DNA metabarcoding reveals metacommunity dynamics in a threatened boreal wetland wilderness.

The complexity and natural variability of ecosystems present a challenge for reliable detection of change due to anthropogenic influences. This issue is exacerbated by necessary trade-offs that reduce the quality and resolution of survey data for assessments at large scales. The Peace-Athabasca Delta (PAD) is a large inland wetland complex in northern Alberta, Canada. Despite its geographic isolation, the PAD is threatened by encroachment of oil sands mining in the Athabasca watershed and hydroelectric dams in the Peace watershed. Methods capable of reliably detecting changes in ecosystem health are needed to evaluate and manage risks. Between 2011 and 2016, aquatic macroinvertebrates were sampled across a gradient of wetland flood frequency, applying both microscope-based morphological identification and DNA metabarcoding. By using multispecies occupancy models, we demonstrate that DNA metabarcoding detected a much broader range of taxa and more taxa per sample compared to traditional morphological identification and was essential to identifying significant responses to flood and thermal regimes. We show that family-level occupancy masks high variation among genera and quantify the bias of barcoding primers on the probability of detection in a natural community. Interestingly, patterns of community assembly were nearly random, suggesting a strong role of stochasticity in the dynamics of the metacommunity. This variability seriously compromises effective monitoring at local scales but also reflects resilience to hydrological and thermal variability. Nevertheless, simulations showed the greater efficiency of metabarcoding, particularly at a finer taxonomic resolution, provided the statistical power needed to detect change at the landscape scale.

Immunoadjuvant and Humoral Immune Responses of Garlic (Allium sativum L.) Lectins upon Systemic and Mucosal Administration in BALB/c Mice.

Dietary food components have the ability to affect immune function; following absorption, specifically orally ingested dietary food containing lectins can systemically modulate the immune cells and affect the response to self- and co-administered food antigens. The mannose-binding lectins from garlic (Allium sativum agglutinins; ASAs) were identified as immunodulatory proteins in vitro. The objective of the present study was to assess the immunogenicity and adjuvanticity of garlic agglutinins and to evaluate whether they have adjuvant properties in vivo for a weak antigen ovalbumin (OVA). Garlic lectins (ASA I and ASA II) were administered by intranasal (50 days duration) and intradermal (14 days duration) routes, and the anti-lectin and anti-OVA immune (IgG) responses in the control and test groups of the BALB/c mice were assessed for humoral immunogenicity. Lectins, co-administered with OVA, were examined for lectin-induced anti-OVA IgG response to assess their adjuvant properties. The splenic and thymic indices were evaluated as a measure of immunomodulatory functions. Intradermal administration of ASA I and ASA II had showed a four-fold and two-fold increase in anti-lectin IgG response, respectively, vs. the control on day 14. In the intranasal route, the increases were 3-fold and 2.4-fold for ASA I and ASA II, respectively, on day 50. No decrease in the body weights of animals was noticed; the increases in the spleen and thymus weights, as well as their indices, were significant in the lectin groups. In the adjuvanticity study by intranasal administration, ASA I co-administered with ovalbumin (OVA) induced a remarkable increase in anti-OVA IgG response (~six-fold; p < 0.001) compared to the control, and ASA II induced a four-fold increase vs. the control on day 50. The results indicated that ASA was a potent immunogen which induced mucosal immunogenicity to the antigens that were administered intranasally in BALB/c mice. The observations made of the in vivo study indicate that ASA I has the potential use as an oral and mucosal adjuvant to deliver candidate weak antigens. Further clinical studies in humans are required to confirm its applicability.

Determinants of Soil Bacterial and Fungal Community Composition Toward Carbon-Use Efficiency Across Primary and Secondary Forests in a Costa Rican Conservation Area.

