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opencc-zeroDec 2012View details →

Data from: A DNA metabarcoding study of a primate dietary diversity and plasticity across its entire fragmented range

In tropical regions, most primary ecosystems have been replaced by mosaic landscapes in which species must cope with a large shift in the distribution of their habitat and associated food resources. Primates are particularly vulnerable to habitat modifications. Most species persist in small fragments surrounded by complex human-mediated matrices whose structure and connectivity may strongly influence their dispersal and feeding behavior. Behavioral plasticity appears to be a crucial parameter governing the ability of organisms to exploit the resources offered by new matrix habitats and thus to persist in fragmented habitats. In this study, we were interested in the dietary plasticity of the golden-crowned sifaka (Propithecus tattersalli), an endangered species of lemur, found only in the Daraina region in north-eastern Madagascar. We used a DNA-based approach combining the barcoding concept and Illumina next-generation sequencing to (i) describe the species diet across its entire range and (ii) evaluate the influence of landscape heterogeneity on diet diversity and composition. Faeces from 96 individuals were sampled across the entire species range and their contents were analyzed using the trnL metabarcoding approach. In parallel, we built a large DNA reference database based on a checklist of the plant species of the Daraina region. Our results suggest that golden-crowned sifakas exhibit remarkable dietary diversity with at least 130 plant species belonging to 80 genera and 49 different families. We highlighted an influence of both habitat type and openness on diet composition suggesting a high flexibility of foraging strategies. Moreover, we observed the presence of numerous cultivated and naturalized plants in the faeces of groups living in forest edge areas. Overall, our findings support our initial expectation that P. tattersalli is able to cope with the current level of alteration of the landscape and confirm our previous results on the distribution and the dispersal ability of this species.

opencc-zeroDec 2011View details →

Data from: New environmental metabarcodes for analysing soil DNA: potential for studying past and present ecosystems

Metabarcoding approaches use total and typically degraded DNA from environmental samples to analyse biotic assemblages and can potentially be carried out for any kinds of organisms in an ecosystem. These analyses rely on specific markers, here called metabarcodes, which should be optimized for taxonomic resolution, minimal bias in amplification of the target organism group and short sequence length. Using bioinformatic tools, we developed metabarcodes for several groups of organisms: fungi, bryophytes, enchytraeids, beetles and birds. The ability of these metabarcodes to amplify the target groups was systematically evaluated by (1) in silico PCRs using all standard sequences in the EMBL public database as templates, (2) in vitro PCRs of DNA extracts from surface soil samples from a site in Varanger, northern Norway, and (3) in vitro PCRs of DNA extracts from permanently frozen sediment samples of late-Pleistocene age (~ 16 000–50 000 yr BP) from two Siberian sites, Duvanny Yar and Main River. Comparison of the results from the in silico PCR with those obtained in vitro showed that the in silico approach offered a reliable estimate of the suitability of a marker. All target groups were detected in the environmental DNA, but we found large variation in the level of detection among the groups and between modern and ancient samples. Success rates for the Pleistocene samples were highest for fungal DNA, whereas bryophyte, beetle and bird sequences could also be retrieved, but to a much lesser degree. The metabarcoding approach has considerable potential for biodiversity screening of modern samples and also as a paleoecological tool.

Data from: Replication levels, false presences, and the estimation of presence / absence from eDNA metabarcoding data

Environmental DNA (eDNA) metabarcoding is increasingly used to study the present and past biodiversity. eDNA analyses often rely on amplification of very small quantities or degraded DNA. To avoid missing detection of taxa that are actually present (false negatives), multiple extractions and amplifications of the same samples are often performed. However, the level of replication needed for reliable estimates of the presence/absence patterns remains an unaddressed topic. Furthermore, degraded DNA and PCR/sequencing errors might produce false positives. We used simulations and empirical data to evaluate the level of replication required for accurate detection of targeted taxa in different contexts and to assess the performance of methods used to reduce the risk of false detections. Furthermore, we evaluated whether statistical approaches developed to estimate occupancy in the presence of observational errors can successfully estimate true prevalence, detection probability and false-positive rates. Replications reduced the rate of false negatives; the optimal level of replication was strongly dependent on the detection probability of taxa. Occupancy models successfully estimated true prevalence, detection probability and false-positive rates, but their performance increased with the number of replicates. At least eight PCR replicates should be performed if detection probability is not high, such as in ancient DNA studies. Multiple DNA extractions from the same sample yielded consistent results; in some cases, collecting multiple samples from the same locality allowed detecting more species. The optimal level of replication for accurate species detection strongly varies among studies and could be explicitly estimated to improve the reliability of results.

