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Milk thistle is one of the most popular hepatoprotectants, and is often sold in combination with other ingredients. Botanical supplements are known to be vulnerable to contamination and adulteration, and emerging technologies show promise to improve their quality control. Untargeted and semi-targeted metabolomics based on UHPLC-QTOF-ESI+MS techniques and DNA metabarcoding using Illumina MiSeq were used to authenticate eighteen milk thistle botanical preparations (teas, capsules, tablets, emulsion). Untargeted metabolomics separated 217 molecules and using multivariate analysis discrimination of the different groups was established. Semi-targeted metabolomics focused on 62 phytochemicals, mainly silymarin flavolignans that act as putative biomarkers of authenticity. All formulations contained molecules from silymarin complexes at different levels. The quantitative evaluation of silybins was done using in parallel UV spectrometry and UHPLC-QTOF-ESI+MS and their correlations were compared. DNA metabarcoding detected milk thistle in eleven out of sixteen retained preparations, whereas two others had incomplete evidence of milk thistle despite metabolomics validating its presence. Metabolomics enabled the identification and quantification of marker compounds in the samples, while DNA metabarcoding provided insights into the total species composition allowing the interpretation of the results in a broad context. Our study emphasizes that combining spectroscopic, chromatographic, and genetic techniques brings complementary information to guarantee the quality of the botanical formulations.

1. Several studies have shown the potential of eDNA-based proxies for plant identification, but little is known about their spatial and temporal resolution. This limits its use for plant biodiversity assessments and monitoring of vegetation responses to environmental changes. Here we calibrate the temporal and spatial plant signals detected with soil eDNA surveys by comparing with a standard visual above-ground vegetation survey. 2. Our approach compares vegetation in an old-growth boreal forest in southern Norway, surveyed in 100 permanent 1-m2 plots seven times over a 30-year period, with a single soil eDNA metabarcoding-based survey from soil samples collected at the same 100 plots in the year of the last vegetation survey. 3. On average, 60% and 10% of the vascular plants and bryophytes recorded across all vegetation surveys were detected by soil eDNA. Taxa detected by soil eDNA were more representative for the local taxa pool than for the specific plot, and corresponded to those surveyed over the 30-year period although most closely matched the current taxa composition. Soil eDNA detected abundant taxa better than rare ones although both rare taxa and taxa unrecorded by the visual survey were detected. 4. Our study highlights the potential of soil eDNA assessments for monitoring of vegetation responses over broad spatial and temporal scales. The method's ability to detect abundant taxa makes it suitable for assessment of vegetation composition in a specific area and for broad-scale plant diversity assessments.

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., Molecular Ecology, 30 (19), ISSN:0962-1083, ISSN:1365-294X

Highlights: • A chiral HPLC method was validated to determine usnic acid (UA) enantiomer ratios. • Molecular dynamics simulation revealed chiral chromatographic mechanisms. • MS imaging was used for spatial distribution of UA in lichen cross sections. • Fluorescence microscopy was used for spatial imaging of UA in lichen cross sections. Abstract: Usnic acid is an antibiotic metabolite produced by a wide variety of lichenized fungal lineages. The enantiomers of usnic acid have been shown to display contrasting bioactivities, and hence it is important to determine their spatial distribution, amounts and enantiomeric ratios in lichens to understand their roles in nature and grasp their pharmaceutical potential. The overall aim of the study was to characterise the spatial distribution of the predominant usnic acid enantiomer in lichens by combining spatial imaging and chiral chromatography. Specifically, separation and quantification of usnic acid enantiomers in four common lichens in Iceland was performed using a validated chiral chromatographic method. Molecular dynamics simulation was carried out to rationalize the chiral separation mechanism. Spatial distribution of usnic acid in the lichen thallus cross-sections were analysed using Desorption Electrospray Ionization-Imaging Mass Spectrometry (DESI-IMS) and fluorescence microscopy. DESI-IMS confirmed usnic acid as a cortical compound, and revealed that usnic acid can be more concentrated around the algal vicinity. Fluorescence microscopy complemented DESI-IMS by providing more detailed distribution information. By combining results from spatial imaging and chiral separation, we were able to visualize the distribution of the predominant usnic acid enantiomer in lichen cross-sections: (+)-usnic acid in Cladonia arbuscula and Ramalina siliquosa, and (−)-usnic acid in Alectoria ochroleuca and Flavocetraria nivalis. This study provides an analytical foundation for future environmental and functional studies of usnic acid enantiomers in lichens.