Tropical secondary forests currently represent over half of the world's remaining tropical forests and are critical candidates for maintaining global biodiversity and enhancing potential carbon-use efficiency (CUE) and, thus, carbon sequestration. However, these ecosystems can exhibit multiple successional pathways, which have hindered our understanding of the soil microbial drivers that facilitate improved CUE. To begin to address this, we examined soil % C; % N; C:N ratio; soil microbial biomass C (Cmic); NO3-; NH4+; pH; % moisture; % sand, silt, and clay; and elevation, along with soil bacterial and fungal community composition, and determined which soil abiotic properties structure the soil Cmic and the soil bacterial and fungal communities across a primary forest, 33-year-old secondary forest, and 22-year-old young secondary in the Northern Zone of Costa Rica. We provide evidence that soil microbial communities were mostly distinct across the habitat types and that these habitats appear to have affected the soil ectomycorrhizal fungi and the soil microbial groups associated with the degradation of complex carbon compounds. We found that soil Cmic levels increased along the management gradient from young, to old secondary, to primary forest. In addition, the changes in soil Cmic and soil fungal community structure were significantly related to levels of soil NO3-. Our analyses showed that even after 33 years of natural forest regrowth, the clearing of tropical forests can have persistent effects on soil microbial communities and that it may take a longer time than we realized for secondary forests to develop carbon-utilization efficiencies similar to that of a primary forest. Our results also indicated that forms of inorganic N may be an important factor in structuring soil Cmic and the soil microbial communities, leading to improved CUE in regenerating secondary forests. This study is the first in the region to highlight some of the factors which appear to be structuring the soil Cmic and soil microbial communities such that they are more conducive for enhanced CUE in secondary forests.

Using metagenomics to show the efficacy of forest restoration in the New Jersey Pine Barrens.

The Franklin Parker Preserve within the New Jersey Pine Barrens contains 5000 acres of wetlands habitat, including old-growth Atlantic white cedar (or AWC; Chamaecyparis thyoides) swamps, cranberry bogs, and former cranberry bogs undergoing restoration into AWC forests. This study showed that the C-use efficiency was greater in the old-growth AWC soils than in soils from 8-year-old mid-stage restored AWC stands, which were greater than found in soil from 4-year-old AWC stands-the latter two stands being restored from long-term cranberry bogs. A metagenomic analysis of eDNA extracted from these soils showed that the C-cycle trends were associated with increases in the relative numbers of DNA sequences from several copiotrophic bacterial groups (Bacteroidetes and Proteobacteria), complex C-decomposing fungal groups (Sordiomycetes, Mortierellales, and Thelephorales), and collembolan and formicid invertebrates. All groups are indicators of successionally more advanced soils, and critical for soil C-cycle activities. These data suggest that the restoration activities studied are enhancing critical guilds of soil biota, and increasing C-use efficiency in the soils of restored habitats, and that the use of metagenomic analysis of soil eDNA can be used in the development of assessment models for soil recovery of wetlands following restoration.

Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics.

Conventional assessments of ecosystem sample composition are based on morphology-based or DNA barcode identification of individuals. Both approaches are costly and time-consuming, especially when applied to the large number of specimens and taxa commonly included in ecological investigations. Next-generation sequencing approaches can overcome the bottleneck of individual specimen isolation and identification by simultaneously sequencing specimens of all taxa in a bulk mixture. Here we apply multiple parallel amplification primers, multiple DNA barcode markers, 454-pyrosequencing, and Illumina MiSeq sequencing to the same sample to maximize recovery of the arthropod macrobiome and the bacterial and other microbial microbiome of a bulk arthropod sample. We validate this method with a complex sample containing 1,066 morphologically distinguishable arthropods from a tropical terrestrial ecosystem with high taxonomic diversity. Multiamplicon next-generation DNA barcoding was able to recover sequences corresponding to 91% of the distinguishable individuals in a bulk environmental sample, as well as many species present as undistinguishable tissue. 454-pyrosequencing was able to recover 10 more families of arthropods and 30 more species than did conventional Sanger sequencing of each individual specimen. The use of other loci (16S and 18S ribosomal DNA gene regions) also added the detection of species of microbes associated with these terrestrial arthropods. This method greatly decreases the time and money necessary to perform DNA-based comparisons of biodiversity among ecosystem samples. This methodology opens the door to much cheaper and increased capacity for ecological and evolutionary studies applicable to a wide range of socio-economic issues, as well as a basic understanding of how the world works.

Mitochondrial and nuclear phylogenetic analysis with Sanger and next-generation sequencing shows that, in Área de Conservación Guanacaste, northwestern Costa Rica, the skipper butterfly named Urbanus belli (family Hesperiidae) comprises three morphologically cryptic species.

