Application of environmental DNA metabarcoding to identify fish community characteristics in subtropical river systems.

Fish are vital in river ecosystems; however, traditional investigations of fish usually cause ecological damage. Extracting DNA from aquatic environments and identifying DNA sequences offer an alternative, noninvasive approach for detecting fish species. In this study, the effects of environmental DNA (eDNA), coupled with PCR and next-generation sequencing, and electrofishing for identifying fish community composition and diversity were compared. In three subtropical rivers of southern China, fish specimens and eDNA in water were collected along the longitudinal (upstream-downstream) gradient of the rivers. Both fish population parameters, including species abundance and biomass, and eDNA OTU richness grouped 38 sampling sites into eight spatial zones with significant differences in local fish community composition. Compared with order-/family-level grouping, genus-/species-level grouping could more accurately reveal the differences between upstream zones I-III, midstream zones IV-V, and downstream zones VI-VIII. From the headwaters to the estuary, two environmental gradients significantly influenced the longitudinal distribution of the fish species, including the first gradient composed of habitat and physical water parameters and the second gradient composed of chemical water parameters. The high regression coefficient of alpha diversity between eDNA and electrofishing methods as well as the accurate identification of dominant, alien, and biomarker species in each spatial zone indicated that eDNA could characterize fish community attributes at a level similar to that of traditional approaches. Overall, our results demonstrated that eDNA metabarcoding can be used as an effective tool for revealing fish composition and diversity, which is important for using the eDNA technique in aquatic field monitoring.

A set of nematode rRNA cistron databases and a primer assessment tool to enable more flexible and comprehensive metabarcoding.

The ITS-2-rRNA has been particularly useful for nematode metabarcoding but does not resolve all phylogenetic relationships, and reference sequences are not available for many nematode species. This is a particular issue when metabarcoding complex communities such as wildlife parasites or terrestrial and aquatic free-living nematode communities. We have used markerDB to produce four databases of distinct regions of the rRNA cistron: the 18S rRNA gene, the 28S rRNA gene, the ITS-1 intergenic spacer and the region spanning ITS-1_5.8S_ITS-2. These databases comprise 2645, 254, 13,461 and 10,107 unique full-length sequences representing 1391, 204, 1837 and 1322 nematode species, respectively. The comparative analysis illustrates the complementary value but also reveals a better representation of Clade III, IV and V than Clade I and Clade II nematodes in each case. Although the ITS-1 database includes the largest number of unique full-length sequences, the 18S rRNA database provides the widest taxonomic coverage. We also developed PrimerTC, a tool to assess primer sequence conservation across any reference sequence database, and have applied it to evaluate a large number of previously published rRNA cistron primers. We identified sets of primers that currently provide the broadest taxonomic coverage for each rRNA marker across the nematode phylum. These new resources will facilitate more comprehensive metabarcoding of nematode communities using either short-read or long-read sequencing platforms. Further, PrimerTC is available as a simple WebApp to guide or assess PCR primer design for any genetic marker and/or taxonomic group beyond the nematode phylum.

Validation and development of eDNA metabarcoding primers for comprehensive assessment of Chinese amphibians.

Environmental DNA (eDNA) metabarcoding has emerged as a powerful, non-invasive tool for biodiversity assessments. However, the accuracy and limitations of these assessment techniques are highly dependent on the choice of primer pairs being used. Although several primer sets have been used in eDNA metabarcoding studies of amphibians, there are few comparisons of their reliability and efficiency. Here, we employed lab- and field-tested sets of publicly available and de novo-designed primers in amplifying 83 species of amphibian from all three orders (Anura, Caudata, and Gymnophiona) and 13 families present in China to evaluate the versatility and specificity of these primers sets in amphibian eDNA metabarcoding studies. Three pairs of primers were highly effective, as they could successfully amplify all the major clades of Chinese amphibians in our study. A few non-amphibian taxa were also amplified by these primers, which implies that further optimization of amphibian-specific primers is still needed. The simultaneous use of three primer sets can completely cover all the species obtained by conventional survey methods and has even effectively distinguished quite a number of species (n = 20) in the Wenshan National Nature Reserve. No single primer set could individually detect all of the species from the studied region, indicating that multiple primers might be necessary for a comprehensive survey of Chinese amphibians. Besides, seasonal variations in amphibian species composition were also revealed by eDNA metabarcoding, which was consistent with traditional survey methods. These results indicate that eDNA metabarcoding has the potential to be a powerful tool for studying spatial and temporal community changes in amphibian species richness.