opencc-zeroMay 2020View details →

Data from: Reconstructing hyperdiverse food webs: gut content metabarcoding as a tool to disentangle trophic interactions on coral reefs

1. Anthropogenic stressors have strong impacts on ecosystems. To understand their influence, detailed knowledge about trophic relationships among species is critical. However, this requires both exceptional resolution in dietary assessments and sampling breadth within communities, especially for highly-diverse, tropical ecosystems. 2. We used gut content metabarcoding across a broad range of coral reef fishes (8 families, 22 species) in Mo'orea, French Polynesia to test whether this technique has the potential to capture the structure of a hyperdiverse marine food web. Moreover, we explored whether taxonomic groups (families) and traditional, broad-scale trophic assignments explained fish diet across four different metrics of quantifying predator-prey interactions. 3. Metabarcoding yielded a large number (4,341) of unique operational taxonomic units (i.e. prey) with high-resolution taxonomic assignments (i.e. often to the level of genus or species). We demonstrate that across multiple metrics, taxonomic group at the family level is a consistently better, albeit still weak, predictor of empirical trophic relationships than frequently-used, broad-scale functional assignments. Our method also reveals a complex trophic network with fine-scale partitioning among species, further emphasizing the importance of examining fish diets beyond broad trophic categories. 4. We demonstrate the capacity of metabarcoding to reconstruct diverse and complex food webs with exceptional resolution, a significant advancement from traditional food web reconstruction. Further, this method allows us to pinpoint the trophic niche of species with niche-based modelling, even across hyperdiverse species assemblages such as coral reefs. In conjunction with complementary techniques such as stable isotope analysis, applying metabarcoding to whole communities will provide unparalleled information about energy and nutrient fluxes and inform their susceptibility to disturbances even in the world's most diverse ecosystems.

opencc-zeroOct 2019View details →

Data from: Metabarcoding reveals diet diversity in an ungulate community in Thailand

<p>Asian dry forests contain diverse and abundant large herbivore communities whose diet breadth is largely unstudied. We examined the diet composition of eight ungulate species in a dry tropical forest using metabarcoding to determine if the diet separation of the diverse community was structured and if and obvious attributes (i.e. body size, phylogeny or ecology) can explain the structure. We collected fecal samples from the ungulates in Huai Kha Khaeng Wildlife Sanctuary in the western forest complex of Thailand. The fecal collections occurred around a Large Forest Tree Plot (ForestGeo) where all woody species were codified within a genetic barcode library and used in this study. This DNA library was supplemented with samples from forb and grass species known to be consumed by ungulates in the region. Of 333 plant species tested, at least 92 were found within the fecal samples of the ungulates tested. Over half of the identified species were not previously identified by experts as forage species. All ungulate species showed a strong consumption of grasses and forbs. The pattern of forage consumption by these species did not consistently cluster according to obvious paradigms of body size or taxonomy, with significant differences found in diet selection of two similar-sized bovids (guar and banteng), and with the diet of sambar being more similar to bovids than to the other deer species in the community. We conclude Asian ungulate communities do differentiate in their forage consumption and that metabarcoding techniques should allow for testing of diet shifts in response to seasonal rains and fires which dominate the phenology of Asian dry forests. </p>