The study of faecal samples to reconstruct the diets and habitats of extinct megafauna has traditionally relied on pollen and macrofossil analysis. DNA metabarcoding has emerged as a valuable tool to complement and refine these proxies. While published studies have compared the results of these three proxies for sediments, this comparison is currently lacking for permafrost preserved mammal faeces. Moreover, most metabarcoding studies have focused on a single plant-specific DNA marker region. In this study, we target both the commonly used chloroplast trnL P6 loop as well as nuclear ribosomal ITS (nrITS). The latter can increase taxonomic resolution of plant identifications but requires DNA to be relatively well preserved because of the target length (∼300–500 bp). We compare DNA results to pollen and macrofossil analyses from permafrost and ice-preserved faeces of Pleistocene and Holocene megafauna. Samples include woolly mammoth, horse, steppe bison as well as Holocene and extant caribou. Most plant identifications were found using DNA, likely because the studied faeces contained many vegetative remains that could not be identified using macrofossils or pollen. Several taxa were, however, identified to lower taxonomic levels uniquely with macrofossil and pollen analysis. The nrITS marker provides species level taxonomic resolution for commonly encountered plant families that are hard to distinguish using the other proxies (e.g. Asteraceae, Cyperaceae and Poaceae). Integrating the results from all proxies, we are able to accurately reconstruct known diets and habitats of the extant caribou. Applying this approach to the extinct mammals, we find that the Holocene horse and steppe bison were not strict grazers but mixed feeders living in a marshy wetland environment. The mammoths showed highly varying diets from different non-analogous habitats. This confirms the presence of a mosaic of habitats in the Pleistocene ‘mammoth steppe’ that mammoths could fully exploit due to their flexibility in food choice.

River and sea ecosystem services contribute to the rapid and intensive development of delta social-ecological systems. This development, alongside other global change drivers, threatens the biodiversity of these deltas and in turn the ecosystem services that this biodiversity supports. However, biodiversity can itself mediate anthropogenic impacts by increasing ecosystem resilience. Linkages between biodiversity and ecosystem services are increasingly established, but we lack understanding of whether the mediating effects of biodiversity are global and ubiquitous, and whether they mediate global change drivers in deltas.Here, we examine the potential for biodiversity to mediate the relationships between five anthropogenic indicators and global change drivers (population, infrastructure, land use change, climate change in temperature and precipitation) and 19 ecosystem properties and services. We assess these relationships across a global dataset of 235 large deltas. We find that in 89% of cases, greater biodiversity (species richness and the intactness of biodiversity) is connected to a weakened or reversed association between anthropogenic drivers and ecosystem services. Such weaker or reversed associations were found across different ecosystem services (e.g. food production, carbon sequestration, soil regulation), most commonly with climate change and population.We then investigated the contribution of biodiversity and abiotic and anthropogenic drivers to delta ecosystem service supply. Ecosystem service supply was most strongly and consistently associated with abiotic drivers (mostly climatic), but biodiversity and anthropogenic drivers were also important to individual services (productivity and crop-related services respectively). Deltas showed fewer than expected associations between biotic, abiotic and anthropogenic indicators and ecosystem services, yet weakened or reversed associations were more frequent than in other social-ecological systems. Our results empirically show how biodiversity can both act as a resource and mediate social-ecological relationships, but that both of these roles could be compromised as deltas become more modified.

Current knowledge about Paleolithic human plant use is limited by the rare survival of identifiable plant remains as well as the availability of methods for plant detection and identification. By analyzing DNA preserved in cave sediments, we can identify organisms in the absence of any visible remains, opening up new ways to study details of past human behavior, including plant use. Aghitu-3 Cave contains a 15,000-yearlong record (from ∼39,000 to 24,000 cal BP) of Upper Paleolithic human settlement and environmental variability in the Armenian Highlands. Finds from this cave include stone artifacts, faunal remains, bone tools, shell beads, charcoal, and pollen, among others. We applied sedimentary ancient DNA (sedaDNA) metabarcoding to the Aghitu-3 sedimentary sequence and combined this with pollen data to obtain a temporal reconstruction of plant assemblages. Our results reveal a stratification of plant abundance and diversity where sedaDNA reflects periods of human occupation, showing higher diversity in layers with increased human activity. Low pollen concentrations combined with high sedaDNA abundance indicate plant remains may have been brought into the cave by animals or humans during the deposition of the lower two archaeological horizons. Most of the recovered plants are reported to be useful for food, flavor, medicine, and/or technical purposes, demonstrating the potential of the environment around Aghitu-3 Cave to support humans during the Upper Paleolithic. Moreover, we identified several specific plant taxa that strengthen previous findings about Upper Paleolithic plant use in this region (i.e., for medicine and the manufacturing and dyeing of textiles). This study represents the first application of plant sedaDNA analysis of cave sediments for the investigation of potential plant use by prehistoric humans.