BACKGROUND: Skipper butterflies (Hesperiidae) are a relatively well-studied family of Lepidoptera. However, a combination of DNA barcodes, morphology, and natural history data has revealed several cryptic species complexes within them. Here, we investigate three DNA barcode lineages of what has been identified as Urbanus belli (Hesperiidae, Eudaminae) in Área de Conservación Guanacaste (ACG), northwestern Costa Rica. RESULTS: Although no morphological traits appear to distinguish among the three, congruent nuclear and mitochondrial lineage patterns show that "Urbanus belli" in ACG is a complex of three sympatric species. A single strain of Wolbachia present in two of the three cryptic species indicates that Urbanus segnestami Burns (formerly Urbanus belliDHJ01), Urbanus bernikerni Burns (formerly Urbanus belliDHJ02), and Urbanus ehakernae Burns (formerly Urbanus belliDHJ03) may be biologically separated by Wolbachia, as well as by their genetics. Use of parallel sequencing through 454-pyrosequencing improved the utility of ITS2 as a phylogenetic marker and permitted examination of the intra- and interlineage relationships of ITS2 variants within the species complex. Interlineage, intralineage and intragenomic compensatory base pair changes were discovered in the secondary structure of ITS2. CONCLUSION: These findings corroborate the existence of three cryptic species. Our confirmation of a novel cryptic species complex, initially suggested by DNA barcode lineages, argues for using a multi-marker approach coupled with next-generation sequencing for exploration of other suspected species complexes.

Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1.

The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the ~12-Mb genome of CAT-1, when compared with the reference S228c genome, contains ~36,000 homozygous and ~30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains.

Next-generation sequencing technologies for environmental DNA research.

Since 2005, advances in next-generation sequencing technologies have revolutionized biological science. The analysis of environmental DNA through the use of specific gene markers such as species-specific DNA barcodes has been a key application of next-generation sequencing technologies in ecological and environmental research. Access to parallel, massive amounts of sequencing data, as well as subsequent improvements in read length and throughput of different sequencing platforms, is leading to a better representation of sample diversity at a reasonable cost. New technologies are being developed rapidly and have the potential to dramatically accelerate ecological and environmental research. The fast pace of development and improvements in next-generation sequencing technologies can reflect on broader and more robust applications in environmental DNA research. Here, we review the advantages and limitations of current next-generation sequencing technologies in regard to their application for environmental DNA analysis.

Assessing biodiversity of a freshwater benthic macroinvertebrate community through non-destructive environmental barcoding of DNA from preservative ethanol.

BACKGROUND: Characterizing biodiversity in a habitat or in targeted taxonomically or socioeconomically important groups remains a challenge. Standard DNA-based biodiversity identification tools such as DNA barcoding coupled with high-throughput Next-Generation Sequencing (NGS) technologies are rapidly changing the landscape of biodiversity analysis by targeting various habitats and a wide array of organisms. However, effective use of these technological advances requires optimized protocols and benchmarking against traditional tools. Here we investigate the use of commonly used preservative ethanol as a non-destructive and inexpensive source of DNA for NGS biodiversity analysis of benthic macroinvertebrates. We used the preservative ethanol added to field collected organisms (live sorted bulk benthic samples) as a source of community DNA for NGS environmental barcoding. We directly compare this approach with a DNA barcode library generated using Sanger sequencing of all individuals separated from abenthic sample as well as with NGS environmental barcoding of DNA extracted from mixed/homogenized tissue specimens of the same benthic sample. We also evaluate a multiplex PCR strategy, as compared to commonly used single amplicon workflow, using three newly designed primer sets targeting a wide array of benthic macroinvertebrate taxa. RESULTS: Our results indicate the effectiveness of ethanol-based DNA in providing sequence information from 87% of taxa identified individually from mixture as compared to 89% in conventional tissue extracted DNA. Missing taxa in both DNA sources were from species with the lowest abundance (e.g. 1 individual) in the benthic mixture. Interestingly, we achieved 100% detection for taxa represented with more than 1% individuals in the mixture in both sources of DNA. Our multiplex amplification regime increased the detection as compared to any single primer set indicating the usefulness of using multiple primer sets in initial amplification of target genes. CONCLUSIONS: Although NGS approaches have significantly increased the potential of using DNA information in biodiversity analysis, robust methods are needed to provide reliable data and alleviate sample-processing bottlenecks. Here we coupled non-destructive DNA access and a multiplex PCR approach in NGS environmental barcoding for effective data generation from benthic live-sorted samples collected in bulk and preserved in ethanol. Our study provides a possible solution to sampling and vouchering challenges in using benthic samples through next-generation environmental barcoding and facilitates wider utility of DNA information, especially species-specific DNA barcodes, in ecological and environmental studies and real-world applications such as biomonitoring programs.