opencc-zeroDec 2017View details →

Data from: The use of metabarcoding for meiofauna ecological patterns assessment

Marine meiofauna comprises up to 22 phyla. Its morphological identification requires time and taxonomists' expertise, and molecular tools can make this task faster. We aim to disentangle meiofaunal diversity patterns at Araçá Bay by applying a model selection approach and estimating the effectiveness of metabarcoding (18S rDNA) and morphological methods for estimating the response of meiofauna diversity in small-scale interactions with environmental variables. A rarefaction curve indicated that ten samples were sufficient for estimating the total number of meiofauna OTUs in a tidal flat. In both approaches, richness was predicted by mean sand percentage, sediment sorting, and bacteria concentration. Nematode genera composition differed significantly between approaches, the result of taxonomic mismatch in the genetic database. The similarity between the model selected for diversity descriptors, the richness of nematode genera and meiofauna composition emphasized the utility of predictive models for metabarcoding estimates to detect small-scale interactions of these organisms.

opencc-zeroDec 2012View details →

Data from: A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents

Introduction: The PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile ("universal") COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups. Results: We first design a new PCR primer within the highly variable mitochondrial COI region, the "mlCOIintF" primer. We then show that this newly designed forward primer combined with the "jgHCO2198" reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment. Conclusions: The molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.

Data from: Estimating and mitigating amplification bias in qualitative and quantitative arthropod metabarcoding

Amplicon based metabarcoding promises rapid and cost-efficient analyses of species composition. However, it is disputed whether abundance estimates can be derived from metabarcoding due to taxon specific PCR amplification biases. PCR-free approaches have been suggested to mitigate this problem, but come with considerable increases in workload and cost. Here, we analyze multilocus datasets of diverse arthropod communities, to evaluate whether amplification bias can be countered by (1) targeting loci with highly degenerate primers or conserved priming sites, (2) increasing PCR template concentration, (3) reducing PCR cycle number or (4) avoiding locus specific amplification by directly sequencing genomic DNA. Amplification bias is reduced considerably by degenerate primers or targeting amplicons with conserved priming sites. Surprisingly, a reduction of PCR cycles did not have a strong effect on amplification bias. The association of taxon abundance and read count was actually less predictable with fewer cycles. Even a complete exclusion of locus specific amplification did not exclude bias. Copy number variation of the target loci may be another explanation for read abundance differences between taxa, which would affect amplicon based and PCR free methods alike. As read abundance biases are taxon specific and predictable, the application of correction factors allows abundance estimates.

Data from: DNA metabarcoding reveals that 200-μm-size-fractionated filtering is unable to discriminate between planktonic microbial and large eukaryotes

Microeukaryotic plankton (0.2–200 μm) are critical components of aquatic ecosystems and key players in global ecological processes. High-throughput sequencing is currently revolutionizing their study on an unprecedented scale. However, it is currently unclear whether we can accurately, effectively and quantitatively depict the microeukaryotic plankton communities using traditional size-fractionated filtering combined with molecular methods. To address this, we analysed the eukaryotic plankton communities both with, and without, prefiltering with a 200 μm pore-size sieve –by using SSU rDNA-based high-throughput sequencing on 16 samples with three replicates in each sample from two subtropical reservoirs sampled from January to October in 2013. We found that ~25% reads were classified as metazoan in both size groups. The species richness, alpha and beta diversity of plankton community and relative abundance of reads in 99.2% eukaryotic OTUs showed no significant changes after prefiltering with a 200 μm pore-size sieve. We further found that both >0.2 μm and 0.2–200 μm eukaryotic plankton communities, especially the abundant plankton subcommunities, exhibited very similar, and synchronous, spatiotemporal patterns and processes associated with almost identical environmental drivers. The lack of an effect on community structure from prefiltering suggests that environmental DNA from larger metazoa is introduced into the smaller size class. Therefore, size-fractionated filtering with 200 μm is insufficient to discriminate between the eukaryotic plankton size groups in metabarcoding approaches. Our results also highlight the importance of sequencing depth, and strict quality filtering of reads, when designing studies to characterize microeukaryotic plankton communities.

opencc-zeroDec 2015View details →

Data from: Spatial representativeness of environmental DNA metabarcoding signal for fish biodiversity assessment in a natural freshwater system

