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Supplementary material 3 from: Nugent CM, Adamowicz SJ (2020) Alignment-free classification of COI DNA barcode data with the Python package Alfie. Metabarcoding and Metagenomics 4: e55815. https://doi.org/10.3897/mbmg.4.55815

File S3 – The parameters utilized in the grid search for each of the five machine learning algorithms tested in the design of the Alfie package

Supplementary material 4 from: Nugent CM, Adamowicz SJ (2020) Alignment-free classification of COI DNA barcode data with the Python package Alfie. Metabarcoding and Metagenomics 4: e55815. https://doi.org/10.3897/mbmg.4.55815

File S4 – Jupyter notebook with tutorial demonstrating how to apply the Alfie classifier in the Python programming language, and how to train custom alignment-free classifiers using the Alfie training module

Supplementary material 2 from: Macher J-N, Drakou K, Papatheodoulou A, Hoorn B, Vasquez M (2020) The mitochondrial genomes of 11 aquatic macroinvertebrate species from Cyprus. Metabarcoding and Metagenomics 4: e58259. https://doi.org/10.3897/mbmg.4.58259

Supplementary tables showing reads numbers, coverage and length of mitochondrial genomes, and length and blast results of 18S and 28S rRNAs

Plant DNA metabarcoding records from three cores of lacustrine lakes along a open larch forest-forest tundra-tundra transect at the Omoloy region, north-eastern Siberia

Here, we provide the raw plant DNA metabarcoding data archived in three Siberian lake sediment cores spanning the mid-Holocene to the present (7.6-0 cal ka BP), from northern typical tundra to southern open larch forest in the Omoloy region. There are three cores: <ol> <li> 14-OM-20B, Lat. / °: 70.53, Lon. / °: 132.91, Ele. / m a.s.l.: 52, Modern vegetation: open larch forest, Lake area / km2: 0.26, Maximal depth / m: 3.4</li> <li> 14-OM-02B, Lat. / °: 70.72, Lon. / °: 132.67, Ele. / m a.s.l.: 58, Modern vegetation: forest tundra, Lake area / km2: 0.08, Maximal depth / m: 3.5</li> <li> 14-OM-12A, Lat. / °: 70.96, Lon. / °: 132.57, Ele. / m a.s.l.: 60, Modern vegetation: tundra, Lake area / km2: 0.09, Maximal depth / m: 4.5</li> </ol> Three lake sediment cores, 14OM12A (33 cm long), 14OM02B (49.5 cm long) and 14OM20B (86 cm long), were recovered from three sites using a UWITEC gravity corer (6 cm internal diameter) equipped with a hammer tool in July 2014. From the three cores, 16 bulk organic carbon samples were selected because of the lack of macrofossil remains and radiocarbon dated using accelerator mass spectrometry (AMS) at Poznań radiocarbon laboratory of Adam Mickiewicz University, Poland. In addition, 30 freeze-dried samples per core at 0.25 or 0.5 cm intervals between 0 and 15 cm were analysed for 210Pb/137Cs at the Liverpool University Environmental Radioactivity Laboratory. (The dating table has been submitted to PANGAEA) In this project, we analyse pollen (has been submitted to PANGAEA, doi: 10.1594/PANGAEA.922550) and sedaDNA from three lake sediment cores from the Omoloy region in north-eastern Siberia (northern Yakutia), which are currently surrounded by different vegetation types ranging from typical tundra to open larch forest. First, our aim is to compare sedaDNA with the pollen data to see whether both methods track the same pattern with respect to compositional changes and diversity changes across the northern Russian treeline zone or are complementary to each other. Second, we reconstruct the mid- to late-Holocene changes of vegetation composition along a north–south transect. Third, we use the sedaDNA data to reconstruct variations in species richness and relate this to vegetation and climate change.