Nutrients Uptake and Accumulation in Plant Parts of Fragrant Rosa Species Irrigated with Treated and Untreated Wastewater.

Water scarcity has critically augmented the need for the exploration of alternative irrigation sources mainly in water-scarce regions. This water scarcity has put tremendous pressure on the agri-based economy of countries such as Pakistan. The reuse of sewage wastewater has been appearing as the only alternative water source, which can lessen our dependence upon freshwater (FW). The current study aimed to scrutinize the influence of treated wastewater (TWW) and untreated wastewater (UTWW) irrigation on the nutrient (N, P, K, Ca, and Na) concentration in different plant parts, i.e., roots, stems, leaves, and flowers, of four scented Rosa species (R. bourboniana, R. centifolia, R. Gruss-an-telpitz, and R. damascena) during the first week of 2018 to the last week of 2019. The experiment was arranged according to the two-factor factorial arrangement i.e., factor I was the irrigation source, while factor II was the Rosa species. The experimental water analysis showed that mineral and chemical concentrations in FW and TWW were within permissible limits of national environmental quality standards (NEQSs) for wastewater. The UTWW of this study possessed a higher electrical conductivity (EC), chemical oxygen demand (COD), biological oxygen demand (BOD), total nitrogen (TN), and metals (Cd, Co, and Pb) than recommended levels. The results revealed that P, K, Ca, and Na contents significantly increased in all studied plant parts of Rosa species as the duration of irrigation with TWW and UTWW increased and vice versa in the case of N contents, while the ratio of N content elevation by applying TWW and UTWW were also not increased compared to other studied nutrients. The nutrients (except Ca) were found as maximum in all plant parts with UTWW compared to FW and TWW irrigation in roses. These stimulations were accredited to the presence of higher essential nutrients and some metals in UTWW. This experiment confirmed the disparities in nutrient contents of scented Rosa species due to the different absorbability of each element in every plant part. Regarding the nutrient accumulation in rose plant tissues, the results of the present study confirm that untreated wastewater must be treated to some extent to grow scented roses where water is scarce.

Spatial distribution and identification of potential risk regions to rice blast disease in different rice ecosystems of Karnataka.

Rice is a globally important crop and highly vulnerable to rice blast disease (RBD). We studied the spatial distribution of RBD by considering the 2-year exploratory data from 120 sampling sites over varied rice ecosystems of Karnataka, India. Point pattern and surface interpolation analyses were performed to identify the spatial distribution of RBD. The spatial clusters of RBD were generated by spatial autocorrelation and Ripley's K function. Further, inverse distance weighting (IDW), ordinary kriging (OK), and indicator kriging (IK) approaches were utilized to generate spatial maps by predicting the values at unvisited locations using neighboring observations. Hierarchical cluster analysis using the average linkage method identified two main clusters of RBD severity. From the Local Moran's I, most of the districts were clustered together (at I > 0), except the coastal and interior districts (at I < 0). Positive spatial dependency was observed in the Coastal, Hilly, Bhadra, and Upper Krishna Project ecosystems (p > 0.05), while Tungabhadra and Kaveri ecosystem districts were clustered together at p < 0.05. From the kriging, Hilly ecosystem, middle and southern parts of Karnataka were found vulnerable to RBD. This is the first intensive study in India on understanding the spatial distribution of RBD using geostatistical approaches, and the findings from this study help in setting up ecosystem-specific management strategies against RBD.

Impact of Safe Rock® Minerals, Mineral Fertilizers, and Manure on the Quantity and Quality of the Wheat Yield in the Rice-Wheat Cropping System.