In the last few years, the study of environmental DNA (eDNA) has drawn attention for many reasons, including its advantages for monitoring and conservation purposes. So far, in aquatic environments, most of eDNA research has focused on the detection of single species using species-specific markers. Recently, species inventories based on the analysis of a single generalist marker targeting a larger taxonomic group (eDNA metabarcoding) have proven useful for bony fish and amphibian biodiversity surveys. This approach involves in situ filtering of large volumes of water followed by amplification and sequencing of a short discriminative fragment from the 12S rDNA mitochondrial gene. In this study, we went one step further by investigating the spatial representativeness (i.e. ecological reliability and signal variability in space) of eDNA metabarcoding for large-scale fish biodiversity assessment in a freshwater system including lentic and lotic environments. We tested the ability of this approach to characterize large-scale organization of fish communities along a longitudinal gradient, from a lake to the outflowing river. First, our results confirm that eDNA metabarcoding is more efficient than a single traditional sampling campaign to detect species presence, especially in rivers. Second, the species list obtained using this approach is comparable to the one obtained when cumulating all traditional sampling sessions since 1995 and 1988 for the lake and the river, respectively. In conclusion, eDNA metabarcoding gives a faithful description of local fish biodiversity in the study system, more specifically within a range of a few kilometers along the river in our study conditions, i.e. longer than a traditional fish sampling site.

Data from: Ecosystem biomonitoring with eDNA: metabarcoding across the tree of life in a tropical marine environment

Effective marine management requires comprehensive data on the status of marine biodiversity. However, efficient methods that can document biodiversity in our oceans are currently lacking. Environmental DNA (eDNA) sourced from seawater offers a new avenue for investigating the biota in marine ecosystems. Here, we investigated the potential of eDNA to inform on the breadth of biodiversity present in a tropical marine environment. Directly sequencing eDNA from seawater using a shotgun approach resulted in only 0.34% of 22.3 million reads assigning to eukaryotes, highlighting the inefficiency of this method for assessing eukaryotic diversity. In contrast, using 'tree of life' (ToL) metabarcoding and 20-fold fewer sequencing reads, we could detect 287 families across the major divisions of eukaryotes. Our data also show that the best performing 'universal' PCR assay recovered only 44% of the eukaryotes identified across all assays, highlighting the need for multiple metabarcoding assays to catalogue biodiversity. Lastly, focusing on the fish genus Lethrinus, we recovered intra- and inter-specific haplotypes from seawater samples, illustrating that eDNA can be used to explore diversity beyond taxon identifications. Given the sensitivity and low cost of eDNA metabarcoding we advocate this approach be rapidly integrated into biomonitoring programs.

Data from: DNA metabarcoding diet analysis for species with parapatric versus sympatric distribution: a case study on subterranean rodents

Closely related sympatric species commonly develop different ecological strategies to avoid competition. Ctenomys minutus and C. flamarioni are subterranean rodents parapatrically distributed in the southern Brazilian coastal plain, showing a narrow sympatric zone. To gain understanding on food preferences and possible competition for food resources, we evaluated their diet composition performing DNA metabarcoding analyzes of 67 C. minutus and 100 C. flamarioni scat samples, collected along the species geographical ranges. Thirteen plant families, mainly represented by Poaceae, Araliaceae, Asteraceae and Fabaceae, were identified in the diet of C. minutus. For C. flamarioni, 10 families were recovered, with a predominance of Poaceae, Araliaceae and Asteraceae. A significant correlation between diet composition and geographical distance was detected in C. minutus, whereas the diet of C. flamarioni was quite homogeneous throughout its geographical distribution. No significant differences were observed between males and females of each species. However, differences in diet composition between species were evident according to multivariate analysis. Our results suggest some level of diet partitioning between C. flamarioni and C. minutus in the sympatric region. While the first species is more specialized on few plant items, the second showed a more varied and heterogeneous diet pattern among individuals. These differences might have been developed to avoid competition in the region of co-occurrence. Resource availability in the environment also seems to influence food choices. Our data indicate that C. minutus and C. flamarioni are generalist species, but that some preference for Poaceae, Asteraceae and Araliaceae families can be suggested for both rodents.

