe interaction can be useful in identifying the microbial drivers of plant invasions. It is in this context that we explored the diversity of endophytic microbes from leaves of Anthemis cotula, an annual plant that is highly invasive in Kashmir Himalaya. We also tried to establish the role of endophytes in the invasiveness of this alien species. We collected and processed leaf samples from three populations at three different sites. A total of 902 endophytic isolates belonging to 4 bacterial and 2 fungal phyla were recovered that belonged to 27 bacterial and 14 fungal genera. Firmicutes (29.1%), Proteobacteria (24.1%), Ascomycota (22.8%) and Actinobacteria (19%) were dominant across all samples. Plant growth promoting traits, such as Ammonia production, Indole Acetic Acid (IAA) production, Phosphate solubilization and biocontrol activity of these endophytes were also studied and most of the isolates (74.68%) were positive for ammonia production. IAA production, phosphate solubilization and biocontrol activity was present in 39.24%, 36.70% and 20.26% isolates, respectively. Furthermore, Botrytis cinerea, a pathogen of A. cotula in its native range, though present in Kashmir Himalaya does not affect A. cotula probably due to the presence of leaf endophytic microbial antagonists. Our results highlight that the beneficial plant growth promoting interactions and enemy suppression by leaf endophytes of A. cotula, may be contributing to its survival and invasion in the Kashmir Himalaya.

Insights into the seed microbiome and its ecological significance in plant life.

In recent years, the microbiome has attracted much attention because of the multiple roles and functions that microbes play in plants, animals, and human beings. Seed-associated microbes are of particular interest in being the initial microbial inoculum that affects the critical early life stages of a plant. The seed-microbe interactions are also known to improve nutrient acquisition, resilience against pathogens, and resistance against abiotic stresses. Despite these diverse roles, the seed microbiome has received little attention in plant ecology. Thus, we review the current knowledge on seed microbial diversity, community structure, and functions obtained through culture-dependent and culture-independent approaches. Furthermore, we present a comprehensive synthesis of the ecological literature on seed-microbe interactions to better understand the impact of these interactions on plant health and productivity. We suggest that future research should focus on the role of the seed microbiome in the establishment, colonization and spread of plant species in their native and non-native ranges as it may provide new insights into conservation biology and invasion ecology.

Rapid upwards spread of non-native plants in mountains across continents.

High-elevation ecosystems are among the few ecosystems worldwide that are not yet heavily invaded by non-native plants. This is expected to change as species expand their range limits upwards to fill their climatic niches and respond to ongoing anthropogenic disturbances. Yet, whether and how quickly these changes are happening has only been assessed in a few isolated cases. Starting in 2007, we conducted repeated surveys of non-native plant distributions along mountain roads in 11 regions from 5 continents. We show that over a 5- to 10-year period, the number of non-native species increased on average by approximately 16% per decade across regions. The direction and magnitude of upper range limit shifts depended on elevation across all regions. Supported by a null-model approach accounting for range changes expected by chance alone, we found greater than expected upward shifts at lower/mid elevations in at least seven regions. After accounting for elevation dependence, significant average upward shifts were detected in a further three regions (revealing evidence for upward shifts in 10 of 11 regions). Together, our results show that mountain environments are becoming increasingly exposed to biological invasions, emphasizing the need to monitor and prevent potential biosecurity issues emerging in high-elevation ecosystems.

Phyllosphere microbiome: Diversity and functions.

Phyllosphere or aerial surface of plants represents the globally largest and peculiar microbial habitat that inhabits diverse and rich communities of bacteria, fungi, viruses, cyanobacteria, actinobacteria, nematodes, and protozoans. These hyperdiverse microbial communities are related to the host's specific functional traits and influence the host's physiology and the ecosystem's functioning. In the last few years, significant advances have been made in unravelling several aspects of phyllosphere microbiology, including diversity and microbial community composition, dynamics, and functional interactions. This review highlights the current knowledge about the assembly, structure, and composition of phyllosphere microbial communities across spatio-temporal scales, besides functional significance of different microbial communities to the plant host and the surrounding environment. The knowledge will help develop strategies for modelling and manipulating these highly beneficial microbial consortia for furthering scientific inquiry into their interactions with the host plants and also for their useful and economic utilization.

Anthropogenic activities and geographic locations regulate microbial diversity, community assembly and species sorting in Canadian and Indian freshwater lakes.

Freshwater lakes are important reservoirs and sources of drinking water globally. However, the microbiota, which supports the functionality of these ecosystems is threatened by the influx of nutrients, heavy metals and other toxic chemical substances from anthropogenic activities. The influence of these factors on the diversity, assembly mechanisms and co-occurrence patterns of bacterial communities in freshwater lakes is not clearly understood. Hence, samples were collected from six different impacted lakes in Canada and India and examined by 454-pyrosequencing technology. The trophic status of these lakes was determined using specific chemical parameters. Our results revealed that bacterial diversity and community composition was altered by both the lake water chemistry and geographic distance. Anthropogenic activities pervasively influenced species distribution. Dispersal limitation (32.3%), homogenous selection (31.8%) and drift (20%) accounted for the largest proportions of the bacterial community assembly mechanisms. Homogenous selection increased in lakes with higher nutrient concentration, while stochasticity reduced. Community functional profiles revealed that deterministic processes dominated the assembly mechanisms of phylotypes with higher potential for biodegradation, while stochasticity dominated the assembly of phylotypes with potential for antimicrobial resistance. Bacteroidota (44%) and Proteobacteria (34%) were the most abundant phyla. Co-occurrence network analysis revealed that complexity increased in more impacted lakes, while competition and the nature of anthropogenic activity contributed to species sorting. Overall, this study demonstrates that bacterial community changes in freshwater lakes are linked to anthropogenic activities, with corresponding consequences on the distribution of phylotypes of environmental and human health interest.

Think globally, measure locally: The MIREN standardized protocol for monitoring plant species distributions along elevation gradients.

Climate change and other global change drivers threaten plant diversity in mountains worldwide. A widely documented response to such environmental modifications is for plant species to change their elevational ranges. Range shifts are often idiosyncratic and difficult to generalize, partly due to variation in sampling methods. There is thus a need for a standardized monitoring strategy that can be applied across mountain regions to assess distribution changes and community turnover of native and non-native plant species over space and time. Here, we present a conceptually intuitive and standardized protocol developed by the Mountain Invasion Research Network (MIREN) to systematically quantify global patterns of native and non-native species distributions along elevation gradients and shifts arising from interactive effects of climate change and human disturbance. Usually repeated every five years, surveys consist of 20 sample sites located at equal elevation increments along three replicate roads per sampling region. At each site, three plots extend from the side of a mountain road into surrounding natural vegetation. The protocol has been successfully used in 18 regions worldwide from 2007 to present. Analyses of one point in time already generated some salient results, and revealed region-specific elevational patterns of native plant species richness, but a globally consistent elevational decline in non-native species richness. Non-native plants were also more abundant directly adjacent to road edges, suggesting that disturbed roadsides serve as a vector for invasions into mountains. From the upcoming analyses of time series, even more exciting results can be expected, especially about range shifts. Implementing the protocol in more mountain regions globally would help to generate a more complete picture of how global change alters species distributions. This would inform conservation policy in mountain ecosystems, where some conservation policies remain poorly implemented.