Plants are highly sensitive to the water status of the soil in which they grow, with too little soil moisture causing drought stress whereas too much soil moisture causing flooding stress. This stress in response to opposing water conditions can be understood from the fact that plant growth demands both sufficient water uptake from the soil and rapid gas exchange with the environment. Given that drought and flooding events can occur in the same system and even consecutively, a single model simulating plant responses to a continuum of soil water conditions from drought to flooding would be attractive. However, as far as we know, such a model with sufficient mechanistic biological details currently does not exist. In this study we propose a theoretical framework of an integrated mechanistic model that is capable of describing plant responses to both flooding and drought, building on the biophysics of plant water transport and gas exchange and its dependence on environmental conditions. Since the restricted root water uptake and stomatal activity that limits gas exchange through photosynthesis are essential processes in both scenarios, we propose using a combined SPAC-Farquhar model that describes these processes as a “backbone” of the envisioned model framework. Further we propose to add processes related to oxygen dynamics and hormonal signaling, as oxygen deficit serves as the main driver of flooding stress to which hormonal signaling plays an essential role in plant response. The model aims to mimic various responsive strategies by different plant species. These strategies include isohydric and anisohydric strategies for drought response, and “escape” and “quiescence” strategies for flooding response. Stomatal activities of isohydric plants are reported to be more sensitive to leaf water potential but less sensitive to abscisic acid compared to those of anisohydric plants. This can be achieved by assigning different sensitivity coefficients. Plants that are tolerant and adaptive under flooding stress can generally switch between the “escape” and “quiescence” strategy, depending on shoot ethylene concentration. This integrated model framework is envisioned to mimic the complex behavior of plant responses to consecutive drought and flooding events, as the physiological processes involved occur on various time scales, ranging from sub-hours to weeks. Moreover, hormones and certain irreversible morphological changes can serve as “memory factors”, leading to the history-dependent nature of plant responsive behavior. We hope that the proposed theoretical model framework will serve as a basis for model research on resilience to combined drought and flooding in both agriculture and natural vegetation systems.

The promotion of responsible and sustainable trade in biological resources is widely proposed as one solution to mitigate currently high levels of global biodiversity loss. Various molecular identification methods have been proposed as appropriate tools for monitoring global supply chains of commercialized animals and plants. We demonstrate the efficacy of target capture genomic barcoding in identifying and establishing the geographic origin of samples traded as Anacyclus pyrethrum, a medicinal plant assessed as globally vulnerable in the IUCN Red List of Threatened Species. Samples collected from national and international supply chains were identified through target capture sequencing of 443 low-copy nuclear makers and compared to results derived from genome skimming of plastome, standard plastid barcoding regions and ITS. Both target capture and genome skimming provided approximately 3.4 million reads per sample, but target capture largely outperformed standard plant DNA barcodes and entire plastid genome sequences. Despite the difficulty of distinguishing among closely related species and infraspecific taxa of Anacyclus using conventional taxonomic methods, we succeeded in identifying 89 of 110 analysed samples to subspecies level without ambiguity through target capture. Of the remaining samples, we determined that eleven contained plant material from other genera and families and ten were unidentifiable regardless of the method used. Furthermore, we were able to discern the geographical origin of Anacyclus samples collected in Moroccan, Indian and Sri Lankan markets, differentiating between plant materials originally harvested from diverse populations in Algeria and Morocco. With a recent drop in the cost of analysing samples, target capture offers the potential to routinely identify commercialized plant species and determine their geographic origin. It promises to play an important role in monitoring and regulation of plant species in trade, supporting biodiversity conservation efforts, and in ensuring that plant products are unadulterated, contributing to consumer protection.

Cultural Effects on Sorghum Varieties Grown, Traits Preferred, and Seed Management Practices in Northern Ethiopia. Agrobiodiversity is fundamentally shaped by farmers’ preferences and management practices, and these are again shaped by the farmers’ social and cultural background. This study investigates variety preferences and seed management practices in the crop sorghum (Sorghum bicolor) among the Kunama and the Tigrayan ethnolinguistic groups living side by side in Northern Ethiopia. Surveys were conducted in 10 villages located in two districts inhabited by the two ethnolinguistic groups and analyzed using descriptive and multinominal analysis. We find important differences in varieties grown across the ethnolinguistic groups, but we also find that ethnicity and geographic proximity interact and affect trait preference and seed management practices. Altogether, 22 varieties are cultivated, and few farmers cultivated improved varieties, especially among the Kunama. Respondents considered use traits as important as agronomic traits when selecting sorghum varieties. Notably, preferred use traits were not limited to food (e.g., construction materials were important for the Kunama), and preferred agronomic traits were not focused on drought resistance. Timing of seed selection, seed selection criteria (e.g., panicle size and color), and seed storage practices differed among the ethnic groups. Understanding cultural and social preferences towards sorghum varieties, their traits, and the criteria used for seed management is crucial for the success of crop breeding programs, climate change adaptation policies, and development interventions.

Plant responses to abiotic stresses are complex and dynamic, and involve changes in different traits, either as the direct consequence of the stress, or as an active acclimatory response. Abiotic stresses frequently occur simultaneously or in succession, rather than in isolation. Despite this, most studies have focused on a single stress and single or few plant traits. To address this gap, our study comprehensively and categorically quantified the individual and combined effects of three major abiotic stresses associated with climate change (flooding, progressive drought and high temperature) on 12 phenotypic traits related to morphology, development, growth and fitness, at different developmental stages in four

International audience; Global vegetation and land-surface models embody interdisciplinary scientific understanding of the behaviour of plants and ecosystems, and are indispensable to project the impacts of environmental change on vegetation and the interactions between vegetation and climate. However, systematic errors and persistently large differences among carbon and water cycle projections by different models highlight the limitations of current process formulations. In this review, focusing on core plant functions in the terrestrial carbon and water cycles, we show how unifying hypotheses derived from eco-evolutionary optimality (EEO) principles can provide novel, parameter-sparse representations of plant and vegetation processes. We present case studiesthat demonstrate how EEO generate parsimonious representations of core, leaf-level processes that are individually testable and supported by evidence. EEO approaches to photosynthesis and primary production, dark respiration, and stomatal behaviour are ripe for implementation in global models. EEO approaches to other important traits, including the leaf economics spectrum and applications of EEO at the community level are active research areas. Independently tested modules emerging from EEO studies could profitably be integrated into modelling frameworks that account for the multiple time scales on which plants and plant communities adjust toenvironmental change.

