Metabarcoding Analysis Research Articles

Chloroplast genome evolution of Berberis (Berberidaceae): Implications for phylogeny and metabarcoding

Berberidis Radix (Sankezhen), a typical multi-origin Chinese medicinal material, originates from the dried roots of plants of the Berberis genus and is used to treat various ailments. These species have similar morphologies, potentially leading to misidentifications that can impact medicine efficacy. Therefore, developing suitable molecular markers to identify medicinal species is imperative. Furthermore, discrepancies exist in the taxonomy of the Berberis genus. In the present study, we de novo assembled the chloroplast genomes of six Berberis species (Berberis woomungensis C. Y. Wu, Berberis pruinosa Franch., Berberis thunbergii DC., Berberis chinensis Poir., Berberis wilsoniae Hemsl., and Berberis sp.) that commonly constitute Berberidis Radix and compared them with previously reported genomes. Our comparative analysis revealed similarities in genome structure, relative synonymous codon usage, amino acid frequency, repeats, and substitutions. Higher synonymous substitutions, indicative of predominant purifying selection on protein-coding genes, were observed compared to non-synonymous substitutions. However, positive selection was identified in six genes across 29 Berberis species—accD, matK, ndhD, rbcL, ycf1, and ycf2—highlighting their potential roles in adaptive responses to specific environmental conditions within the genus. Inverted repeats expansion and contraction affected the rate of mutations and were associated with the phylogenetic classification of Berberis. Our phylogenetic analysis supported the division of the Berberis complex into four genera, which corroborates previous studies involving extensive sampling. We identified the ndhD-ccsA region as the most polymorphic region and applied this region to Chinese patent medicines containing Berberidis Radix through metabarcoding. The metabarcoding analysis confirmed that five Berberis species commonly constitute Berberidis Radix in Chinese patent medicines. In conclusion, this study provides insight into the molecular evolution of the chloroplast genome and the phylogeny of the Berberis genus. In addition, metabarcoding provides insight into the species composition of Berberidis Radix in Chinese patent medicines.

Enhanced degradation performance toward para-nitrophenol of adapted immobilized microbial community on coconut coir

Nitrophenol pollutants, including para-nitrophenol (p-NP), are known for their harmful environmental impact due to their persistence, toxicity, and widespread distribution in water sources. While biodegradation generally offers a more effective removal of organic pollutants compared to chemical or physical methods, degrading persistent and toxic compounds like p-NP remains challenging. In this study, a microbial community derived from food processing wastewater was immobilized on coconut coir and adapted to p-NP before being employed for p-NP biodegradation. The spectroscopic analysis demonstrates the effective biodegradation performance of the adapted microbial community, achieving 99% degradation of 50 mg L⁻1 p-NP in 38 min and 250 mg L⁻1 p-NP in 4.65 h. The degradation ability of immobilized cells was determined across a broad range of stirring speeds, temperatures, pH levels, and p-NP solution volumes. Complete mineralization of p-NP was confirmed by chemical oxygen demand (COD) measurements of the treated solution and in-situ CO2 generation. Notably, the p-NP degradation performance of the adapted immobilized microbial community remained stable for the first 40 days, with only a slight decrease observed after 47 days of cold preservation at 4 °C. An average p-NP degradation rate of 0.75 mg L⁻1 min⁻1 was maintained over 54 consecutive runs. Significant alterations in microbial diversity were identified through 16S metabarcoding analysis. The unadapted microbial community comprised a diverse range of genera, while the adapted community showed reduced diversity with an enrichment of specific genera known for p-NP degradation, such as unidentified members of the Micrococcaceae family, Paenarthrobacter spp., and Zoogloea spp.