Data from: Carrion fly-derived DNA metabarcoding is an effective tool for mammal surveys: evidence from a known tropical mammal community

Metabarcoding of vertebrate DNA derived from carrion flies has been proposed as a promising tool for biodiversity monitoring. To evaluate its efficacy, we conducted metabarcoding surveys of carrion flies on Barro Colorado Island (BCI), Panama, which has a well-known mammal community, and compared our results against diurnal transect counts and camera-trapping. We collected 1084 flies in 29 sampling days, conducted metabarcoding with mammal-specific (16S) and vertebrate-specific (12S) primers, and sequenced amplicons on Illumina MiSeq. For taxonomic assignment, we compared BLAST with the new program PROTAX, and we found that PROTAX improved species identifications. We detected 20 mammal, four bird, and one lizard species from carrion fly metabarcoding, all but one of which are known from BCI. Fly metabarcoding detected more mammal species than concurrent transect counts (29 sampling days, 13 species) and concurrent camera-trapping (84 sampling days, 17 species), and detected 67% of the number of mammal species documented by eight years of transect counts and camera-trapping combined, although fly metabarcoding missed several abundant species. This study demonstrates that carrion fly metabarcoding is a powerful tool for mammal biodiversity surveys, and has the potential to detect a broader range of species than more commonly used methods.

opencc-zeroSep 2019View details →

Data from: Non-specific amplification compromises environmental DNA metabarcoding with COI

1. Metabarcoding extra-organismal DNA from environmental samples is now a key technique in aquatic biomonitoring and ecosystem health assessment. However, choice of genetic marker and primer set is a critical consideration when designing experiments, especially so when developing community standards and legislative frameworks. Mitochondrial cytochrome c oxidase subunit I (COI), the standard DNA barcode marker for animals, with its extensive reference library, taxonomic discriminatory power, and predictable sequence variation, is the natural choice for many metabarcoding applications such as the bulk sequencing of invertebrates. However, the overall utility of COI for environmental sequencing of targeted taxonomic groups has yet to be fully scrutinised. 2. Here, by using a case study of marine and freshwater fishes from the British Isles, we quantify the in silico performance of twelve mitochondrial primer pairs from COI, cytochrome b, 12S and 16S, in terms of reference library coverage, taxonomic discriminatory power, and primer universality. We subsequently test in vitro three COI primer pairs and one 12S pair for their specificity, reproducibility, and congruence with independent datasets derived from traditional survey methods at five estuarine and coastal sites in the English Channel and North Sea coast. 3. Our results show that for aqueous extra-organismal DNA at low template concentrations, both metazoan and fish-targeted COI primers perform poorly in comparison to 12S, exhibiting low levels of reproducibility due to non-specific amplification of prokaryotic and non-target eukaryotic DNAs. 4. An ideal metabarcode would have an extensive reference library for which custom primer sets can be designed for either broad assessments of biodiversity or taxon specific surveys, but unfortunately, low primer specificity hinders the use of COI, while the paucity of reference sequences is problematic for 12S. The latter, however, can be mitigated by expanding the concept of DNA barcodes to include whole mitochondrial genomes generated by genome-skimming existing tissue collections.

opencc-zeroDec 2012View details →

Data from: DNA metabarcoding multiplexing and validation of data accuracy for diet assessment: application to omnivorous diet

Ecological understanding of the role of consumer-resource interactions in natural food webs is limited by the difficulty of accurately and efficiently determining the complex variety of food types animals have eaten in the field. We developed a method based on DNA metabarcoding multiplexing and next-generation sequencing to uncover different taxonomic groups of organisms from complex diet samples. We validated this approach on 91 faeces of a large omnivorous mammal, the brown bear, using DNA metabarcoding markers targeting the plant, vertebrate, and invertebrate components of the diet. We included internal controls in the experiments and performed PCR replication for accuracy validation in post-sequencing data analysis. Using our multiplexing strategy, we significantly simplified the experimental procedure and accurately and concurrently identified different prey DNA corresponding to the targeted taxonomic groups, with ≥60% of taxa of all diet components identified to genus/species level. The systematic application of internal controls and replication was a useful and simple way to evaluate the performance of our experimental procedure, standardize the selection of sequence filtering parameters for each marker data, and validate the accuracy of the results. Our general approach can be adapted to the analysis of dietary samples of various predator species in different ecosystems, for a number of conservation and ecological applications entailing large-scale population level diet assessment through cost effective screening of multiple DNA metabarcodes, and the detection of fine dietary variation among samples or individuals and of rare food items.