Airborne pollen monitoring is of global socio-economic importance as it provides information on presence and prevalence of allergenic pollen in ambient air. Traditionally, this task has been performed by microscopic investigation, but novel techniques are being developed to automate this process. Among these, DNA metabarcoding has the highest potential of increasing the taxonomic resolution, but uncertainty exists about whether the results can be used to quantify pollen abundance. In this study, it is shown that DNA metabarcoding using trnL and nrITS2 provides highly improved taxonomic resolution for pollen from aerobiological samples from the Netherlands. A total of 168 species from 143 genera and 56 plant families were detected, while using a microscope only 23 genera and 22 plant families were identified. NrITS2 produced almost double the number of OTUs and a much higher percentage of identifications to species level (80.1%) than trnL (27.6%). Furthermore, regressing relative read abundances against the relative abundances of microscopically obtained pollen concentrations showed a better correlation for nrITS2 (R2 = 0.821) than for trnL (R2 = 0.620). Using three target taxa commonly encountered in early spring and fall in the Netherlands (Alnus sp., Cupressaceae/Taxaceae and Urticaceae) the nrITS2 results showed that all three taxa were dominated by one or two species (Alnus glutinosa/incana, Taxus baccata and Urtica dioica). Highly allergenic as well as artificial hybrid species were found using nrITS2 that could not be identified using trnL or microscopic investigation (Alnus × spaethii, Cupressus arizonica, Parietaria spp.). Furthermore, perMANOVA analysis indicated spatiotemporal patterns in airborne pollen trends that could be more clearly distinguished for all taxa using nrITS2 rather than trnL. All results indicate that nrITS2 should be the preferred marker of choice for molecular airborne pollen monitoring., Graphical abstract Unlabelled Image, Highlights • DNA successfully extracted from microscopic pollen slides and Burkard-collected tapes. • nrITS2 and trnL DNA metabarcoding improves taxonomic resolution of airborne pollen. • Relative read abundance nrITS2 shows higher correlation with pollen counts than trnL. • Finer scale spatiotemporal patterns in pollen trends detected using nrITS2. • Detection of artificial hybrid (Alnus × spaethii) that significantly prolongs hay fever season

Monitoring of airborne pollen concentrations provides an important source of information for the globally increasing number of hay fever patients. Airborne pollen is traditionally counted under the microscope, but with the latest developments in image recognition methods, automating this process has become feasible. A challenge that persists, however, is that many pollen grains cannot be distinguished beyond the genus or family level using a microscope. Here, we assess the use of Convolutional Neural Networks (CNNs) to increase taxonomic accuracy for airborne pollen. As a case study we use the nettle family (Urticaceae), which contains two main genera (Urtica and Parietaria) common in European landscapes which pollen cannot be separated by trained specialists. While pollen from Urtica species has very low allergenic relevance, pollen from several species of Parietaria is severely allergenic. We collect pollen from both fresh as well as from herbarium specimens and use these without the often used acetolysis step to train the CNN model. The models show that unacetolyzed Urticaceae pollen grains can be distinguished with > 98% accuracy. We then apply our model on before unseen Urticaceae pollen collected from aerobiological samples and show that the genera can be confidently distinguished, despite the more challenging input images that are often overlain by debris. Our method can also be applied to other pollen families in the future and will thus help to make allergenic pollen monitoring more specific.

Estimating time-dependent rates of speciation and extinction from dated phylogenetic trees of extant species (timetrees), and determining how and why they vary, is key to understanding how ecological and evolutionary processes shape biodiversity. Due to an increasing availability of phylogenetic trees, a growing number of process-based methods relying on the birth-death model have been developed in the last decade to address a variety of questions in macroevolution. However, this methodological progress has regularly been criticised such that one may wonder how reliable the estimations of speciation and extinction rates are. In particular, using lineages-through-time (LTT) plots, a recent study (Louca and Pennell, 2020) has shown that there are an infinite number of equally likely diversification scenarios that can generate any timetree. This has lead to questioning whether or not diversification rates should be estimated at all. Here we summarize, clarify, and highlight technical considerations on recent findings regarding the capacity of models to disentangle diversification histories. Using simulations we demonstrate the characteristics of newly-proposed “pulled rates” and their utility. We recognize that the recent findings are a step forward in understanding the behavior of macroevolutionary modelling, but they in no way suggest we should abandon diversification modelling altogether. On the contrary, the study of macroevolution using phylogenetic trees has never been more exciting and promising than today. We still face important limitations in regard to data availability and methodological shortcomings, but by acknowledging them we can better target our joint efforts as a scientific community.

