Pollen DNA metabarcoding—marker‐based genetic identification of potentially mixed‐species pollen samples—has applications across a variety of fields. While basic species‐level pollen identification ...using standard DNA barcode markers is established, the extent to which metabarcoding (a) correctly assigns species identities to mixes (qualitative matching) and (b) generates sequence reads proportionally to their relative abundance in a sample (quantitative matching) is unclear, as these have not been assessed relative to known standards. We tested the quantitative and qualitative robustness of metabarcoding in constructed pollen mixtures varying in species richness (1–9 species), taxonomic relatedness (within genera to across class) and rarity (5%–100% of grains), using Illumina MiSeq with the markers rbcL and ITS2. Qualitatively, species composition determinations were largely correct, but false positives and negatives occurred. False negatives were typically driven by lack of a barcode gap or rarity in a sample. Species richness and taxonomic relatedness, however, did not strongly impact correct determinations. False positives were likely driven by contamination, chimeric sequences and/or misidentification by the bioinformatics pipeline. Quantitatively, the proportion of reads for each species was only weakly correlated with its relative abundance, in contrast to suggestions from some other studies. Quantitative mismatches are not correctable by consistent scaling factors, but instead are context‐dependent on the other species present in a sample. Together, our results show that metabarcoding is largely robust for determining pollen presence/absence but that sequence reads should not be used to infer relative abundance of pollen grains.
Summary
Disturbances have altered community dynamics in boreal forests with unknown consequences for belowground ecological processes. Soil fungi are particularly sensitive to such disturbances; ...however, the individual response of fungal guilds to different disturbance types is poorly understood.
Here, we profiled soil fungal communities in lodgepole pine forests following a bark beetle outbreak, wildfire, clear‐cut logging, and salvage‐logging. Using Illumina MiSeq to sequence ITS1 and SSU rDNA, we characterized communities of ectomycorrhizal, arbuscular mycorrhizal, saprotrophic, and pathogenic fungi in sites representing each disturbance type paired with intact forests. We also quantified soil fungal biomass by measuring ergosterol.
Abiotic disturbances changed the community composition of ectomycorrhizal fungi and shifted the dominance from ectomycorrhizal to saprotrophic fungi compared to intact forests. The disruption of the soil organic layer with disturbances correlated with the decline of ectomycorrhizal and the increase of arbuscular mycorrhizal fungi. Wildfire changed the community composition of pathogenic fungi but did not affect their proportion and diversity. Fungal biomass declined with disturbances that disrupted the forest floor.
Our results suggest that the disruption of the forest floor with disturbances, and the changes in C and nutrient dynamics it may promote, structure the fungal community with implications for fungal biomass–C.
See also the Commentary on this article by Marín & Kohout, 229: 656–658.
The biostimulant potential of three different organic acids (OAs) present in the rhizosphere, specifically lactic, oxalic, and citric acids, have been studied. The results showed a rapid and complete ...metabolism of these three acids with soil microorganisms using them as a source of carbon and energy. Biostimulation was confirmed by soil biochemical studies which showed an increase in enzymatic activities, such as dehydrogenase and phosphatase, lactic and citric acids being those that produced the greatest biostimulation. With regard to microbiota composition, amplicon sequencing of the 16S rRNA gene showed changes in the structure of soil microbial communities. Applying OAs produced a decrease in richness and diversity indices, inducing specific changes in the structure of the microbiological communities. Applying lactic acid induced rapid changes in microbiota composition at both phylum and family taxonomic levels, favoring the proliferation of microorganisms involved in its degradation and soil fertility, such as the genus
Bacillus
and the family Micrococcaceae. Once the lactic acid was degraded, the biodiversity tended to return to similar phyla, but specific distinctive families and genera remained, leaving a pattern of induction of taxa described as plant growth-promoting bacteria (PGPB), such as the
Sinorhizobium
and
Lysobacter
genera, and the Pseudomonaceae family. Similar behavior was found with citric acid, which favored the proliferation and dominance of microorganisms of the Clostridiaceae family, involved in its degradation, as well as microorganisms of both the Micrococcaceae and Pseudomonadaceae families which were found on day 7, leaving a similar pattern of induction as that found after the mineralization of lactic acid. On the other hand, oxalic acid induced long-lasting changes in the bacterial community composition. This was characterized by an increase in the proportion of the Burkholderiales order, which includes microorganisms involved in the degradation of this acid and microorganisms described as PGPB. This study presents evidence supporting the use of OAs as potential soil fertility inducers, due both to their effects in enhancing the dominance of taxa described as PGPB and to their stimulating soil microbial activity.