Money spider dietary choice in pre- and post-harvest cereal crops using metabarcoding

<ol> <li>Money spiders (Linyphiidae) are an important component of conservation biological control in cereal crops, but they rely on alternative prey when pests are not abundant, such as between cropping cycles. To optimally benefit from these generalist predators, prey choice dynamics must first be understood.</li> <li>Money spiders and their locally available prey were collected from cereal crops two weeks pre- and post-harvest. Spider gut DNA was amplified with two novel metabarcoding primer pairs designed for spider dietary analysis, and sequenced.</li> <li>The combined general and spider-exclusion primers successfully identified prey from 15 families in the guts of the 46 linyphiid spiders screened, whilst avoiding amplification of Erigone spp. The primers show promise for application to the diets of other spider families such as Agelenidae and Pholcidae.</li> <li>Distinct invertebrate communities were identified pre- and post-harvest, and changes in spider diet and, to a lesser extent, prey choice reflected this. Spiders were found to consume one another more than expected, indicating their propensity toward intraguild predation, but also consumed common pest families.</li> <li>Changes in spider prey choice may redress prey community changes to maintain a consistent dietary intake. Consistent provision of alternative prey via permanent refugia should be considered to sustain effective conservation biocontrol.</li> </ol>

opencc-zeroDec 2019View details →

Evaluating the genetic variation of the COI gene of Insecta: Implications for DNA barcoding, metabarcoding and species delimitation studies

The genetic variation of the COI gene has a great effect on the final results of the species delimitation studies. However, little research has comprehensively investigated the genetic divergence in COI among Insecta. The fast-growing COI data in BOLD provide an opportunity for comprehensively appraising the genetic variation in COI among Insecta. We calculated the K2P distance of 64,414 insect species downloaded from BOLD. The match ratios of the clustering analysis based on different thresholds were compared among 4,288 genera (35,068 species). Besides, we also compared the match ratios obtained from two species delimitation methods: the clustering analysis (distance-based method) and the bPTP analysis (tree-based method). Furthermore, the effectiveness of two different results of the bPTP analysis: bPTP_h and bPTP_ml was also tested. Approximately one-quarter of the species of Insecta showed high intraspecific genetic variation (> 3%), and a conservative estimate of this value is 12.05-22.58%. The application of empirical thresholds (e.g., 2% and 3%) in the clustering analysis may result in the overestimation of species diversity. In metabarcoding studies, a threshold of 3% can only be used to estimate the insect diversity roughly. As for the clustering analysis, the "threshOpt" or "localMinima" algorithms can provide a priori value for the researcher. Nevertheless, if the minimum interspecific genetic distance of congeneric species was greater than or equal to 2%, it is possible to avoid overestimating the species diversity based on the empirical thresholds. Besides, the match ratios of the bPTP_ml results were higher than those of the bPTP_h results. As for the bPTP analysis, the bPTP_ml results were recommended. If a proper threshold was selected, the clustering analysis may outperform the bPTP analysis.

opencc-zeroNov 2020View details →

Environmental DNA metabarcoding of cow dung reveals taxonomic and functional diversity of invertebrate assemblages

Insects and other terrestrial invertebrates are declining in species richness and abundance. This includes the invertebrates associated with herbivore dung, which have been negatively affected by grazing abandonment and the progressive loss of large herbivores since the Late Pleistocene. Importantly, traditional monitoring of these invertebrates is time-consuming and requires considerable taxonomic expertise, which is becoming increasingly scarce. In this study, we investigated the potential of environmental DNA (eDNA) metabarcoding of cow dung samples for biomonitoring of dung-associated invertebrates. From eight cow pats we recovered eDNA from 12 orders, 29 families, and at least 54 species of invertebrates (mostly insects), representing several functional groups. Furthermore, species compositions differed between the three sampled habitats of dry grassland, meadow, and forest. These differences were in accordance with the species' ecology; for instance, several species known to be associated with humid conditions or lower temperatures were found only in the forest habitat. We discuss potential caveats of the method, as well as directions for future study and perspectives for implementation in research and monitoring.

opencc-zeroNov 2020View details →

The applicability of eDNA metabarcoding approaches for sessile benthic surveying in the Kimberley region, north-western Australia