A 1 Ma sedimentary ancient DNA (sedaDNA) record of catchment vegetation changes and the developmental history of tropical Lake Towuti (Sulawesi, Indonesia).

Studying past ecosystems from ancient environmental DNA preserved in lake sediments (sedaDNA) is a rapidly expanding field. This research has mainly involved Holocene sediments from lakes in cool climates, with little known about the suitability of sedaDNA to reconstruct substantially older ecosystems in the warm tropics. Here, we report the successful recovery of chloroplast trnL (UAA) sequences (trnL-P6 loop) from the sedimentary record of Lake Towuti (Sulawesi, Indonesia) to elucidate changes in regional tropical vegetation assemblages during the lake's Late Quaternary paleodepositional history. After the stringent removal of contaminants and sequence artifacts, taxonomic assignment of the remaining genuine trnL-P6 reads showed that native nitrogen-fixing legumes, C3 grasses, and shallow wetland vegetation (Alocasia) were most strongly associated with >1-million-year-old (>1 Ma) peats and silts (114-98.8 m composite depth; mcd), which were deposited in a landscape of active river channels, shallow lakes, and peat-swamps. A statistically significant shift toward partly submerged shoreline vegetation that was likely rooted in anoxic muddy soils (i.e., peatland forest trees and wetland C3 grasses (Oryzaceae) and nutrient-demanding aquatic herbs (presumably Oenanthe javanica)) occurred at 76 mcd (~0.8 Ma), ~0.2 Ma after the transition into a permanent lake. This wetland vegetation was most strongly associated with diatom ooze (46-37 mcd), thought to be deposited during maximum nutrient availability and primary productivity. Herbs (Brassicaceae), trees/shrubs (Fabaceae and Theaceae), and C3 grasses correlated with inorganic parameters, indicating increased drainage of ultramafic sediments and laterite soils from the lakes' catchment, particularly at times of inferred drying. Downcore variability in trnL-P6 from tropical forest trees (Toona), shady ground cover herbs (Zingiberaceae), and tree orchids (Luisia) most strongly correlated with sediments of a predominantly felsic signature considered to be originating from the catchment of the Loeha River draining into Lake Towuti during wetter climate conditions. However, the co-correlation with dry climate-adapted trees (i.e., Castanopsis or Lithocarpus) plus C4 grasses suggests that increased precipitation seasonality also contributed to the increased drainage of felsic Loeha River sediments. This multiproxy approach shows that despite elevated in situ temperatures, tropical lake sediments potentially comprise long-term archives of ancient environmental DNA for reconstructing ecosystems, which warrants further exploration.

Microbial response to a port fuel spill: Community dynamics and potential for bioremediation.

Following a fuel leakage inside a Portuguese maritime port, we conducted parallel 30-day experiments using contaminated seawater and fuel, sampled five days after the incident. This study aimed to (i)survey the native microbial community response to the spilled fuel and (ii)evaluate the efficacy of bioremediation, both biostimulation and bioaugmentation with a lyophilized bacterial consortium (Rhodococcus erythropolis, Pseudomonas sp.), in accelerating hydrocarbon degradation. Metabarcoding analysis revealed a shift in microbial communities, with increased abundance of hydrocarbon-degraders (e.g. Alcanivorax, Thalassospira). Ninety-five hydrocarbonoclastic bacteria were isolated, including key groups from the enriched communities. The lyophilized bacteria added in bioaugmentation, enhanced the abundance of hydrocarbon-degraders over time and were recovered throughout time. Bioremediation treatments favoured biodegradation, achieving over 60 % removal of total petroleum hydrocarbons after 15 days, contrasting with natural attenuation where almost no TPH was removed. This work highlights the potential of bioremediation technologies to accelerate hydrocarbon-degrading activity, for oil spills inside ports.

Restoration age affects microbial-herbaceous plant interactions in an oak woodland.