Dietary differences in males and females of a strongly sexually dimorphic ungulate

Detailed knowledge of dietary preferences in wild species are important both for understanding evolutionary processes as well as for their conservation. By means of DNA metabarcoding analysis of faecal samples, we investigated the diet of Alpine ibex (Capra ibex), a mountain ungulate characterised by strong sexual dimorphism and segregation, and analysed differences between male and female diet. Despite a low level of selectivity, we found differences in the diet of males and females that consumed different plant in different quantities. Differences were maintained during both spring and summer. Males tended to feed more on plants richer in fibres (i.e., grasses and sedges) and showed stronger seasonal differences while females consumed more digestible plant families (i.e., forbs). Differences in diet between male and female Alpine ibex are compatible with the strong sexual dimorphism and segregation typical of the species and with female reproductive constraints. These results also show that conservation actions have to take into account different ecological needs of males and females in species characterized by sexual dimorphism and segregation.

Feeding ecology of the urchin symbiont Dactylopleustes yoshimurai (Amphipoda) revealed by DNA metabarcoding

The nature of symbiotic relationships between organisms can be difficult to assess and may range from commensalism, to mutualism, and parasitism. Trophic linkage and feeding ecology are essential to disentangle symbiont-host relationships/interactions. Amphipods of the genus Dactylopleustes are known as urchin symbionts. Though their ecology remains largely unknown, Dactylopleustes was recently reported to aggregate on diseased hosts, suggesting that Dactylopleustes feeds on diseased urchins’ tissues and uses urchins as both a habitat and prey. We investigated by DNA metabarcoding analyses, the feeding ecology of Dactylopleustes yoshimurai in relation to growth and disease status of the host (Strongylocentrotus intermedius). Contrary to our hypothesis, sequence reads from the gut contents were dominated by planktonic copepods regardless of body size or host disease status. These results suggest that they mainly feed on copepod fecal pellets deposited on sediments, and do not have a strong trophic linkage with their host. Large individuals on diseased urchins feed more on urchins than those on healthy urchins. However, their main prey still remains copepods, implying that host disease has a limited effect on the feeding behavior. In conclusion, our study indicates that this species is mainly commensal, but also may parasitize its host depending on the situation.

Postharvest blown-arc plasma treatment did not reduce Fusarium graminearum or its mycotoxins in field-infested wheat grain

The increasing prevalence of Fusarium graminearum, a major causative pathogen of Fusarium head blight in cereal crops, leads to contamination of grain with mycotoxins, which necessitates the development of effective management strategies. This study investigated whether postharvest Blown-Arc plasma treatment could reduce F. graminearum and associated mycotoxins in field-infested grain. Postharvest Blown-Arc plasma treatment for 60 or 180 s was trialled to reduce Fusarium graminearum and deoxynivalenol (DON) mycotoxins in field-infested wheat grain. Changes in fungal components of the grain microbiome following the treatment were assessed using metabarcoding analysis of the nuclear ribosomal internal transcribed spacer gene region, and liquid chromatography mass spectrophotometry was used to measure changes in concentrations of the mycotoxins deoxynivalenol (DON), DON-3-glucoside (DON3-G) and 3-acetyl-DON (3-ADON). Fusarium spp. were the most common taxa in the grain over two seasons, with the relative abundance being higher in 2020 (72%) compared to 2021 (45%). Postharvest plasma treatment of field-infested grain for 60 or 180 s did not significantly lower Fusarium abundance. Alpha and beta diversity of the natural mycobiota in the grain were also unaffected. The concentrations of mycotoxins were not significantly different after the treatments, except that in the 2021 trial, the 180 s treatment caused a significant increase in DON concentration. We concluded that the postharvest plasma treatment used in this study did not reduce Fusarium contamination or the concentration of mycotoxins, DON, DON3-G and 3-ADON in field-infested grain. The results highlight the importance of using field-infested grain in experiments, as opposed to grain inoculated under laboratory conditions, which provides only superficial infection. This approach is crucial for accurately understanding the dynamics of treatment efficacy.