The application of environmental DNA technologies is a promising new approach to rapidly audit biodiversity across large-scale, remote regions. Here, we examine the efficacy of a dual-assay eDNA metabarcoding approach for sessile benthic bioassessments in the turbid waters of the Lalang-garram <a>Marine Parks</a>, in the inshore Kimberley region, north-western Australia. We ask three principal questions: 1. Is the eDNA released by sessile benthic taxa (i.e. hard and soft corals, sponges and tunicates) locally detectable? 2. What level of taxonomic resolution is afforded by eDNA metabarcoding using the ITS2 region? and 3. How well does eDNA metabarcoding compare to conventional benthic survey techniques, such as belt and point-intercept transects? We report that a dual-assay eDNA metabarcoding approach is capable of detecting approximately 70% of the local benthic taxa (i.e. at a species, genus level etc) identified at the surveyed locations. It is, however, not as effective at the individual/population level, detecting only approximately 40% of unique amplicon sequence variant (ASV) signals released by an array of individual benthic organisms at the surveyed locations. In examining the efficacy and resolution of the applied ITS2 metabarcoding markers for bioassessments, we report large gaps in the variety of publicly available benthic ITS2 reference sequence data, limiting our ability to provide robust taxonomic assignments. These findings highlight the need to extend ITS2 databases for greater regional representation. Until this is adequately addressed, we recommend that investigating taxonomic assignments to a genus-level is the most robust approach to benthic monitoring using eDNA. Lastly, we found eDNA metabarcoding and conventional belt-transect surveys each detected numerous unique hard coral genera, indicating that a combined approach provides the most effective way to audit benthic biodiversity. Furthermore, eDNA metabarcoding had the power to distinguish similar diversity trends between sites to that determined by the belt-transect methodology, validating the application of eDNA metabarcoding as either a stand-alone, or complementary technique for assessing sessile benthic taxa.

opencc-zeroNov 2020View details →

Data from: All is fish that comes to the net: metabarcoding for rapid fisheries catch assessment

AbstractMonitoring marine resource exploitation is a key activity in fisheries science and biodiversity conservation. Since research surveys are time-consuming and costly, fishery-dependent data (i.e. derived directly from fishing vessels) are increasingly credited with a key role in expanding the reach of ocean monitoring. Fishing vessels may be seen as widely ranging data-collecting platforms, which could act as a fleet of sentinels for monitoring marine life, in particular exploited stocks. Here, we investigate the possibility of assessing catch composition of single hauls carried out by trawlers by applying DNA metabarcoding to the "slush" collected from fishing nets just after the end of hauling operations. We assess the performance of this approach in portraying β-diversity and examining the quantitative relationship between species abundances in the catch and DNA amount in the slush (reads counts generated by amplicon sequencing). We demonstrate that the assemblages identified using DNA in the slush mirror those returned by visual inspection of net content and detect a strong relationship between read counts and species abundances in the catch. We therefore argue that this approach could be upscaled to serve as a powerful source of information on the structure of demersal assemblages and the impact of fisheries.

opencc-zeroDec 2020View details →

The influence of intraspecific sequence variation during DNA metabarcoding: A case study of eleven fungal species

DNA metabarcoding has become a powerful approach for analyzing complex communities from environmental samples, but there are still methodological challenges limiting its full potential. While conserved DNA markers, like 16S and 18S, often are not able to discriminate among closely related species, other more variable markers – like the fungal ITS region, may include considerable intraspecific variation, which can lead to over-splitting of species during DNA metabarcoding analyses. Here we assess the effects of intraspecific sequence variation in DNA metabarcoding, by analyzing local populations of eleven fungal species. We investigated the allelic diversity of ITS2 haplotypes using both Sanger sequencing and high throughput sequencing (HTS) coupled with error correction with the software dada2. All focal species, except one, included some level of intraspecific variation in the ITS2 region. Overall, we observed a high correspondence between haplotypes generated by Sanger sequencing and HTS, with the exception of a few additional haplotypes detected using either approach. These extra haplotypes, often occurring in low frequencies, were likely due to PCR and sequencing errors or intragenomic variation in the rDNA region. The presence of intraspecific (and possibly intragenomic) variation in ITS2 suggest that haplotypes (or ASVs) should not be used as basic units in ITS-based fungal community analyses, but an extra clustering step is needed to reach species-level resolution.