In degraded ecosystems, soil microbial communities (SMCs) may influence the outcomes of ecological restoration. Restoration practices can affect SMCs, though it is unclear how variation in the onset of restoration activities in woodlands affects SMCs, how those SMCs influence the performance of hard-to-establish woodland forbs, and how different woodland forbs shape SMCs. In this study, we quantified soil properties and species abundances in an oak woodland restoration chronosequence (young, intermediate, and old restorations). We measured the growth of three woodland forb species when inoculated with live whole-soil from young, intermediate, or old restorations. We used DNA metabarcoding to characterize SMCs of each inoculum treatment and the soil after conditioning by each plant species. Our goals were to (1) understand how time since the onset of restoration affected soil abiotic properties, plant communities, and SMCs in a restoration chronosequence, (2) test growth responses of three forb species to whole-soil inoculum from restoration sites, and (3) characterize changes in SMCs before and after conditioning by each forb species. Younger restored woodlands had greater fire-sensitive tree species and lower concentrations of soil phosphorous than intermediate or older restored woodlands. Bacterial and fungal soil communities varied significantly among sites. Forbs exhibited the greatest growth in soil from the young restoration. Each forb species developed a unique soil microbial community. Our results highlight how restoration practices affect SMCs, which can in turn affect the growth of hard-to-establish forb species. Our results also highlight that the choice of forb species can alter SMCs, which could have long-term potential consequences for restoration success.

Increasing marine trophic web knowledge through DNA analyses of fish stomach content: a step towards an ecosystem-based approach to fisheries research.

Multispecies and ecosystem models, which are key for the implementation of ecosystem-based approaches to fisheries management, require extensive data on the trophic interactions between marine organisms, including changes over time. DNA metabarcoding, by allowing the simultaneous taxonomic identification of the community present in hundreds of samples, could be used for speeding up large-scale stomach content data collection. Yet, for DNA metabarcoding to be routinely implemented, technical challenges should be addressed, such as the potentially complicated sampling logistics, the detection of a high proportion of predator DNA, and the inability to provide reliable abundance estimations. Here, we present a DNA metabarcoding assay developed to examine the diet of five commercially important fish, which can be feasibly incorporated into routinary samplings. The method is devised to speed up the analysis process by avoiding the stomach dissection and content extraction steps, while preventing the amplification of predator DNA by using blocking primers. Tested in mock samples and in real stomach samples, the method has proven effective and shows great effectiveness discerning diet variations due to predator ecology or prey availability. Additionally, by applying our protocol to mackerel stomachs previously analyzed by visual inspection, we showcase how DNA metabarcoding could complement visually based data by detecting overlooked prey by the visual approach. We finally discuss how DNA metabarcoding-based data can contribute to trophic data collection. Our work reinforces the potential of DNA metabarcoding for the study and monitoring of fish trophic interactions and provides a basis for its incorporation into routine monitoring programs, which will be critical for the implementation of ecosystem-based approaches to fisheries management.

Comparative analysis of bacterial diversity in accumulated snow and exposed sediments across Antarctic Islands.

The Antarctic continent hosts exceptional niches, making it an ideal environment for studying polyextremophilic microorganisms. These organisms are uniquely shaped by the geographic niches and variations in soil types. Here we present, a culture-independent approach using DNA metabarcoding to assess the bacterial communities associated with accumulated snow and exposed sediments across different Antarctic islands situated in the Larsemann Hills, Antarctica. The exposed sediments (ES) were found to be more diverse than the accumulated snow (AS) sediments as represented by the alpha diversity metrics. Out of the total 303 amplicon sequence variants (ASVs) found at the genus level, 93 were unique to accumulated snow sediments and 97 were unique to exposed sediments. The bacterial community composition in accumulated snow was dominated by the phylum Actinobacteriota (24.7%). However, Pseudonocardia (11.9%), Crossiella (11%), and Rhodanobacter (9.1%) were the predominant genera. In contrast, in the exposed sediments, Bacteroidota (24.6%) was the most prevalent phylum, with Crossiella (17.1%), Rhodanobacter (11.1%), and Blastocatella (10%) as the most abundant genera. Metagenomic imputations revealed the abundance of gene families responsible for carbon metabolism, coping with environmental stresses through DNA repair mechanisms, and carbon fixation.

Comparison of qPCR and metabarcoding for environmental DNA surveillance of a freshwater parasite.