Growing fishing pressure worldwide has led to an increase in the exploitation of cephalopod products in fish markets where these taxa were not traditionally consumed. Squid catches have surged to meet that demand, yet for many species little is known about their role in food webs. Doryteuthis sanpaulensis is an important squid species in southeastern Brazilian fisheries. Despite many previous efforts at morphological analysis of its diet, few demersal and benthic species of fishes, crustaceans and mollusks have been identified to species level because the food is consumed in small digestible fragments. Here we used metabarcoding to analyze the diet of adult D. sanpaulensis caught as bycatch in the southern Brazilian sardine fishery. MOTUs generated from COI amplicons were assigned to taxa by matching against the NCBI nt database. Fishes constitute the majority of the diet of the analyzed samples. Considerable variability in the relative read abundance and frequency of occurrence of prey items across samples indicates the importance of increased sample sizes in analyses investigating ontogenetic, spatial, or temporal variation in diet. The results elucidate the rich diet of D. sanpaulensis off the Brazilian coast, and specifically that its varied diet includes more neritic diversity than previous studies have indicated.

Ginseng traditional medicines and food supplements are the globally top selling herbal products. Panax ginseng, Panax quinquefolius and Panax notoginseng are the main commercial ginseng species in herbal medicine. Prices of ginseng products vary widely based on the species, quality, and purity of the used ginseng, and this provides a strong driver for intentional adulteration. Our systematic literature search has reviewed the authenticity results of 507 ginseng-containing commercial herbal products sold in 12 countries scattered across six continents. The analysis of the botanical and chemical identity of all these products shows that 76% are authentic while 24% were reported as adulterated. The number of commercial products as well as the percentage of adulteration varies significantly between continents, being highest in South America (100%) and Australia (75%), and lower in Europe (35%), North America (23%), Asia (21%) and Africa (0%). At a national level, from the five countries for which more than 10 products have been successfully authenticated, the highest percentage of adulterated ginseng products were purchased from Taiwan (49%), followed by Italy (37%), China (21%), and USA (12%), while all products bought in South Korea were reported to be authentic. In most cases, labeled Panax species were substituted with other Panax species, but substitution of ginseng root, the medicinally recommended plant part, with leaves, stems or flowers was also reported. Efficient and practical authentication using biomarkers to distinguish the main ginseng varieties and secondary metabolite spectra for age determination are essential to combat adulteration in the global marketplace.

Water is an essential resource for human life and the environment. The widespread use of chemicals in daily life has led to significant water quality concerns. During the production phase, the use phase as well as after use, (residues of) these chemicals can enter the environment and water systems. Furthermore, the use and production of chemicals are increasing rapidly, driven by mainly population growth, urbanisation and economic growth. Increased use leads to further emissions of chemicals to water, posing significant water quality concerns. Henceforth there is an urgent need to understand the linkage between society and production and consumption of chemicals to explore possible changes in water quality.Socio-economic scenario analysis is a useful tool to investigate the long-term consequences of future change and mitigation options. While scenarios have been broadly applied to understand air pollution, this not yet the case for chemical pollution to surface waters. In this work, we propose a general framework to develop scenarios for the future emissions of chemicals to water by using the Shared Socio-economic Pathways (SSP). The framework follows the basic elements of the scenario development process by defining the current system, describe the changes in emissions with scenario drivers and elaboration to the future. The framework is then tested on a set of selected ‘example’ chemicals that represent broader chemical groups of pharmaceuticals (Ibuprofen and Diclofenac), pesticides (Terbuthylazine) and industrial chemicals (Cadmium and Di-ethyl phthalate). Chemical emissions to water over the past years were used to understand their yearly trends and patterns over the European countries. Lastly, the emission scenarios for chemicals for 2050 were developed by using SSP drivers from the IMAGE Integrated assessment model as an input to the empirical emission models. The three SSP scenarios: SSP1 ("Sustainability"), SSP2 ("Middle of the Road") and SSP3 ("Regional Rivalry") focusing on Europe were included. Additionally, the developed scenarios also describe mitigation efforts.The results of emission scenarios displayed an increase in emissions up to 2050 for the exemplary chemicals in Western Europe for all three scenarios SSP1, SSP2 and SSP3. While the emissions of chemicals linearly decreased in Eastern Europe for the same period. SSP3 showed the highest emissions in 2050 except for cadmium emissions from wastewater treatment plants. The results showed that the framework helps in understanding the possible influence of socio-economic changes on use and emissions of chemicals which can be a part of future risk assessments. While the framework can be extended similarly to other pharmaceuticals and pesticides, it requires a detailed understanding of complex emission sources for industrial chemicals.