opencc-zeroJan 2021View details →

Supplementary material 1 from: Basset Y, Donoso DA, Hajibabaei M, Wright MTG, Perez KHJ, Lamarre GPA, De León LF, Palacios-Vargas JG, Castaño-Meneses G, Rivera M, Perez F, Bobadilla R, Lopez Y, Ramirez JA, Barrios H (2020) Methodological considerations for monitoring soil/litter arthropods in tropical rainforests using DNA metabarcoding, with a special emphasis on ants, springtails and termites. Metabarcoding and Metagenomics 4: e58572. https://doi.org/10.3897/mbmg.4.58572

Methodological considerations for monitoring soil/litter arthropods in tropical rainforests using DNA metabarcoding, with a special emphasis on ants, springtails and termites

opencc-zeroJan 2021View details →

Supplementary material 1 from: Weigand AM, Desquiotz N, Weigand H, Szucsich N (2021) Application of propylene glycol in DNA-based studies of invertebrates. Metabarcoding and Metagenomics 5: e57278. https://doi.org/10.3897/mbmg.5.57278

Overview of DNA-based studies of invertebrates applying propylene glycol, sorted by year and taxonomic group

opencc-zeroJan 2021View details →

Datasets from: Validated removal of nuclear pseudogenes and sequencing artefacts from mitochondrial metabarcode

Metabarcoding of Metazoa using mitochondrial genes may be confounded by both the accumulation of PCR and sequencing artefacts and the co-amplification of nuclear mitochondrial pseudogenes (NUMTs). The application of read abundance thresholds and denoising methods is efficient in reducing noise accompanying authentic mitochondrial amplicon sequence variants (ASVs). However, these procedures do not fully account for the complex nature of concomitant sequences and the highly variable DNA contribution of specimens in a metabarcoding sample. We propose, as a complement to denoising, the metabarcoding Multidimensional Abundance Threshold Evaluation (metaMATE) framework, a novel approach that allows comprehensive examination of multiple dimensions of abundance filtering and the evaluation of the prevalence of unwanted concomitant sequences in denoised metabarcoding datasets. metaMATE requires a denoised set of ASVs as input, and designates a subset of ASVs as being either authentic (mtDNA haplotypes) or non-authentic ASVs (NUMTs and erroneous sequences) by comparison to external reference data and by analysing nucleotide substitution patterns. metaMATE (i) facilitates the application of read abundance filtering strategies, which are structured with regard to sequence library and phylogeny and applied for a range of increasing abundance threshold values, and (ii) evaluates their performance by quantifying the prevalence of non-authentic ASVs and the collateral effects on the removal of authentic ASVs. The output from metaMATE facilitates decision-making about required filtering stringency and can be used to improve the reliability of intraspecific genetic information derived from metabarcode data. The framework is implemented in the metaMATE software, available at https://github.com/tjcreedy/metamate).

opencc-zeroDec 2016View details →

Data from: Pellets of proof: first glimpse of the dietary composition of adult odonates as revealed by metabarcoding of feces

Recent advances in molecular techniques allow us to resolve the diet of unstudied taxa. Odonates are potentially important top-down regulators of many insects. Yet, to date, our knowledge of odonate prey use is based mainly on limited observations of odonates catching or eating their prey. In this study, we examine the potential use of metabarcoding in establishing the diet of three adult odonate species (Lestes sponsa, Enallagma cyathigerum, and Sympetrum danae) at a site in southwestern Finland. To this purpose, we compared three different methods for extracting DNA from fecal samples: the Macherey-Nagel Nucleospin XS kit, a traditional salt extraction, and the Zymo Research Fecal Microprep kit. From these extracts, we amplified group-specific mitochondrial markers (COI and 16S rRNA) from altogether 72 odonate individuals, and compared them to comprehensive reference libraries. The three odonate species show major overlap in diet, with no significant differences between individuals of different size and/or gender, reflecting opportunistic foraging of adult odonates. Of a total of 41 different prey species detected, the most frequently consumed ones were Diptera, with additional records of six other orders. Based on our data, the best DNA extraction method is the traditional salt extraction, as it provides the most information on prey content while also being the most economical. To our knowledge, this is the first study to resolve the species-level diet of adult odonates. Armed with the appropriate methodological caveats, we are ready to examine the ecological role of odonates in both terrestrial and aquatic food webs, and in transferring subsidies between these two realms.