Analysis of environmental DNA (eDNA) has been successfully used across freshwater ecological parasitology to inform management of ecologically and economically important species. However, most studies have used species-specific quantitative polymerase chain reaction (qPCR) assays to detect target taxa. While generally effective, this approach limits the amount of community and management-supporting data that can be obtained from eDNA samples. If eDNA metabarcoding could be conducted with the same accuracy of a single species approach, researchers could simultaneously detect a target species while obtaining vast community data from eDNA samples. We sampled 38 freshwater sites on Fort McCoy, Wisconsin and compared qPCR to metabarcoding for eDNA detection of the ectoparasitic gill louse Salmincola edwardsii, an obligate parasite of Salvelinus fishes (chars). We found no evidence to suggest S. edwardsii occupancy or detection probabilities differed between qPCR and metabarcoding. Further, we found that the number of S. edwardsii reads from metabarcoding were negatively predictive of C T values from qPCR (C T value indicates cycle a significant amount of target eDNA is detected, with lower C Ts indicative of more DNA), demonstrating that our metabarcoding reads positively predicted qPCR DNA quantities. However, the number of reads was not predictive of overall qPCR score (number of positive qPCR replicates). In addition to S. edwardsii, metabarcoding led to the detection of a vast community of over 2600 invertebrate taxa. We underscore the necessity for conducting similar analyses across environments and target species, as the ecology of eDNA will vary on a per-study basis. Our results suggest that eDNA metabarcoding provides a highly sensitive and accurate method for detecting parasitic gill lice while also illuminating the broader biological community and co-occurrence of species in the environment.

Deciphering microbial communities of three Savoyard raw milk cheeses along ripening and regarding the cheese process.

Different Savoyard cheeses are granted with PDO (Protected Designation or Origin) and PGI (Protected Geographical Indication) which guarantees consumers compliance with strict specifications. The use of raw milk is known to be crucial for specific flavor development. To unravel the factors influencing microbial ecosystems across cheese making steps, according to the seasonality (winter and summer) and the mode of production (farmhouse and dairy factory ones), gene targeting on bacteria and fungus was used to have a full picture of 3 cheese making technologies, from the raw milk to the end of the ripening. Our results revealed that Savoyard raw milks are a plenteous source of biodiversity together with the brines used during the process, that may support the development of specific features for each cheese. It was shown that rinds and curds have very contrasted ecosystem diversity, composition, and evolution. Ripening stage was selective for some bacterial species, whereas fungus were mainly ubiquitous in dairy samples. All ripening stages are impacted by the type of cheese technologies, with a higher impact on bacterial communities, except for fungal rind communities, for which the technology is the more discriminant. The specific microorganism's abundance for each technology allow to see a real bar-code, with more or less differences regarding bacterial or fungal communities. Bacterial structuration is shaped mainly by matrices, differently regarding technologies while the influence of technology is higher for fungi. Production types showed 10 differential bacterial species, farmhouses showed more ripening taxa, while dairy factory products showing more lactic acid bacteria. Meanwhile, seasonality looks to be a minor element for the comprehension of both microbial ecosystems, but the uniqueness of each dairy plant is a key explicative feature, more for bacteria than for fungus communities.

Environmental DNA as a tool to reconstruct catch composition for longline fisheries vessels.

Global wild-capture fisheries are a large and diverse sector requiring various tools for fisheries-dependant data collection and effective Monitoring, Control and Surveillance (MCS). Here we present a novel protocol to collect eDNA from brine tanks onboard commercial longline vessels to reconstruct catch composition. We collected samples from nine vessels operating out of the Eastern Tuna Billfish Fishery, Australia, validating eDNA results with reliable catch data consisting of seven target and bycatch species. Environmental DNA was highly effective for detecting species retained on vessels without contamination or false positives. For four vessels, logbook data and eDNA were consistent with detections of all species. The remaining vessels detected all species except for rare catches of short-billed spearfish (Tetrapturus angustirostris). Similarities between rank abundance distributions of catch and eDNA reads were observed with logbook data mirrored when eDNA sequences were organised into rank order abundance. The method was effective at identifying highly abundant taxa retained in brine tanks- tuna (Thunnus spp.), swordfish (Xiphias gladius), marlin (Kajijia audax), and Atlantic Pomfret (Brama brama). Further research is required to validate how eDNA and other molecular monitoring tools can be scaled and applied to provide solutions for monitoring challenges in the fisheries sector.