Culture-Independent Characterization of Citrus Rhizospheric Bacterial and Fungal Microbiota from Piura, Peru

The “limón sutil” (Citrus aurantifolia) has been widely cultivated and well established for many years in Piura, northwestern Peru, because of its exceptional climate and soil conditions. However, decline and death of C. aurantifolia trees caused by different phytopathogens remain a common problem which has been observed in the last decades. It is known that the microbiota of soil plays an important role with their host and could be the starting point to understand the causes of citrus decline. In this study, we identified through culture-independent methods the bacterial and fungal microbiota associated to C. aurantifolia, C. jambhiri and C. volkameriana rhizospheres in the main areas of Piura. By using a 16S rRNA and ITS-metabarcoding analysis, we evaluated the taxonomic diversity between healthy trees and with decline symptoms and how this diversity could influence the health status of citrus trees. More than 600 and 200 bacterial and fungal ASVs were identified, respectively. Our metabarcoding analysis was able to identify Proteobacteria, Cyanobacteria, Firmicutes, Bacteroidota and Acidobacteriota prokaryotic phyla, while fungal phyla included Ascomycota, Basidiomycota and Glomeromycota. In addition, there were differences between microbial diversity indices in rhizospheres evaluated. Finally, bacterial and fungal genera were shared among the different citrus rhizospheres. These results have allowed us to obtain a preliminary identification of microbiota in the citrus rhizospheres of healthy trees and with decline symptoms.

Metabarcoding study of potential pathogens and zoonotic risks associated with dog feces in Seoul, South Korea.

A significant portion of South Korea's population, approximately a quarter, owns pets, with dogs being the most popular choice among them. However, studies analyzing the fecal organism communities of dogs in South Korea are lacking, and limited efforts have been exerted to identify pathogens with potential zoonotic implications. Therefore, this study aimed to investigate potential pathogens using metabarcoding analysis and evaluate the risk of zoonotic diseases in dog feces in Seoul, South Korea. Fecal samples were collected from both pet and stray dogs in the Mapo district of Seoul. Next-generation sequencing (NGS) was utilized, employing 16S rRNA amplicon sequencing to identify prokaryotic pathogens, and 18S rRNA amplicon sequencing for eukaryotic pathogens. The data obtained from the QIIME2 pipeline were subjected to various statistical analyses to identify different putative pathogens and their compositions. Significant variations in microbiota composition were found between stray and pet dogs, and putative prokaryotic and eukaryotic pathogens were identified. The most prevalent putative bacterial pathogens were Fusobacterium, Helicobacter, and Campylobacter. The most prevalent putative eukaryotic pathogens were Giardia, Pentatrichomonas, and Cystoisospora. Interestingly, Campylobacter, Giardia, and Pentatrichomonas were found to be significantly more prevalent in stray dogs than in pet dogs. The variation in the prevalence of potential pathogens in dog feces could be attributed to environmental factors, including dietary variances and interactions with wildlife, particularly in stray dogs. These factors likely contributed to the observed differences in pathogen occurrence between stray and pet dogs. This study offers valuable insights into the zoonotic risks associated with dog populations residing in diverse environments. By identifying and characterizing putative pathogens in dog feces, this research provides essential information on the impact of habitat on dog-associated pathogens, highlighting the importance of public health planning and zoonotic risk management.