Rice-wheat (RW) rotation is the largest agriculture production system in South Asia with a multifaceted role in maintaining the livelihood of people. The customary practices and indiscriminate use of synthetic fertilizers have culminated in the decline of its productivity and profitability during the past two decades, thus affecting the sustainability of wheat. Safe Rock® Minerals (SRM) is a multi-nutrient rich natural rock mineral with great potential to manage soil degradation, reducing the input of fertilizers, improving soil fertility, and plant health. Thus, a field trial was conducted at the research farm of ICAR-Indian Agricultural Research Institute, New Delhi from 2016 to 2018 to evaluate the impact of Safe Rock® Minerals (SRM) on biometric parameters, productivity, quality, and nutrient uptake by conventional wheat and System of Wheat Intensification (SWI) in the wheat-rice cropping system. The results indicate that SWI performed better in terms of growth, yield, and quality parameters than conventional wheat. Among nutrient management practices; the highest growth, yield, and yield attributes of wheat were achieved with the use of SRM application 250 kg ha-1 + 100% Recommended Dose of Fertilizer (RDF). SRM application also increased grain protein content significantly. In conclusion, the integrated use of SRM with organic manures can serve as an eco-friendly approach for sustainable wheat production.

Antimicrobial activities of different solvent extracts from stem and seeds of Peganum Harmala L.

Wild medicinal herbs have been used as folk and traditional medicines all across the world since well before recorded history. This present study was designed to test the antimicrobial activities of five different solvent extracted samples (n-hexane, n-butanol, ethyl acetate, methanol, and water) of Peganum harmala using stems and seeds. Two different strains of Gram-negative bacteria (Escherichia coli and Klebsiella pneumonia), two Gram-positive bacteria (Bacillus subtilus and Staphylococcus aureus), and one fungal strain (Candida albicans) were used. The antimicrobial activities were measured using a disc diffusion assay. Two concentrations of the extracts (1 and 2mgDisc-1) were used. Ethyl acetate fraction was found more affective among the tested solvents and showed maximum activity (zone of inhibition) against S. aureus (65.53 and 81.10%), E. coli (46.22 and 61.29%) while n-butanol and water fractions gave maximum activity against S. aureus (78.86 and 70.00%) and K. pneumonia (57.00 and 61.39%) respectively. Water fraction showed maximum activity against C. albicans (60.00 and 81.88%). In the case of the stem, Ethyl acetate again showed more activity against B. subtilus (38.57 and 42.10%) and S. aureus (36.66 and 46.66%) while n-butanol showed maximum activity against K. pneumonia (24.55 and 32.44%) and E. coli (27.93 and 37.61%). Methanol was found more effective against C. albicans (25.71 and 43.80%). Seed extracted samples were found more effective compared to the stem. Ethyl acetate, butanol, and aqueous extracted samples showed good activity against the tested microbes, so these fractions are recommended for study their mechanism of actions and isolation of bioactive metabolites responsible for antimicrobial activities. The P. harmala should be evaluated for their bioactive compounds to be used in future studies. Our objective is to provide the framework for future study on the roles of P. harmala as traditional medicines.

Antioxidant, Scavenging, Reducing, and Anti-Proliferative Activities of Selected Tropical Brown Seaweeds Confirm the Nutraceutical Potential of Spatoglossum asperum.

Brown seaweeds have shown high potential of bioactivity and provide health benefits as an important functional food ingredient. Therefore, four abundantly growing tropical brown seaweeds-Iyengaria stellata, Spatoglossum asperum, Sargassum linearifolium, and Stoechospermum polypodioides-were collected from the Saurashtra Coast of the Arabian Sea. They were analyzed for metabolite profiling, biochemical activities (including total antioxidant, reducing, scavenging, and anti-proliferative characteristics), and total phenolic and flavonoid contents. A concentration-dependent antioxidant, reducing, and scavenging activities were observed for all four brown seaweeds. The S. asperum and I. stellata extracts showed maximum total antioxidant activity. S. asperum also showed high scavenging and reducing activities compared to other studied brown seaweeds. Further, S. asperum contained high total phenolic and flavonoid content compared to other brown seaweeds collected from the same coast. A multivariate correlation study confirmed a positive correlation between total phenolic and flavonoid contents, and biochemical activities (total antioxidant, scavenging and reducing) for all brown seaweeds. About 35% anti-proliferative activity was observed with S. asperum extract on Huh7 cells; in contrast S. polypodioide showed about 44% proliferation inhibition of Huh7 cells. Similarly, 26% proliferation inhibition of HeLa cells was observed with S. asperum extract. Overall, S. asperum possesses high total flavonoid and phenolic amounts, and showed potential antioxidant, scavenging and reducing characteristics. The study confirmed the nutraceutical potential of S. asperum and that it could be a promising functional food ingredient.