Deciphering mycobiota and its functional dynamics in root hairs of Rhododendron campanulatum D. Don through Next-gen sequencing.

The Himalayas provide unique opportunities for the extension of shrubs beyond the upper limit of the tree. However, little is known about the limitation of the biotic factors belowground of shrub growth at these cruising altitudes. To fill this gap, the present study deals with the documentation of root-associated microbiota with their predicted functional profiles and interactions in the host Rhododendron campanulatum, a krummholz species. While processing 12 root samples of R. campanulatum from the sites using Omics we could identify 134 root-associated fungal species belonging to 104 genera, 74 families, 39 orders, 17 classes, and 5 phyla. The root-associated microbiota members of Ascomycota were unambiguously dominant followed by Basidiomycota. Using FUNGuild, we reported that symbiotroph and pathotroph as abundant trophic modes. Furthermore, FUNGuild revealed the dominant prevalence of the saptroptroph guild followed by plant pathogens and wood saprotrophs. Alpha diversity was significantly different at the sites. The heatmap dendrogram showed the correlation between various soil nutrients and some fungal species. The study paves the way for a more in-depth exploration of unidentified root fungal symbionts, their interactions and their probable functional roles, which may serve as an important factor for the growth and conservation of these high-altitude ericaceous plants.

Experimental evidence that root-associated fungi improve plant growth at high altitude.

Unravelling how species communities change along environmental gradients requires a dual understanding: the direct responses of the species to their abiotic surroundings and the indirect variation of these responses through biotic interactions. Here, we focus on the interactive relationships between plants and their symbiotic root-associated fungi (RAF) along stressful abiotic gradients. We investigate whether variations in RAF community composition along altitudinal gradients influence plant growth at high altitudes, where both plants and fungi face harsher abiotic conditions. We established a translocation experiment between pairs of Bistorta vivipara populations across altitudinal gradients. To separate the impact of shifting fungal communities from the overall influence of changing abiotic conditions, we used a root barrier to prevent new colonization by RAF following translocation. To characterize the RAF communities, we applied DNA barcoding to the root samples. Through the utilization of joint species distribution modelling, we assessed the relationship between changes in plant functional traits resulting from experimental treatments and the corresponding changes in the RAF communities. Our findings indicate that RAF communities influence plant responses to stressful abiotic conditions. Plants translocated from low to high altitudes grew more when they were able to associate with the resident high-altitude RAF compared to those plants that were not allowed to associate with the resident RAF. We conclude that interactions with RAF impact how plants respond to stressful abiotic conditions. Our results provide experimental support that interactions with RAF improve plant stress tolerance to altitudinal stressors such as colder temperatures and less nutrient availability.

Three steps towards comparability and standardization among molecular methods for characterizing insect communities.

Molecular methods are currently some of the best-suited technologies for implementation in insect monitoring. However, the field is developing rapidly and lacks agreement on methodology or community standards. To apply DNA-based methods in large-scale monitoring, and to gain insight across commensurate data, we need easy-to-implement standards that improve data comparability. Here, we provide three recommendations for how to improve and harmonize efforts in biodiversity assessment and monitoring via metabarcoding: (i) we should adopt the use of synthetic spike-ins, which will act as positive controls and internal standards; (ii) we should consider using several markers through a multiplex polymerase chain reaction (PCR) approach; and (iii) we should commit to the publication and transparency of all protocol-associated metadata in a standardized fashion. For (i), we provide a ready-to-use recipe for synthetic cytochrome c oxidase spike-ins, which enable between-sample comparisons. For (ii), we propose two gene regions for the implementation of multiplex PCR approaches, thereby achieving a more comprehensive community description. For (iii), we offer guidelines for transparent and unified reporting of field, wet-laboratory and dry-laboratory procedures, as a key to making comparisons between studies. Together, we feel that these three advances will result in joint quality and calibration standards rather than the current laboratory-specific proof of concepts. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Brute force prey metabarcoding to explore the diets of small invertebrates.