Background and aims – Many Aloe species are globally threatened due to overharvesting for trade and habitat destruction. CITES regulates their international trade. In Tanzania, 50% of all existing Aloe species had previously been assessed, though some of these assessments were Data Deficient. For those with sufficient data, an update is required as the rate of decline has rapidly increased over the last years. Material and methods – We estimated Area of Occupancy (AOO), Extent of Occurrence (EOO), and number of locations for 22 Tanzanian Aloe species using the Geospatial Conservation Assessment software (GeoCAT). We assessed the reasons leading to their decline based on direct field observations and community perceptions. Key results – We revised the conservation status of 22 Aloe species; two were assessed as Critically Endangered, ten as Endangered, five as Vulnerable, and five as Least Concern. We re-discovered the Critically Endangered Aloe boscawenii, which had not been seen in Tanzania for more than six decades. We propose to downgrade the endemic Aloe dorotheae, Aloe leptosiphon, and Aloe flexilifolia from Critically Endangered to a lower threat level. The community perception on Aloe species availability did not accurately reflect their categorisation based on the IUCN criteria B. We identified agricultural activities and climate change effects as the two main threats to Tanzanian Aloe species.Conclusion – We conclude that overall numbers are declining for 22 Aloe species in Tanzania, mainly due to human activities. We recommend the implementation of laws and policies to protect their natural habitats.

Monitoring of airborne pollen concentrations provides an important source of information for the globally increasing number of hay fever patients. Airborne pollen are traditionally counted under the microscope, but with the latest developments in image recognition methods, automating this process has become feasible. A challenge that persists, however, is that many pollen grains cannot be distinguished beyond the genus or family level using a microscope. Here, we assess the use of a Convolutional Neural Network (CNN) to increase taxonomic accuracy for airborne pollen. As a case study we use the nettle family (Urticaceae), which contains two main genera (Urtica and Parietaria) common in European landscapes which pollen cannot be separated by trained specialists. While pollen from Urtica species have very low allergenic relevance, those from several species of Parietaria are severely allergenic. We collect pollen from both fresh as well as from herbarium specimens and use these to train the CNN model VGG16. The model shows that Urticaceae pollen can be distinguished with 98.3% accuracy. We then apply our model on Urticaceae pollen collected from aerobiological samples and show that the genera can be confidently distinguished, despite the more challenging input images that are often overlain by debris. Our method can also be applied to other pollen families in the future and will thus help to make allergenic pollen monitoring more specific.

Silene (Caryophyllaceae) is distributed predominantly in the northern Hemisphere, where it is most diverse around the Mediterranean Basin. The genus is also well represented in North Africa, extending into tropical, sub-Saharan and southern Africa. Eight native species are recognized in southern Africa, taxonomically placed in two sections: Elisanthe and Silene s.l. Although the taxonomy of the southern African taxa has recently been revised, their phylogenetic relationships and biogeographic history remain unclear. This study aims to infer the phylogenetic position and geographic origins of the southern African taxa. We generated DNA sequences of nuclear and plastid loci from several individuals belonging to all eight species of Silene recognized from southern Africa, and combined our DNA sequences with existing data representing species from major clades (i.e. sections) based on the recently revised Silene infrageneric taxonomy. We used a Bayesian coalescent species tree continuous diffusion approach to co-estimate the species tree and the ancestral areas of representative members of the genus. Our results show that the perennial southern African members of section Elisanthe form a strongly-supported clade with the Eurasian annual S. noctiflora and the Central Asian perennial S. turkestanica. The rest of the perennial species form a strongly-supported clade together with the annual S. aethiopica, which is nested in a larger Mediterranean clade comprising mostly annual species classified in section Silene s.l. Estimates of ancestral areas indicate a late Pleistocene dispersal to southern Africa from central and East Africa for the sub-Saharan members of section Silene s.l. The Elisanthe clade is inferred to have colonized southern Africa through long-distance dispersal from Eurasia during the late Pleistocene. Our findings support the hypothesis of a relatively recent colonization into southern Africa resulting from two independent dispersal events during the Pleistocene.

Background Stomata analysis using microscope imagery provides important insight into plant physiology, health and the surrounding environmental conditions. Plant scientists are now able to conduct automated high-throughput analysis of stomata in microscope data, however, existing detection methods are sensitive to the appearance of stomata in the training images, thereby limiting general applicability. In addition, existing methods only generate bounding-boxes around detected stomata, which require users to implement additional image processing steps to study stomata morphology. In this paper, we develop a fully automated, robust stomata detection algorithm which can also identify individual stomata boundaries regardless of the plant species, sample collection method, imaging technique and magnification level. Results The proposed solution consists of three stages. First, the input image is pre-processed to remove any colour space biases occurring from different sample collection and imaging techniques. Then, a Mask R-CNN is applied to estimate individual stomata boundaries. The feature pyramid network embedded in the Mask R-CNN is utilised to identify stomata at different scales. Finally, a statistical filter is implemented at the Mask R-CNN output to reduce the number of false positive generated by the network. The algorithm was tested using 16 datasets from 12 sources, containing over 60,000 stomata. For the first time in this domain, the proposed solution was tested against 7 microscope datasets never seen by the algorithm to show the generalisability of the solution. Results indicated that the proposed approach can detect stomata with a precision, recall, and F-score of 95.10%, 83.34%, and 88.61%, respectively. A separate test conducted by comparing estimated stomata boundary values with manually measured data showed that the proposed method has an IoU score of 0.70; a 7% improvement over the bounding-box approach. Conclusions The proposed method shows robust performance across multiple microscope image datasets of different quality and scale. This generalised stomata detection algorithm allows plant scientists to conduct stomata analysis whilst eliminating the need to re-label and re-train for each new dataset. The open-source code shared with this project can be directly deployed in Google Colab or any other Tensorflow environment.