A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing.

Multiplexed high-throughput pyrosequencing is currently limited in complexity (number of samples sequenced in parallel), and in capacity (number of sequences obtained per sample). Physical-space segregation of the sequencing platform into a fixed number of channels allows limited multiplexing, but obscures available sequencing space. To overcome these limitations, we have devised a novel barcoding approach to allow for pooling and sequencing of DNA from independent samples, and to facilitate subsequent segregation of sequencing capacity. Forty-eight forward-reverse barcode pairs are described: each forward and each reverse barcode unique with respect to at least 4 nt positions. With improved read lengths of pyrosequencers, combinations of forward and reverse barcodes may be used to sequence from as many as n(2) independent libraries for each set of 'n' forward and 'n' reverse barcodes, for each defined set of cloning-linkers. In two pilot series of barcoded sequencing using the GS20 Sequencer (454/Roche), we found that over 99.8% of obtained sequences could be assigned to 25 independent, uniquely barcoded libraries based on the presence of either a perfect forward or a perfect reverse barcode. The false-discovery rate, as measured by the percentage of sequences with unexpected perfect pairings of unmatched forward and reverse barcodes, was estimated to be <0.005%.

Watered-down biodiversity? A comparison of metabarcoding results from DNA extracted from matched water and bulk tissue biomonitoring samples.

Biomonitoring programs have evolved beyond the sole use of morphological identification to determine the composition of invertebrate species assemblages in an array of ecosystems. The application of DNA metabarcoding in freshwater systems for assessing benthic invertebrate communities is now being employed to generate biological information for environmental monitoring and assessment. A possible shift from the extraction of DNA from net-collected bulk benthic samples to its extraction directly from water samples for metabarcoding has generated considerable interest based on the assumption that taxon detectability is comparable when using either method. To test this, we studied paired water and benthos samples from a taxon-rich wetland complex, to investigate differences in the detection of arthropod taxa from each sample type. We demonstrate that metabarcoding of DNA extracted directly from water samples is a poor surrogate for DNA extracted from bulk benthic samples, focusing on key bioindicator groups. Our results continue to support the use of bulk benthic samples as a basis for metabarcoding-based biomonitoring, with nearly three times greater total richness in benthic samples compared to water samples. We also demonstrated that few arthropod taxa are shared between collection methods, with a notable lack of key bioindicator EPTO taxa in the water samples. Although species coverage in water could likely be improved through increased sample replication and/or increased sequencing depth, benthic samples remain the most representative, cost-effective method of generating aquatic compositional information via metabarcoding.

Fecal source tracking and eDNA profiling in an urban creek following an extreme rain event.

Fecal contamination of recreational waters (i.e. lakes, rivers, beaches) poses an on-going problem for environmental and public health. Heavy rainfall can exacerbate existing problems with fecal contamination. As there could be variable sources of fecal contamination, identifying the source is critical for remediation efforts. This study utilized microbial source tracking (MST), chemical source tracking (CST) markers and environmental DNA (eDNA) metabarcoding to profile sampling areas and identify sources of fecal contamination in creek, stormwater outfall and beach sites in the Etobicoke Creek watershed (Toronto, ON). Water samples were collected before and immediately following an extreme rain event. MST and CST identified stormwater outfalls as an important source of human fecal contamination during dry and wet conditions. eDNA metabarcoding allowed for potential identification of additional sources of fecal contamination and provided additional evidence of human fecal contamination. The extreme rainfall event altered the eDNA profiles, causing creek and beach sites to reflect a greater diversity of mammal and bird eDNA sequences. The profiles provided by eDNA metabarcoding provide a proof of concept suggesting that eDNA metabarcoding can be a useful tool to complement MST and CST methods for profiling sources of fecal contamination and studying impacts of extreme rain events.

Performance of amplicon and shotgun sequencing for accurate biomass estimation in invertebrate community samples.