Prey metabarcoding has become a popular tool in molecular ecology for resolving trophic interactions at high resolution, from various sample types and animals. To date, most predator-prey studies of small-sized animals (<1 mm) have met the problem of overabundant predator DNA in dietary samples by adding blocking primers/peptide nucleic acids. These primers aim to limit the PCR amplification and detection of the predator DNA but may introduce bias to the prey composition identified by interacting with sequences that are similar to those of the predator. Here we demonstrate the use of an alternative method to explore the prey of small marine copepods using whole-body DNA extracts and deep, brute force metabarcoding of an 18S rDNA fragment. After processing and curating raw data from two sequencing runs of varying depths (0.4 and 5.4 billion raw reads), we isolated 1.3 and 52.2 million prey reads, with average depths of ~15,900 and ~120,000 prey reads per copepod individual, respectively. While data from both sequencing runs were sufficient to distinguish dietary compositions from disparate seasons, locations, and copepod species, greater sequencing depth led to better separation of clusters. As computation and sequencing are becoming ever more powerful and affordable, we expect the brute force approach to become a general standard for prey metabarcoding, as it offers a simple and affordable solution to consumers that is impractical to dissect or unknown to science.

Nemabiome metabarcoding shows a high prevalence of Haemonchus contortus and predominance of Camelostrongylus mentulatus in alpaca herds in the northern UK.

Gastrointestinal nematodes (GINs) are a common threat faced by pastoral livestock. Since their major introduction to the UK in the early 1990s, South American camelids have been cograzed with sheep, horses, and other livestock, allowing exposure to a range of GIN species. However, there have been no molecular-based studies to investigate the GIN populations present in these camelids. In the current study, we sampled nine alpaca herds from northern England and southern Scotland and used high-throughput metabarcoded sequencing to describe their GIN species composition. A total of 71 amplicon sequence variants (ASVs) were identified representing eight known GIN species. Haemonchus contortus was the most prevalent species found in almost all herds in significant proportions. The identification of H. contortus in other livestock species is unusual in the northern UK, implying that alpacas may be suitable hosts and potential reservoirs for infection in other hosts. In addition, the camelid-adapted GIN species Camelostrongylus mentulatus was identified predominantly in herds with higher faecal egg counts. These findings highlight the value of applying advanced molecular methods, such as nemabiome metabarcoding to describe the dynamics of gastrointestinal nematode infections in novel situations. The results provide a strong base for further studies involving cograzing animals to confirm the potential role of alpacas in transmitting GIN species between hosts.

Environmental DNA-based biodiversity profiling along the Houdong River in north-eastern Taiwan.

Background: This paper describes two datasets: species occurrences, which were determined by environmental DNA (eDNA) metabarcoding and their associated DNA sequences, originating from a research project which was carried out along the Houdong River (), Jiaoxi Township, Yilan, Taiwan. The Houdong River begins at an elevation of 860 m and flows for approximately 9 km before it empties into the Pacific Ocean. Meandering through mountains, hills, plains and alluvial valleys, this short river system is representative of the fluvial systems in Taiwan. The primary objective of this study was to determine eukaryotic species occurrences in the riverine ecosystem through the use of the eDNA analysis. The second goal was, based on the current dataset, to establish a metabarcoding eDNA data template that will be useful and replicable for all users, particularly the Taiwan community. The species occurrence data are accessible at the Global Biodiversity Information Facility (GBIF) portal and its associated DNA sequences have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI, respectively. A total of 12 water samples from the study yielded an average of 1.5 million reads. The subsequent species identification from the collected samples resulted in the classification of 432 Operational Taxonomic Units (OTUs) out of a total of 2,734. Furthermore, a total of 1,356 occurrences with taxon matches in GBIF were documented (excluding 4,941 incertae sedis, accessed 05-12-2023). These data will be of substantial importance for future species and habitat monitoring within the short river, such as assessment of biodiversity patterns across different elevations, zonations and time periods and its correlation to water quality, land uses and anthropogenic activities. Further, these datasets will be of importance for regional ecological studies, in particular the freshwater ecosystem and its status in the current global change scenarios. New information: The datasets are the first species diversity description of the Houdong River system using either eDNA or traditional monitoring processes.

Exploring the use of metabarcoding to reveal eukaryotic associations with mononchids nematodes.