opencc-zeroDec 2017View details →

Data from: DNA metabarcoding reveals changes in the contents of carnivorous plants along an elevation gradient

Resource variation along abiotic gradients influences subsequent trophic interactions and these effects can be transmitted through entire food webs. Interactions along abiotic gradients can provide clues as to how organisms will face changing environmental conditions, such as future range shifts. However, it is challenging to find replicated systems to study these effects. Phytotelmata, such as those found in carnivorous plants, are isolated aquatic communities and thus form a good model for the study of replicated food webs. Due to the degraded nature of the prey, molecular techniques provide a useful tool to study these communities. We studied the pitcher plant Sarracenia purpurea L. in allochthonous populations along an elevational gradient in the Alps and Jura. We predicted that invertebrate richness in the contents of the pitcher plants would decrease with increasing elevation, reflecting harsher environmental conditions. Using metabarcoding of the COI gene, we sequenced the invertebrate contents of these pitcher plants. We assigned Molecular Operational Taxonomic Units at ordinal level as well as recovering species-level data. We found small but significant changes in community composition with elevation. These recovered sequences could belong to invertebrate prey, rotifer inquilines, pollinators and other animals possibly living inside the pitchers. However, we found no directional trend or site-based differences in MOTU richness with elevational gradient. Use of molecular techniques for dietary or contents analysis is a powerful way to examine numerous degraded samples, although factors such as DNA persistence and the relationship to species presence still have to be completely determined.

opencc-zeroJun 2021View details →

eDNA metabarcoding in lakes to quantify influences of landscape features and human activity on aquatic invasive species prevalence and fish community diversity

Aim: Our goal was to use eDNA metabarcoding to characterize fish community diversity, detect aquatic invasive species (AIS), and assess how measures of community (or AIS) diversity are influenced by lake physical and environmental covariates, measures of hydrological connectivity, and human accessibility. Location: Michigan, USA. Methods: eDNA samples collected from 22 lakes were sequenced using two mitochondrial gene regions (12S and 16S rRNA). Metabarcoding data were compared to traditional fisheries survey data for a subset of lakes, and data from all 22 lakes were combined with environmental information to identify significant associations with community diversity and AIS relative abundance. Results: Occupancy modeling indicated that detection probabilities were generally higher with eDNA than traditional fisheries gear. Measures of connectivity with upstream aquatic habitats were positively associated with both AIS relative abundance and fish species diversity. We also demonstrate the use of spatial interpolation methods to map distributions of species diversity and AIS relative abundance within lakes. Conclusions: eDNA metabarcoding methods provided information on the composition and diversity of fish assemblages and the presence of AIS in freshwater lakes that varied greatly in drainage connectivity and anthropogenic development. Our case study identified associations between environmental covariates and fish diversity or AIS relative abundance across lakes. This information is of particular importance given increasing anthropogenic disturbance, invasive species spread, and associated declines in aquatic biodiversity. Incorporating eDNA metabarcoding as a supplement to traditional fisheries surveys will permit managers to identify greater numbers of taxa, including early detection of AIS, with less field effort and fish mortality. Further, eDNA methods may more accurately identify physical and biological features that correlate with diversity and abundance, and allow agencies to more effectively direct AIS management activities.

opencc-zeroDec 2016View details →

Data from: Metabarcoding for the parallel identification of several hundred predators and their preys: application to bat species diet analysis