Matching animal demand to forage availability is a core principle in sustainable rangeland management. We evaluated the use of interannual flexibility in stocking rates compared with fixed stocking ...at light, moderate, and heavy stocking rates on livestock weight gains and economic responses for 7 yr (2016−2022) in North American northern mixed-grass prairie. The grazing season began in early June each year, so stocking rates in the flexible treatment were calculated on the basis of the amount of forage production predicted from actual precipitation received in April and May combined with long-term mean annual precipitation received at the study site in June, as well as an adjustment in stocking rate based on the amount of residual forage remaining at the end of the previous grazing season. Across years, mean stocking rate for the flexible stocking treatment (32.5 animal unit days AUD ha−1) was between heavy (38.6 AUD ha−1) and moderate (29.7 AUD ha−1) and was twice as high as the light (15.8 AUD ha−1). Cumulative total beef production for the 7 yr was highest with heavy stocking (282.6 kg ha−1), 17% less in the flexible (234.2 kg ha−1), and 19% less in the moderate (229.4 kg ha−1) stocking rates. It was 55% lower with light stocking (128.4 kg ha−1). Crude protein and digestible organic matter, as well as composition of plant functional groups in diets of yearlings, did not differ between the moderate versus the flexible stocking treatments. Compared with moderate stocking, flexible stocking resulted in 6.9% lower cumulative gross ($2 299) and 10.8% lower net ($1 407) economic returns per yearling. We suggest that future evaluations of flexible stocking strategies consider incorporating seasonal forecasts combined with intraseasonal adjustments in stocking rates as the growing season unfolds. Advancements in predictive forage forecasting tools and remote sensing capabilities are needed to support such a strategy.
•Environmental DNA (eDNA) can be obtained from ancient and modern environments.•Coupled to DNA metabarcoding, eDNA is a powerful means of biodiversity monitoring.•eDNA has short degradation time in ...contemporary ecosystems.•We review main findings of eDNA studies and discuss limitations and perspectives.•We focus specifically on eDNA for biodiversity monitoring and conservation.
The continuous decline in Earth’s biodiversity represents a major crisis and challenge for the 21st century, and there is international political agreement to slow down or halt this decline. The challenge is in large part impeded by the lack of knowledge on the state and distribution of biodiversity – especially since the majority of species on Earth are un-described by science. All conservation efforts to save biodiversity essentially depend on the monitoring of species and populations to obtain reliable distribution patterns and population size estimates. Such monitoring has traditionally relied on physical identification of species by visual surveys and counting of individuals. However, traditional monitoring techniques remain problematic due to difficulties associated with correct identification of cryptic species or juvenile life stages, a continuous decline in taxonomic expertise, non-standardized sampling, and the invasive nature of some survey techniques. Hence, there is urgent need for alternative and efficient techniques for large-scale biodiversity monitoring. Environmental DNA (eDNA) – defined here as: genetic material obtained directly from environmental samples (soil, sediment, water, etc.) without any obvious signs of biological source material – is an efficient, non-invasive and easy-to-standardize sampling approach. Coupled with sensitive, cost-efficient and ever-advancing DNA sequencing technology, it may be an appropriate candidate for the challenge of biodiversity monitoring. Environmental DNA has been obtained from ancient as well as modern samples and encompasses single species detection to analyses of ecosystems. The research on eDNA initiated in microbiology, recognizing that culture-based methods grossly misrepresent the microbial diversity in nature. Subsequently, as a method to assess the diversity of macro-organismal communities, eDNA was first analyzed in sediments, revealing DNA from extinct and extant animals and plants, but has since been obtained from various terrestrial and aquatic environmental samples. Results from eDNA approaches have provided valuable insights to the study of ancient environments and proven useful for monitoring contemporary biodiversity in terrestrial and aquatic ecosystems. In the future, we expect the eDNA-based approaches to move from single-marker analyses of species or communities to meta-genomic surveys of entire ecosystems to predict spatial and temporal biodiversity patterns. Such advances have applications for a range of biological, geological and environmental sciences. Here we review the achievements gained through analyses of eDNA from macro-organisms in a conservation context, and discuss its potential advantages and limitations for biodiversity monitoring.