METAPLANTCODE: Harmonizing Plant Metabarcoding Pipelines in Europe

The METAPLANTCODE project is dedicated to advancing and optimizing pan-European case studies on metabarcoding. The project's objectives include providing best practice recommendations, optimizing analysis pipelines for species identification, and creating user-friendly reference databases. To accomplish these objectives, METAPLANTCODE will identify and address gaps in current methodologies, publish best practice documents on FAIR (Findable, Accessible, Interoperable, Reusable) data publishing for plant metabarcode data to GBIF (Global Biodiversity Information Facility) and the INSDC (International Nucleotide Sequence Database Collaboration), and implement ELIXIR-compatible multimodal deep learning (DL) models in novel tools for standalone metabarcoding analyses using various data sources. A significant focus of the project is enhancing species identification accuracy through GBIF records and metadata. This involves mapping regional, national, and international botanical taxonomic checklists, red lists, and floras to the Catalogue of Life (COL) via the COL ChecklistBank. Additionally, taxonomic and floristic literature will be semantically enriched with new entity recognition and relationship extraction modules, supporting the enhanced identification of species through domain-specific descriptive and phenotypic features. An interface will link taxonomic names to treatments, identify homonyms and synonyms, and facilitate the conversion and annotation of floras, red lists, and ecological treatments. All METAPLANTCODE products will adhere to FAIR standards by the project's end. The project emphasizes knowledge transfer from the outset, engaging with associated partners and stakeholders. Key stakeholders will be identified, priorities set, and communication channels established, monitored, and adjusted as necessary. Efforts to enhance stakeholder engagement, training, and outreach will ensure that plant metabarcoding becomes a routine standard for biodiversity monitoring in Europe and beyond. Deep Learning for Plant Metabarcoding Within the METAPLANTCODE project, our team is tasked with improving taxonomic precision by integrating deep learning on metabarcoding data and metadata. Previous studies have demonstrated the applicability of deep learning to non-plant barcoding data and its computational efficiency compared to traditional bioinformatics approaches (Flück et al. 2022). Deep Learning Models for Metabarcoding Data Our approach involves evaluating the efficacy of several deep learning models—such as Convolutional Neural Networks (CNN)(LeCun et al. 2015), Transformer models (Vaswani et al. 2017), Hyena (Poli et al. 2023), and Mamba architectures (Gu et al. 2023)—on plant barcoding datasets. Preliminary results will be presented, highlighting the application of these models and the proposed ensemble method (Mohammed and Kora 2023), which combines multiple barcode sequence representations and learning strategies. The ensemble approach, when integrated with classical machine learning models such as logistic regression and Support Vector Machines (SVM) (Noble 2006), is anticipated to offer improved precision and robustness compared to individual model applications (Fig. 1). Multimodal Refinement of Predictions In the subsequent phase, we aim to refine genetic sequence classifications by employing a multimodal strategy. This approach will integrate genetic information with traditional botanical knowledge. We will utilize biological interaction lists (e.g., species-species, species-habitat) provided by the METAPLANTCODE project to train a large language model (LLM) on relevant scientific literature. This LLM, specifically tailored for plant biodiversity, will incorporate metadata associated with genetic samples (including location, temporality, and climatic conditions). By merging embeddings of both metadata and genetic data, we aim to enhance the accuracy of taxonomic predictions (Fig. 2). Conclusion Through this research, we aim to develop an effective method for integrating genetic data with textual information from various sources. We anticipate that this approach will not only enhance plant metabarcoding but also be applicable to other barcoding fields, such as bacteria, fish, fungi, and more. Additionally, we expect this methodology to find broader applications in genomic research, providing valuable insights and improvements across diverse biological disciplines.

Verifying the Diet of the Slender Racer, <i>Orientocoluber spinalis</i> (Peters, 1866), Rare, Threatened, and Endangered Across Northeast Asian Countries

Understanding the diet of a species provides important information on various aspects of its ecology. Diet studies are few on the reptile species, of which observation frequency is very low, due to the great difficulty of sampling. We investigated the diet of Orientocoluber spinalis, a long and slender small grassland snake with high rarity and a high extinction risk across northeast Asian countries, by applying regurgitated stomach content analysis and metabarcoding analysis using a developed O. spinalis blocking primer. Among 19 diet samples, which were mainly collected on islands along the west coast of the Korean Peninsula, we determined the food sources of 13 samples, which consisted of four small vertebrate species, the Tsushima smooth skink (Scincella vandenburghi), mountain grass lizard (Takydromus wolteri), Japanese keelback (Hebius vibakari), and lesser white-toothed shrew (Crocidura shantungensis). Among them, O. spinalis most frequently fed on S. vandenburghi (10 of 13 cases). Our results show that O. spinalis mainly forages small reptiles and mammals in grasslands adjoining to lowland forests and could be beneficial for conservation in the field and rearing of this vulnerable species in the facilities.