Nematodes play a vital ecological role in soil and marine ecosystems, but there is limited information about their dietary diversity and feeding habits. Due to methodological challenges, the available information is based on inference rather than confirmed observations. The lack of correct dietary requirements also hampers rearing experiments. To achieve insight into the prey of mononchid nematodes, this study employed high-throughput Illumina paired-end sequencing using universal eukaryotic species 18S primers on 10 pooled mononchid nematode species, namely Mylonchulus brachyuris, M. brevicaudatus, Mylonchulus sp., Clarkus parvus, Prionchulus sp. M. hawaiiensis, M. sigmaturellus, M. vulvapapillatus, Anatonchus sp. and Miconchus sp. The results indicate that mononchids are associated with a remarkable diversity of eukaryotes, including fungi, algae, and protists. While the metabarcoding approach, first introduced here for mononchids, proved to be a simple and rapid method, it has several limitations and crucial methodological challenges that should be addressed in future studies. Ultimately, such methods should be able to evaluate the dietary complexity of nematodes and provide a valuable avenue for unraveling the dietary requirements of previously unculturable nematodes. This can contribute to the methodology of understanding their feeding habits and contributions to ecosystem dynamics.

Prevalent arbuscular mycorrhizae in roots and highly variable mycobiome in leaves of epiphytic subtropical fern Ophioderma pendulum.

PREMISE: Endophytic and mycorrhizal fungi are crucial in facilitating plant nutrition acquisition and stress tolerance. In epiphytic habitats, plants face nutrition and water stress, but their roots are mostly nonmycorrhizal and especially lacking in arbuscular mycorrhizal associations. Ophioderma pendulum is an epiphytic fern with a partially mycoheterotrophic lifestyle, likely heavily reliant on symbiotic fungi. To characterize fungal associations in the sporophyte of O. pendulum, we focused on leaves and roots of O. pendulum, seeking to reveal the fungal communities in these organs. METHODS: Roots and leaves from O. pendulum in a subtropical forest were examined microscopically to observe the morphology of fungal structures and determine the percentage of various fungal structures in host tissues. Fungal composition was profiled using metabarcoding techniques that targeted ITS2 of the nuclear ribosomal DNA. RESULTS: Roots were consistently colonized by arbuscular mycorrhizal fungi (Glomeromycota), especially Acaulospora. Unlike previous findings on epiphytic ferns, dark septate endophytes were rare in O. pendulum roots. Leaves were predominantly colonized by Ascomycota fungi, specifically the classes Dothideomycetes (46.88%), Eurotiomycetes (11.51%), Sordariomycetes (6.23%), and Leotiomycetes (6.14%). Across sampling sites, fungal community compositions were similar in the roots but differed significantly in the leaves. CONCLUSIONS: Ophioderma pendulum maintains stable, single-taxon-dominant communities in the roots, primarily featuring arbuscular mycorrhizal fungi, whereas the leaves may harbor opportunistic fungal colonizers. Our study underlines the significance of mycorrhizal fungi in the adaptation of epiphytic ferns.

Dynamics and drivers of mycorrhizal fungi after glacier retreat.

The development of terrestrial ecosystems depends greatly on plant mutualists such as mycorrhizal fungi. The global retreat of glaciers exposes nutrient-poor substrates in extreme environments and provides a unique opportunity to study early successions of mycorrhizal fungi by assessing their dynamics and drivers. We combined environmental DNA metabarcoding and measurements of local conditions to assess the succession of mycorrhizal communities during soil development in 46 glacier forelands around the globe, testing whether dynamics and drivers differ between mycorrhizal types. Mycorrhizal fungi colonized deglaciated areas very quickly (< 10 yr), with arbuscular mycorrhizal fungi tending to become more diverse through time compared to ectomycorrhizal fungi. Both alpha- and beta-diversity of arbuscular mycorrhizal fungi were significantly related to time since glacier retreat and plant communities, while microclimate and primary productivity were more important for ectomycorrhizal fungi. The richness and composition of mycorrhizal communities were also significantly explained by soil chemistry, highlighting the importance of microhabitat for community dynamics. The acceleration of ice melt and the modifications of microclimate forecasted by climate change scenarios are expected to impact the diversity of mycorrhizal partners. These changes could alter the interactions underlying biotic colonization and belowground-aboveground linkages, with multifaceted impacts on soil development and associated ecological processes.