Data from: Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias

Studies of insect assemblages are suited to the simultaneous DNA-based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR-amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers (16S and COI) and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico less than 75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided greater than 90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR-amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 different insect orders and one arachnid. We PCR-amplified the blend using five primers sets, targeting either COI or 16S, with high-throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR, and that researchers should be wary of potential bias when selecting metabarcoding markers.

opencc-zeroDec 2017View details →

Data from: A pipeline for metabarcoding and diet analysis from fecal samples developed for a small semi-aquatic mammal

Metabarcoding allows the genetic analysis of pooled samples of various sources. It is becoming popular in the study of animal diet, especially because it allows the analysis of the composition of feces without the need of handling animals. In this work, we studied the diet of the Pyrenean desman (Galemys pyrenaicus), a small semi-aquatic mammal endemic to the Iberian Peninsula and the Pyrenees, by sequencing COI minibarcodes from feces using next-generation sequencing techniques. For the identification of assembled sequences, we employed a tree-based identification method that used a reference tree of sequences of freshwater organisms. The comparison of freshly collected fecal samples and older samples showed that fresh samples produced significantly more sequencing reads. They also rendered more operational taxonomical units (OTUs), but not significantly. Our analyses of 41 samples identified 224 OTUs corresponding to species of the reference tree. Ephemeroptera, Diptera excl. Chironomidae, and Chironomidae were the most highly represented groups in terms of reads as well as samples. Other groups of freshwater organisms detected were Plecoptera, Trichoptera, Neuropteroida, Coleoptera, Crustacea, and Annelida. Our results are largely in line with previous morphological and genetic studies on the diet of the Pyrenean desman, but allowed the identification of a higher diversity of OTUs in each sample. Additionally, the bioinformatic pipeline we developed for deep sequencing of fecal samples will enable the quantitative analysis of the diet of this and other species, which can be highly useful to determine their ecological requirements.

opencc-zeroDec 2014View details →

Data from: Application of ITS2 metabarcoding to determine the provenance of pollen collected by honey bees in an agroecosystem

Premise of the study: Melissopalynology, the identification of bee-collected pollen, provides insight into the flowers exploited by foraging bees. Information provided by melissopalynology could guide floral enrichment efforts aimed at supporting pollinators, but it has rarely been used because traditional methods of pollen identification are laborious and require expert knowledge. We approach melissopalynology in a novel way, employing a molecular method to study the pollen foraging of honey bees (Apis mellifera) in a landscape dominated by field crops, and compare these results to those obtained by microscopic melissopalynology. Methods: Pollen was collected from honey bee colonies in Madison County, Ohio, USA, during a two-week period in mid-spring and identified using microscopic methods and ITS2 metabarcoding. Results: Metabarcoding identified 19 plant families and exhibited sensitivity for identifying the taxa present in large and diverse pollen samples relative to microscopy, which identified eight families. The bulk of pollen collected by honey bees was from trees (Sapindaceae, Oleaceae, and Rosaceae), although dandelion (Taraxacum officinale) and mustard (Brassicaceae) pollen were also abundant. Discussion: For quantitative analysis of pollen, using both metabarcoding and microscopic identification is superior to either individual method. For qualitative analysis, ITS2 metabarcoding is superior, providing heightened sensitivity and genus-level resolution.

opencc-zeroDec 2015View details →

Data from: A metabarcoding framework for facilitated survey of endolithic phototrophs with tufA