Assessing diet variability is of main importance to better understand the biology of bats and design conservation strategies. Although the advent of metabarcoding has facilitated such analyses, this approach does not come without challenges. Biases may occur throughout the whole experiment, from fieldwork to biostatistics, resulting in the detection of false negatives, false positives or low taxonomic resolution. We detail a rigorous metabarcoding approach based on a short COI minibarcode and two-step PCR protocol enabling the 'all at once' taxonomic identification of bats and their arthropod preys for several hundreds of samples. Our study includes faecal pellets collected in France from 357 bats representing 16 species, as well as insect mock communities that mimic bat meals of known composition, negative and positive controls. All samples were analysed using three replicates. We compare the efficiency of DNA extraction methods and we evaluate the effectiveness of our protocol using identification success, taxonomic resolution, sensitivity, and amplification biases. Our parallel identification strategy of predators and preys reduces the risk of mis-assigning preys to wrong predators and decreases the number of molecular steps. Controls and replicates enable to filter the data and limit the risk of false positives, hence guaranteeing high confidence results for both prey occurrence and bat species identification. We validate 551 COI variants from arthropod including 18 orders, 117 family, 282 genus and 290 species. Our method therefore provides a rapid, resolutive and cost-effective screening tool for addressing evolutionary ecological issues or developing 'chirosurveillance' and conservation strategies.

opencc-zeroDec 2016View details →

Data from: A from-benchtop-to-desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies

The main objective of this work was to develop and validate a robust and reliable 'from benchtop-to-desktop' metabarcoding workflow to investigate the diet of invertebrate-eaters. We applied our workflow to fecal DNA samples of an invertebrate-eating fish species. A fragment of the COI gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq platform. We developed a filtering approach based on a series of non-arbitrary thresholds established from control samples and from molecular replicates in order to address the elimination of cross-contamination, PCR/sequencing errors and mistagging artifacts. This resulted in a conservative and informative metabarcoding dataset. We developed a taxonomic assignment procedure that combines different approaches and that allowed the identification of ~75% of invertebrate COI variants to the species level. Moreover, based on the diversity of the variants, we introduced a semi-quantitative statistic in our diet study, the Minimum Number of Individuals (MNI), which is based on the number of distinct variants in each sample. The metabarcoding approach described in this paper may guide future diet studies that aim to produce robust datasets associated with a fine and accurate identification of prey items.

opencc-zeroDec 2017View details →

Data from: Determining diet from faeces: selection of metabarcoding primers for the insectivore Pyrenean desman (Galemys pyrenaicus)

Molecular techniques allow non-invasive dietary studies from faeces, providing an invaluable tool to unveil ecological requirements of endangered or elusive species. They contribute to progress on important issues such as genomics, population genetics, dietary studies or reproductive analyses, essential knowledge for conservation biology. Nevertheless, these techniques require general methods to be tailored to the specific research objectives, as well as to substrate- and species-specific constraints. In this pilot study we test a range of available primers to optimise diet analysis from metabarcoding of faeces of a generalist aquatic insectivore, the endangered Pyrenean desman (Galemys pyrenaicus, É. Geoffroy Saint-Hilaire, 1811, Talpidae), as a step to improve the knowledge of the conservation biology of this species. Twenty-four faeces were collected in the field, DNA was extracted from them, and fragments of the standard barcode region (COI) were PCR amplified by using five primer sets (Brandon-Mong, Gillet, Leray, Meusnier and Zeale). PCR outputs were sequenced on the Illumina MiSeq platform, sequences were processed, clustered into OTUs (Operational Taxonomic Units) using UPARSE algorithm and BLASTed against the NCBI database. Although all primer sets successfully amplified their target fragments, they differed considerably in the amounts of sequence reads, rough OTUs, and taxonomically assigned OTUs. Primer sets consistently identified a few abundant prey taxa, probably representing the staple food of the Pyrenean desman. However, they differed in the less common prey groups. Overall, the combination of Gillet and Zeale primer sets were most cost-effective to identify the widest taxonomic range of prey as well as the desman itself, which could be further improved stepwise by adding sequentially the outputs of Leray, Brandon-Mong and Meusnier primers. These results are relevant for the conservation biology of this endangered species as they allow a better characterization of its food and habitat requirements.