Wildlife trade is a threat to species survival and wild collected orchids are a notable component of it. In the first systematic patent review of any wild species, we find many innovative and industrial uses of orchids, using the example of traditional product salep made with wild harvested Mediterranean and Central Asian terrestrial orchids. Despite considerable interest in the conservation of these orchids, no systematic analysis of innovation and technology transfer had been conducted for natural products using them. Our systematic review on salep found 244 patent applications, of which 89 were granted, spanning 163 years from 1855 to 2018. Uses included industrial materials and contemporary medicine formulations. This recalibration of the value of salep using patent analysis shows that, rather than being restricted to two specific products of limited regional circulation, these orchids are a rich source of unique materials with myriad applications around the world. In order to conserve wild terrestrial orchids collected for salep, conservation science must engage with the full extent of the utility of and commercial interest in these particular orchids. Additionally we demonstrate how conservationists can use patents as a source of information on incipient commercial interest in wild species.

Ethnopharmalogical relevance In Africa, traditional medicine is important for local healthcare and plants used for these purposes are commonly traded. Identifying medicinal plants sold on markets is challenging, as leaves, barks and roots are often fragmented or powdered. Vernacular names are often homonymic, and identification of material lacking sufficient morphological characters is time-consuming, season-dependent and might lead to incorrect assessments of commercialised species diversity. Aim of the study In this study, we identified cases of vernacular heterogeneity of medicinal plants using a tiered approach of literature research, morphology and DNA barcoding. Material and methods A total of 870 single ingredient medicinal plant samples corresponding to 452 local names were purchased from herbal markets in Dar-es-Salaam and Tanga, Tanzania, and identified using conventional methods as well as DNA barcoding using rbcL, matK and nrITS. Results Using conventional methods, we could identify 70% of samples to at least family level, while 62% yielded a DNA barcode for at least one of the three markers. Combining conventional methods and DNA barcoding, 76% of the samples could be identified to species level, revealing a diversity of at least 175 species in 65 plant families. Analysis of the market samples revealed 80 cases of multilingualism and over- and under-differentiation. Afzelia quanzensis Welw., Zanthoxylum spp., Allophylus spp. and Albizia anthelmintica Brongn. were the most evident cases of multilingualism and over-differentiation, as they were traded under 8–12 vernacular names in up to five local languages. The most obvious case of under-differentiation was mwingajini (Swahili), which matched to eight scientific species in five different plant families. Conclusions Use of a tiered approach increases the identification success of medicinal plants sold in local market and corroborates findings that DNA barcoding can elucidate the identity of material that is unidentifiable based on morphology and literature as well as verify or disqualify these identifications. Results of this study can be used as a basis for quantitative market surveys of fragmented herbal medicine and to investigate conservation issues associated with this trade.

As many ecosystems are under increasing pressure from invasive species, habitat degradation, overgrazing and overharvesting, pollution, and climate change, dietary niche monitoring is gaining importance. The Malai Mahadeshwara Wildlife Sanctuary (MMH) in southern India is home to several long‐standing ethnic and tribal groups and supports a considerable number of domestic herbivores (cattle, goats and water buffalo) as well as a range of wildlife (including several species of deer, bonnet macaque, and Asian elephant). We reconstructed dietary niche partitioning of the herbivores occurring in MMH using eDNA metabarcoding to quantify diet richness, composition, and overlap. In total, we distinguish 134 diet items (molecular operational taxonomic units), covering 31 plant families. Overall, our results indicate 35% overlap in domestic and wild herbivore diet items. The greatest overlap is found for the dietary niches of cattle and sambar deer (Pianka's niche overlap index: 0.68), and the dietary niche of cattle also overlaps considerably with those of Indian hare (0.65) and Asian elephant (0.46). This suggests that these herbivores may compete for these food plants in the case of limited availability, which could lead to exclusion of some herbivore species. Particular concern should go to bonnet macaque and Asian elephant as their below average dietary richness could make them vulnerable to changes in their environment. With increasing pressures on local wildlife from a range of different factors, DNA metabarcoding of fecal samples is a non‐invasive method for monitoring changes in animal diets, providing valuable information for the management of biodiversity in mosaic natural and anthropogenic landscapes.