Evaluating the winter diet of elk using DNA metabarcoding analysis

North American elk Cervus canadensis inhabited portions of the eastern United States until extirpation in the mid‐1800s. From 2000 to 2008, 201 elk were reintroduced to the North Cumberland Wildlife Management Area (NCWMA), Tennessee. The stocking source was Elk Island National Park, Alberta Canada where there are two distinct genetic populations isolated from the north and south. This genetic structure has largely persisted in the population after translocation. Food habits were evaluated in the early stages of restoration, but the population has had approximately 20 years to adapt to the landscape, and current food habits are unknown. To assess diet composition using DNA metabarcoding, we collected fecal pellets of elk from 65 openings within the 79 318 ha NCWMA weekly from February to April of 2019. We targeted the ITS2 region of the nuclear ribosomal DNA to amplify vegetation sequences found in the internal portion of the elk feces. DNA metabarcoding of feces was linked to results from an accompanying elk population genetics study to investigate food habits between sexes from the two different genetic groups. The majority (80.298%) of sequences matched plants from 21 genera. The top genera (> 5.000%) represented were Vaccinium (15.216%), Festuca (8.446%), Rosa (6.358%), Robinia (5.793%), and Eleagnus (5.186%). Elk heavily used woody plants before and after spring green‐up (> 50% of diet). However, the quantity of forbs in their diet more than doubled after emergence in the spring. The sex‐genetic groups consumed similar vegetation in approximately proportionate amounts. Diversity analyses revealed a significant difference in plant genera sequence detection between males from the two genetic groups, although this finding is likely explained by limited sample size. NCWMA elk used a variety of forage in the winter and DNA metabarcoding analysis allows for a comprehensive analysis of food habits useful for monitoring how elk respond dietarily to habitat management.

DNA Metabarcoding Analysis of Arthropod Diversity in Dust from the Natural History Museum, Vienna

This paper introduces a new method for identifying museum pests through the analysis of DNA present in settled dust. Traditionally, the identification of pests in cultural institutions such as museums and depositories has relied on insect trapping (monitoring). They give good results but need time (minimum spring until summer of one year for a complete survey) and face challenges related to the identification of small, rare, or damaged species. Our study presents a non-invasive approach that utilizes metabarcoding analysis of dust samples to identify pests and other arthropods at the species level. We collected dust samples with a handheld vacuum cleaner in summer 2023 from the six different floors of the Natural History Museum in Vienna and compared the results with the insect monitoring. We found over 359 different species of arthropods in the museum and could show how the diversity increases with the elevation of the building floor. This method could be used for rapid and cost-effective screening of pests before monitoring. But the interpretation of results is sometimes difficult (for Lepismatidae, for example), and it cannot replace a continuous monitoring of pests with traps. This investigation might present the highest indoor animal biodiversity ever found in a single building.