The lack of information about plastic pollution in many marine regions hinders firm actions to manage human activities and mitigate their impacts. This study conducted for the first time a ...quali-quantitative evaluation of floating plastics and their associated biota from coastal and oceanic waters in South Brazil. Plastics were collected using a manta net, and were categorized according to their shape, size, malleability and polymer composition. Multi-marker DNA metabarcoding (16S, and 18S V4 and V9 rRNA regions) was performed to identify prokaryotes and eukaryotes associated to plastics. We found 371 likely plastic particles of several sizes, shapes and polymers, and the average concentration of plastics at the region was 4461 items.km-2 (SD ± 3914). Microplastics (0.5 - 5 mm) were dominant in most sampling stations, with fragments and lines representing the most common shapes. Diverse groups of prokaryotes (20 bacteria phyla) and eukaryotes (41 groups) were associated with plastics. Both the community composition and richness of epiplastic organisms were highly variable between individual plastics but, in general, were not influenced by plastic categories. Organisms with potential pathogenicity (e.g. Vibrio species. and Alexandrium tamarense), as well as potential plastic degraders (e.g. Ralstonia, Pseudomonas, and Alcanivorax species), were found. The information generated here is pivotal to support strategies to prevent the input and mitigate the impacts of plastics and their associated organisms on marine environments.
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•An average of 4461 plastic items.km-2 was found in the Western South Atlantic.•Microplastic fragments and lines, of nine colours and seven polymers, were dominant.•Rivers and fisheries are likely major sources of plastics to the region.•Plastics had a high diversity of associated prokaryotes and eukaryotes.•Plastic-associated organisms included potential biodegraders and pathogens.
The freshwater bodies of India are highly biodiverse but still understudied, especially concerning ciliates. Ciliates constitute a significant portion of eukaryotic diversity and play crucial roles ...in microbial loops, nutrient recycling, and ecosystem maintenance. The present study aimed to elucidate ciliate diversity in three freshwater sites in the Delhi region of India: Okhla Bird Sanctuary (OBS), Sanjay Lake (SL), and Raj Ghat pond (RJ). This study represents the first investigation into the taxonomic diversity and richness of freshwater ciliates in India using a high-throughput DNA metabarcoding approach. For the analysis, total environmental DNA was extracted from the three freshwater samples, followed by sequencing of the 18S V4 barcode region and subsequent phylogenetic analyses. Operational taxonomic units (OTU) analyses revealed maximum species diversity in OBS (106), followed by SL (104) and RJ (99) sites. Ciliates from the classes Oligohymenophorea, Prostomatea, and Spirotrichea were dominant in the three sites. The study discusses the ability of the metabarcoding approach to uncover unknown and rare species. The study highlights the need for refined reference databases and cautious interpretation of the high-throughput sequencing-generated data while emphasizing the complementary nature of molecular and morphological approaches in studying ciliate diversity.
With the growing anthropogenic pressure on marine ecosystems, the need for efficient monitoring of biodiversity grows stronger. DNA metabarcoding of bulk samples is increasingly being implemented in ...ecosystem assessments and is more cost‐efficient and less time‐consuming than monitoring based on morphology. However, before raw sequences are obtained from bulk samples, a profound number of methodological choices must be made. Here, we critically review the recent methods used for metabarcoding of marine bulk samples (including benthic, plankton and diet samples) and indicate how potential biases can be introduced throughout sampling, preprocessing, DNA extraction, marker and primer selection, PCR amplification and sequencing. From a total of 64 studies evaluated, our recommendations for best practices include to (a) consider DESS as a fixative instead of ethanol, (b) use the DNeasy PowerSoil kit for any samples containing traces of sediment, (c) not limit the marker selection to COI only, but preferably include multiple markers for higher taxonomic resolution, (d) avoid touchdown PCR profiles, (e) use a fixed annealing temperature for each primer pair when comparing across studies or institutes, (f) use a minimum of three PCR replicates, and (g) include both negative and positive controls. Although the implementation of DNA metabarcoding still faces several technical complexities, we foresee wide‐ranging advances in the near future, including improved bioinformatics for taxonomic assignment, sequencing of longer fragments and the use of whole‐genome information. Despite the bulk of biases involved in metabarcoding of bulk samples, if appropriate controls are included along the data generation process, it is clear that DNA metabarcoding provides a valuable tool in ecosystem assessments.
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