Background : The genus Aloe has long been known for its use in healthcare and cosmetics. In Tanzania, overexploitation is threatening some Aloe species with extinction and yet, little has been documented on the abundance and biocultural uses. Material and Methods: Semi-structured questionnaires were used to obtain ethnobotanical information from 236 respondents across 22 villages in four regions of Tanzania (Kilimanjaro, Tanga, Mara, Katavi-Rukwa). Results: A total of 23 Aloe species were identified, 20 of which were being used locally and were mostly being collected from the wild. We report the uses of A. mzimbana , A. volkensii subsp. volkensii, A. leptosiphon , A. parvidens and A. bicomitum for the first time in East Africa. The most preferred species were A. lateritia, A. duckeri and A. secundiflora which are three common, widely distributed species. Diseases frequently treated with Aloe species include malaria and general stomachache in humans, and Newcastle disease in chickens. Some Aloe species were found to have gone extinct locally due to over-harvesting. Rare Aloe species were perceived to be less preferred as they mostly occurred far away from settlements. Conclusions: We conclude that the genus Aloe is widely used across Tanzania and needs strong conservation measures to prevent individual species from becoming extinct in the wild. Keywords: Distribution; Ethnobotany; Folk taxonomy; Perception; Sustainable harvesting.

The resin of Canarium strictum Roxb. is used for rheumatism and asthma; the bark is used as a mosquito repellent. The major compounds in the resin are triterpenoids, but as no studies have been performed on the bark, this study investigated this economically important resource. Ten folk healers were interviewed about their medicinal uses of C. strictum. Resin and bark were extracted with dichloromethane followed by methanol using accelerated solvent extraction. The extracts were fractionated using different chromatographic methods, and isolated compounds were identified by NMR spectroscopy and GC–MS. Resin and bark extracts were investigated for DPPH radical scavenging, 15-lipoxygenase inhibition, effects on nitric oxide (NO) production in LPS-activated dendritic D2SC/I cells and toxicity against Artemia salina nauplii. Traditional healers used resin to treat colds, airway afflictions and rheumatoid arthritis. α-Amyrin and β-amyrin were identified as the major constituents in the dichloromethane resin extract. From the stem bark, procyanidins, gallic acid, methyl gallate, scopoletin, 3,3′-di-O-methylellagic acid 4-O-α-arabinofuranoside and elephantorrhizol (3,3′,4′,5,6,7,8-heptahydroxyflavan) were isolated and identified. By GC–MS, α-amyrin and β-amyrin and their acetates, lupeol, and taraxasterol were identified. Radical scavenging, 15-lipoxygenase inhibitory activity and inhibition of NO production was observed from resin and bark extracts, and no toxicity towards Artemia salina nauplii was found. Triterpenoids and procyanidins are the major compounds in C. strictum resin and stem bark, respectively. The high content of triterpenoids might contribute to anti-inflammatory effects and give a rationale for the widespread usage of the resin in India.

Herbal medicinal products gain increasing popularity. The growing demand for herbal medicine along with a lack of regulation render herbal products subject to intentional adulteration. The substitution of costly ingredients with unlabelled plant-based fillers of inferior quality has been widely reported. Such fraudulent practices erode consumer trust, but can also pose serious health risks. In this work, 71 herbal medicinal products were randomly purchased from Greek markets and analysed using ITS2 metabarcoding for species identification. The aim was to investigate possible adulterations and assess the efficacy of metabarcoding in plant-based product authentication. Of the 131 detected species in our analyses, 87 were not listed on the product labels. This indicates a high level of adulteration and/or contamination during processing and distribution. Two toxic species, Chelidonium majus and Nicotiana tabacum, were also detected as major ingredients of two herbal mixtures for medicinal purposes. Furthermore, the detection of wheat in eight samples raises concerns for people with gluten intolerance. This study stresses the need for stricter quality control of herbal products. In addition, to overcome the limitations of metabarcoding and augment the approach we used Bar-HRM for the first time as a verification tool. The combination of metabarcoding with species-specific Bar-HRM analysisenhances the reliability of the results.

Introduction: Herbal medicines play an important role globally in the health care sector and in industrialised countries they are often considered as an alternative to mono-substance medicines. Current quality and authentication assessment methods rely mainly on morphology and analytical phytochemistry-based methods detailed in pharmacopoeias. Herbal products however are often highly processed with numerous ingredients, and even if these analytical methods are accurate for quality control of specific lead or marker compounds, they are of limited suitability for the authentication of biological ingredients. / Objective: To review the benefits and limitations of DNA barcoding and metabarcoding in complementing current herbal product authentication. / Method: Recent literature relating to DNA based authentication of medicinal plants, herbal medicines and products are summarised to provide a basic understanding of how DNA barcoding and metabarcoding can be applied to this field. / Results: Different methods of quality control and authentication have varying resolution and usefulness along the value chain of these products. DNA barcoding can be used for authenticating products based on single herbal ingredients and DNA metabarcoding for assessment of species diversity in processed products, and both methods should be used in combination with appropriate hyphenated chemical methods for quality control. / Conclusions: DNA barcoding and metabarcoding have potential in the context of quality control of both well and poorly regulated supply systems. Standardisation of protocols for DNA barcoding and DNA sequence-based identification are necessary before DNA-based biological methods can be implemented as routine analytical approaches and approved by the competent authorities for use in regulated procedures.