Harmonized coexistence of intragenomic variations in diatom Skeletonema strains

Metabarcoding analysis has been demonstrated to be an effective technology for monitoring diversity and dynamics of phytoplankton including Skeletonema species. Although molecular diversity uncovered in metabarcoding projects has generally been interpreted as sum of interspecies diversity and intraspecies diversity, accumulating evidence suggests that it also harbors unprecedentedly high levels of intra-genomic variations (IGVs). As up to thousands of amplicon sequence variants (ASVs) identified in a typical metabarcoding project can be annotated to be Skeletonema species, we hypothesize that substantial portions of these ASVs are contributed by IGVs. Here, the nature of IGVs in Skeletonema species was quantitatively analyzed by carrying out single-strain metabarcoding analysis of 18S rDNA V4 in 49 strains belonging to seven Skeletonema species. Results showed that each Skeletonema strain harbored a high level of IGVs as expected. While many Skeletonema strains each contained one dominant ASV and a substantial number of ASVs displaying much lower relative abundance, other Skeletonema strains each contained multiple ASVs with comparable or nearly equally abundances. Thus the co-existence of multiple dominant ASVs in a single cell indicated a tug-of-war of these variants in evolution, which may eventually result in harmonized coexistence of multiple dominant ASVs. A total of nine dominant ASVs and 652 non-dominant ASVs were found in 49 strains of seven Skeletonema species, indicating rich interspecies and intraspecies variations, and complex evolution of IGVs in genus of Skeletonema. The results confirmed that the extensive degree of IGVs was the main contributor to the high molecular diversity revealed by metabarcoding analysis. This study highlights the importance of quantitative characterization of IGVs in Skeletonema species for accurate interpretation of species diversity in metabarcoding analysis.

The rootstock modifies the arbuscular mycorrhizal community of the root system, while the influence of the scion is limited in grapevines.

Understanding the effects of grapevine rootstock and scion genotypes on arbuscular mycorrhizal fungi (AMF), as well as the roles of these fungi in plant development, could provide new avenues for adapting viticulture to climate change and reducing agrochemical inputs. The root colonization of 10 rootstock/scion combinations was studied using microscopy and metabarcoding approaches and linked to plant development phenotypes. The AMF communities were analysed using 18S rRNA gene sequencing. The 28S rRNA gene was also sequenced for some combinations to evaluate whether the method changed the results. Root colonization indexes measured by microscopy were not significantly different between genotypes. Metabarcoding analyses showed an effect of the rootstock genotype on the β-diversity and the enrichment of several taxa with both target genes, as well as an effect on the Chao1 index with the 18S rRNA gene. We confirm that rootstocks recruit different AMF communities when subjected to the same pedoclimatic conditions, while the scion has little or no effect. Significant correlations were observed between AMF community composition and grapevine development, suggesting that AMF have a positive effect on plant growth. Given these results, it will be important to define consensus methods for studying the role of these beneficial micro-organisms in vineyards.

Unlocking rivers' hidden diversity and ecological status using DNA metabarcoding in Northwest Spain.

Rivers are crucial ecosystems supporting biodiversity and human well-being, yet they face increasing degradation globally. Traditional river biomonitoring methods based on morphological identification of macroinvertebrates present challenges in terms of taxonomic resolution and scalability. This study explores the application of DNA metabarcoding analysis in both bulk and environmental DNA (eDNA) samples for comprehensive assessment of macrozoobenthic biodiversity, detection of invasive and endangered species, and evaluation of river ecological status in northwestern Spain. DNA metabarcoding of homogenized bulk samples and water eDNA revealed a mean of 100 and 87 macrozoobenthos species per sample respectively. However, the specific composition was significantly different with only 27.3% of the total species being shared. It was not possible to identify all the OTUs to species level; only 17.43% and 49.4% of the OTUs generated could be identified to species level in the bulk and eDNA samples, respectively. Additionally, a total of 11 exotic species (two first records for the Iberian Peninsula and another three first records for Asturias region) and one endangered species were detected by molecular tools. Molecular methods showed significant correlations with morphological identification for EQR values (Ecological Quality Ratio) of IBMWP index, yet differences in inferred river ecological status were noted, with bulk samples tending to indicate higher status. Overall, DNA metabarcoding offers a promising approach for river biomonitoring, providing insights into biodiversity, invasive species, and ecological status within a single analysis. Further optimization and intercalibration are required for its implementation in routine biomonitoring programmes, but its scalability and multi-tasking capabilities position it as a valuable tool for integrated monitoring of river ecosystems.