New applications of DNA and RNA sequencing are expanding the field of biodiversity discovery and ecological monitoring, yet questions remain regarding precision and efficiency. Due to primer bias, the ability of metabarcoding to accurately depict biomass of different taxa from bulk communities remains unclear, while PCR-free whole mitochondrial genome (mitogenome) sequencing may provide a more reliable alternative. Here, we used a set of documented mock communities comprising 13 species of freshwater macroinvertebrates of estimated individual biomass, to compare the detection efficiency of COI metabarcoding (three different amplicons) and shotgun mitogenome sequencing. Additionally, we used individual COI barcoding and de novo mitochondrial genome sequencing, to provide reference sequences for OTU assignment and metagenome mapping (mitogenome skimming), respectively. We found that, even though both methods occasionally failed to recover very low abundance species, metabarcoding was less consistent, by failing to recover some species with higher abundances, probably due to primer bias. Shotgun sequencing results provided highly significant correlations between read number and biomass in all but one species. Conversely, the read-biomass relationships obtained from metabarcoding varied across amplicons. Specifically, we found significant relationships for eight of 13 (amplicons B1FR-450 bp, FF130R-130 bp) or four of 13 (amplicon FFFR, 658 bp) species. Combining the results of all three COI amplicons (multiamplicon approach) improved the read-biomass correlations for some of the species. Overall, mitogenomic sequencing yielded more informative predictions of biomass content from bulk macroinvertebrate communities than metabarcoding. However, for large-scale ecological studies, metabarcoding currently remains the most commonly used approach for diversity assessment.

DNA metabarcoding for high-throughput monitoring of estuarine macrobenthic communities.

Morphology-based profiling of benthic communities has been extensively applied to aquatic ecosystems' health assessment. However, it remains a low-throughput, and sometimes ambiguous, procedure. Despite DNA metabarcoding has been applied to marine benthos, a comprehensive approach providing species-level identifications for estuarine macrobenthos is still lacking. Here we report a combination of experimental and field studies to assess the aptitude of COI metabarcoding to provide robust species-level identifications for high-throughput monitoring of estuarine macrobenthos. To investigate the ability of metabarcoding to detect all species present in bulk DNA extracts, we contrived three phylogenetically diverse communities, and applied four different primer pairs to generate PCR products within the COI barcode region. Between 78-83% of the species in the contrived communities were recovered through HTS. Subsequently, we compared morphology and metabarcoding-based approaches to determine the species composition from four distinct estuarine sites. Our results indicate that species richness would be considerably underestimated if only morphological methods were used: globally 27 species identified through morphology versus 61 detected by metabarcoding. Although further refinement is required to improve efficiency and output of this approach, here we show the great aptitude of COI metabarcoding to provide high quality and auditable species identifications in estuarine macrobenthos monitoring.

Large-Scale Monitoring of Plants through Environmental DNA Metabarcoding of Soil: Recovery, Resolution, and Annotation of Four DNA Markers.

In a rapidly changing world we need methods to efficiently assess biodiversity in order to monitor ecosystem trends. Ecological monitoring often uses plant community composition to infer quality of sites but conventional aboveground surveys only capture a snapshot of the actively growing plant diversity. Environmental DNA (eDNA) extracted from soil samples, however, can include taxa represented by both active and dormant tissues, seeds, pollen, and detritus. Analysis of this eDNA through DNA metabarcoding provides a more comprehensive view of plant diversity at a site from a single assessment but it is not clear which DNA markers are best used to capture this diversity. Sequence recovery, annotation, and sequence resolution among taxa were evaluated for four established DNA markers (matK, rbcL, ITS2, and the trnL P6 loop) in silico using database sequences and in situ using high throughput sequencing of 35 soil samples from a remote boreal wetland. Overall, ITS2 and rbcL are recommended for DNA metabarcoding of vascular plants from eDNA when not using customized or geographically restricted reference databases. We describe a new framework for evaluating DNA metabarcodes and, contrary to existing assumptions, we found that full length DNA barcode regions could outperform shorter markers for surveying plant diversity from soil samples. By using current DNA barcoding markers rbcL and ITS2 for plant metabarcoding, we can take advantage of existing resources such as the growing DNA barcode database. Our work establishes the value of standard DNA barcodes for soil plant eDNA analysis in ecological investigations and biomonitoring programs and supports the collaborative development of DNA barcoding and metabarcoding.