Background: In spite of their ecological importance as primary producers and microbioeroders of marine calcium carbonate (CaCO3) substrata, endolithic phototrophs spanning both prokaryotic (the cyanobacteria) and eukaryotic algae lack established molecular resources for their facilitated survey with high throughput sequencing. Here, the development of a metabarcoding framework for the elongation factor EF-Ttu (tufA) was tested on four Illumina-sequenced marine CaCO3 microfloras for the characterization of their endolithic phototrophs, especially the abundant bioeroding Ostreobium spp. (Ulvophyceae). The framework consists of novel tufA degenerate primers and a comprehensive database enabling Operational Taxonomic Unit (OTU) identification at multiple taxonomic ranks with percent identity thresholds determined herein. Results: The newly established tufA database comprises 4057 non-redundant sequences (from 1339 eukaryotic and prokaryotic phototrophs, and 2718 prokaryotic heterotrophs) including 27 classes in 10 phyla of phototrophic diversity summarized from data mining on GenBank®, our barcoding of >150 clones produced from coral reef microfloras, and >300 eukaryotic phototrophs (>230 Ulvophyceae including >100 'Ostreobium' spp., and >70 Florideophyceae, Phaeophyceae and miscellaneous taxa). Illumina metabarcoding with the newly designed primers resulted in 802 robust OTUs including 618 phototrophs and 184 heterotrophs (77 and 23 % of OTUs, respectively). Phototrophic OTUs belonged to 14 classes of phototrophs found in seven phyla, and represented ~98 % of all reads. The phylogenetic profiles of coral reef microfloras showed few OTUs in large abundance (proportion of reads) for the Chlorophyta (Ulvophyceae, i.e. Ostreobium and Phaeophila), the Rhodophyta (Florideophyceae) and Haptophyta (Coccolithophyceae), and a large diversity (richness) of OTUs in lower abundance for the Cyanophyta (Cyanophyceae) and the Ochrophyta (the diatoms, 'Bacillariophyta'). The bioerosive 'Ostreobium' spp. represented four families in a large clade of subordinal divergence, i.e. the Ostreobidineae, and a fifth, phylogenetically remote family in the suborder Halimedineae (provisionally assigned as the 'Pseudostreobiaceae'). Together they harbor 85–95 delimited cryptic species of endolithic microsiphons. Conclusions: The novel degenerate primers allowed for amplification of endolithic phototrophs across a wide phylogenetic breadth as well as their recovery in very large proportions of reads (overall 98 %) and diversity (overall 77 % of OTUs). The established companion tufA database and determined identity thresholds allow for OTU identification at multiple taxonomic ranks to facilitate the monitoring of phototrophic assemblages via metabarcoding, especially endolithic communities rich in bioeroding Ulvophyceae, such as those harboring 'Ostreobium' spp., Phaeophila spp. and associated algal diversity.

Data from: Environmental monitoring through protist NGS metabarcoding: assessing the impact of fish farming on benthic foraminifera communities

The measurement of species diversity represents a powerful tool for assessing the impacts of human activities on marine ecosystems. Traditionally, the impact of fish farming on the coastal environment is evaluated by monitoring the dynamics of macrobenthic infaunal populations. However, taxonomic sorting and morphology-based identification of the macrobenthos demands highly trained specialists and is extremely time-consuming and costly, what makes it unsuitable for large-scale biomonitoring efforts involving numerous samples. Here, we propose to alleviate this laborious task by developing protist metabarcoding tools based on next-generation sequencing (NGS) of environmental DNA and RNA extracted from sediment samples. In this study, we analysed the response of benthic foraminiferal communities to the variation of environmental gradients associated with salmon farms in Scotland. We investigated the foraminiferal diversity based on ribosomal minibarcode sequences generated by the Illumina NGS technology. We compared the molecular data with morphospecies counts and with environmental gradients, including distance to cages and Redox used as a proxy for sediment oxygenation. Our study revealed high variations between foraminiferal communities collected in the vicinity of fish farms and at distant locations. We found evidence for alpha diversity loss in strongly impacted sites, especially visible in the RNA data. We also detected some candidate bioindicator foraminiferal species. Based on this proof-of-concept study, we conclude that NGS metabarcoding using foraminifera and other protists has considerable potential to become a new tool for surveying the impact of aquaculture and other industrial activities in the marine environment.