Environmental DNA metabarcoding for whole community inventories of vertebrates in rivers of the midwestern United States

The application of environmental DNA (eDNA) methods to simultaneously study vertebrate diversity holds promise to accelerate conservation efforts, especially in freshwater systems which are among the most imperiled in the world. Here, using eDNA sampling, we identify patterns of vertebrate biodiversity across different habitats of the Kankakee River watershed, one of the most diverse lotic systems in Illinois, USA. Our eDNA metabarcoding analyses identified 147 different taxa, including 77 fishes, 38 birds, 24 mammals, five amphibians and three reptiles at 11 locations in the watershed, including tributaries and mainstem stretches upstream and within the Kankakee River State Park protected area. When compared to seining, eDNA sampling consistently detected more fish species, including non-native and imperiled species. We also found that vertebrate communities among the different habitats significantly varied in taxonomic composition, showing an upstream-downstream shift along the mainstem river as well as tributary-specific assemblages. Our study demonstrates the ability of single-marker eDNA metabarcoding to simultaneously document aquatic and terrestrial communities across large temperate lotic ecosystems and to monitor diversity patterns across protected areas.

Diet of Andean leaf-eared mice (Phyllotis) living at extreme elevations on Atacama volcanoes: insights from metagenomics, DNA metabarcoding, and stable isotopes.

On the flanks of >6000 m Andean volcanoes that tower over the Atacama Desert, leaf-eared mice (Phyllotis vaccarum) live at extreme elevations that surpass known vegetation limits. What the mice eat in these barren, hyperarid environments has been the subject of much speculation. According to the arthropod fallout hypothesis, sustenance is provided by windblown insects that accumulate in snowdrifts ('aolian deposits'). It is also possible that mice feed on saxicolous lichen or forms of cryptic vegetation that have yet to be discovered at such high elevations. We tested hypotheses about the diet of mice living at extreme elevations on Atacama volcanoes by combining metagenomic and DNA metabarcoding analyses of gut contents with stable-isotope analyses of mouse tissues. Genomic analyses of contents of the gastrointestinal tract of a live-captured mouse from the 6739 m summit of Volcán Llullaillaco revealed evidence for an opportunistic but purely herbivorous diet, including lichens. Although we found no evidence of animal DNA in gut contents of the summit mouse, stable isotope data indicate that mice native to elevations at or near vegetation limits (~5100 m) include a larger fraction of animal prey in their diet than mice from lower elevations. Some plant species detected in the gut contents of the summit mouse are known to exist at lower elevations at the base of the volcano and in the surrounding Altiplano, suggesting that such plants may occur at higher elevations beneath the snowpack or in other cryptic microhabitats.

Spatiotemporal changes of pelagic food webs investigated by environmental DNA metabarcoding and connectivity analysis.

Environmental DNA metabarcoding (eDNA metaB) is fundamental for monitoring marine biodiversity and its spread in coastal ecosystems. We applied eDNA metaB to seawater samples to investigate the spatiotemporal variability of plankton and small pelagic fish, comparing sites with different environmental conditions across a coast-to-offshore gradient at river mouths along the Campania coast (Italy) over 2 years (2020-2021). We found a marked seasonality in the planktonic community at the regional scale, likely owing to the hydrodynamic connection among sampling sites, which was derived from numerical simulations. Nonetheless, spatial variability among plankton communities was detected during summer. Overall, slight changes in plankton and fish composition resulted in the potential reorganization of the pelagic food web at the local scale. This work supports the utility of eDNA metaB in combination with hydrodynamic modelling to study marine biodiversity in the water column of coastal systems. This article is part of the theme issue 'Connected interactions: enriching food web research by spatial and social interactions'.

Spatio-temporal changes of small protist and free-living bacterial communities in a temperate dimictic lake: insights from metabarcoding and machine learning.

Microbial communities, which include prokaryotes and protists, play an important role in aquatic ecosystems and influence ecological processes. To understand these communities, metabarcoding provides a powerful tool to assess their taxonomic composition and track spatio-temporal dynamics in both marine and freshwater environments. While marine ecosystems have been extensively studied, there is a notable research gap in understanding eukaryotic microbial communities in temperate lakes. Our study addresses this gap by investigating the free-living bacteria and small protist communities in Lake Roś (Poland), a dimictic temperate lake. Metabarcoding analysis revealed that both the bacterial and protist communities exhibit distinct seasonal patterns that are not necessarily shaped by dominant taxa. Furthermore, machine learning and statistical methods identified crucial amplicon sequence variants (ASVs) specific to each season. In addition, we identified a distinct community in the anoxic hypolimnion. We have also shown that the key factors shaping the composition of analysed community are temperature, oxygen, and silicon concentration. Understanding these community structures and the underlying factors is important in the context of climate change potentially impacting mixing patterns and leading to prolonged stratification.

Influence of inoculum on process parameters and microbial communities during anaerobic digestion of cattle manure: Insights from metabarcoding analysis

Linking the variety of microbial ecology attributes to system functioning is critical to improve operational strategies and to support microbial pathways, in order to conceive efficient and reliable anaerobic digestion process. In the present study, we conducted a direct comparison between two digesters regarding the process parameters and microbial community dynamics between two digesters: one containing only the substrate (cattle manure) (Digester S), and the other (Digester F) enriched with an inoculum. This study aimed to investigate taxonomic and functional changes in the digester microbiome using a 16S rRNA sequencing approach. Results exhibit that the bacterial richness, diversity, and regularity were higher in Digester F. This later showed also a higher biogas yields (BY) and the process performance indicators compared to Digester S. The BY obtained in digester F was 2.17 times higher than that obtained in digester S. Furthermore, significant correlations were obtained between biogas yields (BY) and microbial communities with Firmicutes/Bacillota (Clostridium, Enterococcus, Streptococcus, Ruminococcaceae, Mogibacterium, and Turicibacter), Actinobacteria (Corynebacterium), and Bacteroidetes (Bacteroides, Paludibacter) in the AD systems. Conversely, in Digester S, biogas yield (BY) was highly significant and positively correlated with the abundance of Actinobacteria (Rhodococcus) and Proteobacteria (Moraxella, Oxalobacter). Moreover, the inoculum in digester F resulted in the enrichment of main bacterial functions involved in biogas production. This study provides results that justify the changes in the structure of the microbial community and the functional strategy used by the most representative micro-organisms in the consortia that carried out this process.

Clubroot-Induced Changes in the Root and Rhizosphere Microbiome of Susceptible and Resistant Canola.

Clubroot is a soilborne disease of canola (Brassica napus) and other crucifers caused by the obligate parasite Plasmodiophora brassicae. In western Canada, clubroot is usually managed by planting-resistant cultivars, but the emergence of resistance-breaking pathotypes of P. brassicae represents a major threat to sustainable canola production. The rhizosphere and root contain beneficial microorganisms that can improve plant health. In this study, we evaluated the effect of two P. brassicae isolates (termed A and B) with different levels of virulence on the root and rhizosphere microbiomes of clubroot-resistant and clubroot-susceptible canola. Additionally, potential biocontrol microorganisms were identified based on taxa antagonistic to clubroot. Although both P. brassicae isolates were classified as pathotype 3A, isolate A caused a higher disease severity index in the resistant canola genotype compared with isolate B. Metabarcoding analysis indicated a shift in the bacterial and fungal communities in response to inoculation with either field isolate. Root endophytic bacterial and fungal communities responded to changes in inoculation, isolate type, sampling time, and canola genotype. In contrast, fungal communities associated with the rhizosphere exhibited significant differences between sampling times, while bacterial communities associated with the rhizosphere exhibited low variability.