2026-10-14T23:10:47Z https://mbmg.pensoft.net/oai.php

10.3897/mbmg.1.19862 2017-09-20 mbmg

Functional and phylogenetic analysis of the core transcriptome of Ochromonadales Graupner,Nadine Boenigk,Jens Bock,Christina Jensen,Manfred Marks,Sabina Rahmann,Sven Beisser,Daniela Chrysophyceae protist expressed sequence tags (EST) evolutionary ecology phylotranscriptomics core genes metabolic pathways Metabarcoding and Metagenomics 1: e19862 BackgroundMost protist lineages consist of members with diverging features e.g. different modes of nutrition and adaptations for life in different habitat types and climatic zones. The nutritional mode is particularly variable in chrysophytes and they are therefore an excellent model group to study the core genes and metabolic pathways of a functionally diverse lineage. The objective of our study is the identification of the joint genetic repertoire expressed in closely related chrysophytes as well as the extent of variation on species and strain level. Therefore, we investigated the transcriptomes of six strains belonging to four species of Ochromonadales. We performed analyses on metabolic pathway level as well as on sequence level.ResultsWe could identify 1,574 core genes shared between all six investigated strains of Ochromonadales. Most of these core genes were affiliated with the primary metabolism. Phylogenetic analysis of 166 protein-coding core genes supported a close relation of Poteriospumella lacustris and Poterioochromonas malhamensis and resolved for more than 50% of investigated genes the relationship of strains affiliated with the species P. lacustris. Further, we found diverging phylogenetic patterns for genes interacting with the environment.ConclusionsIn Ochromonadales, a functionally diverse lineage, the core transcriptome represents only a minor part of the individual transcriptomes. But this small fraction of genes comprises the basal metabolism essential for life in several protist lineages. Phylogenetic analyses of these genes indicate a similar degree of conservation as observed for genes coding for ribosomal proteins. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2017 Research Article text/html info:doi:10.3897/mbmg.1.19862 https://doi.org/10.3897/mbmg.1.19862 https://mbmg.pensoft.net/article/19862/ https://mbmg.pensoft.net/article/19862/download/pdf/ en

10.3897/mbmg.1.14625 2017-09-20 mbmg

Short COI markers for freshwater macroinvertebrate metabarcoding Vamos,Ecaterina Elbrecht,Vasco Leese,Florian metabarcoding COI primers degraded DNA biodiversity assessment freshwater macroinvertebrates in silico Metabarcoding and Metagenomics 1: e14625 Species diversity of metazoan bulk samples can be rapidly assessed using cytochrome c oxidase I (COI) metabarcoding. However, in some applications often only degraded DNA is available, e.g. from poorly conserved museum specimens, environmental DNA (eDNA) filtered from water or gut content analyses. Here universal primer sets targeting only a short COI fragment are advantageous, as they often can still amplify short DNA fragments. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vitro by sequencing previously used freshwater macroinvertebrate mock communities as well as three monitoring samples from Romanian streams of unknown composition. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vitro, detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2+BR2) revealed that detection efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2017 Primer Validation text/html info:doi:10.3897/mbmg.1.14625 https://doi.org/10.3897/mbmg.1.14625 https://mbmg.pensoft.net/article/14625/ https://mbmg.pensoft.net/article/14625/download/pdf/ en

10.3897/mbmg.1.14791 2017-09-20 mbmg

Ready for the deluge! Introducing the new Metabarcoding and Metagenomics (MBMG) journal Leese,Florian Steinke,Dirk Weigand,Alexander M. Penev,Lyubomir Metabarcoding and Metagenomics 1: e14791 info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2017 Editorial text/html info:doi:10.3897/mbmg.1.14791 https://doi.org/10.3897/mbmg.1.14791 https://mbmg.pensoft.net/article/14791/ https://mbmg.pensoft.net/article/14791/download/pdf/ en

10.3897/mbmg.1.13709 2017-10-18 mbmg

Disentangling higher trophic level interactions in the cabbage aphid food web using high-throughput DNA sequencing Lefort,Marie-Caroline Wratten,Stephen Cusumano,Antonino Varennes,Yann-David Boyer,Stephane metabarcoding biological control enemy release hypothesis hyperparasitism parasitoids hyperparasitoids competition Metabarcoding and Metagenomics 1: e13709 The lack of understanding of complex food-web interactions has been a major gap in the history of biological control. In particular, a better understanding of the functioning of pest food-webs and how they vary between native and invaded geographical ranges is of prime interest for biological control research and associated integrated pest management. Technical limitations associated with the deciphering of complex food-webs can now be largely overcome by the use of high throughput DNA sequencing techniques such as Illumina MiSeq. We tested the efficiency of this next generation sequencing technology in a metabarcoding approach, to study aphid food-webs using the cabbage aphid as model. We compared the variations in structure and composition of aphid food-webs in the species’ native range (United Kingdom, UK) and in an invaded range (New Zealand, NZ). We showed that Illumina MiSeq is a well suited technology to study complex aphid food-webs from aphid mummies. We found an unexpectedly high top down pressure in the NZ cabbage aphid food-web, which coupled to a large ratio of consumer species / prey species and a lack of potential inter-specific competition between primary parasitoids, could cause the NZ food-web to be more vulnerable than the UK one. This study also reports for the first time the occurrence of a new hyperparasitoid species in NZ, as well as new associations between hyperparasitoids parasitoids and the cabbage aphid in this country. We conclude that the complexity of aphid food-webs in agricultural systems could often be underestimated, particularly at higher trophic levels; and that the use of high throughput DNA sequencing tools, could largely help to overcome this impediment. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2017 Research Article text/html info:doi:10.3897/mbmg.1.13709 https://doi.org/10.3897/mbmg.1.13709 https://mbmg.pensoft.net/article/13709/ https://mbmg.pensoft.net/article/13709/download/pdf/ en

10.3897/mbmg.1.22262 2017-12-15 mbmg

Combining NCBI and BOLD databases for OTU assignment in metabarcoding and metagenomic datasets: The BOLD_NCBI _Merger Macher,Jan-Niklas Macher,Till-Hendrik Leese,Florian Biodiversity High-throughput sequencing Operational taxonomic unit software MEGAN script taxonomic assignment Metabarcoding and Metagenomics 1: e22262 Metabarcoding and metagenomic approaches are becoming routine techniquesfor use in biodiversity assessment and in ecological studies. The assignment of taxonomic information to millions of sequences obtained via high-throughput sequencing is challenging, as many DNA reference libraries are lacking information on certain taxonomic groups and can contain erroneous sequences. Combining different reference databases is therefore a promising approach for maximising taxonomic coverage and reliability of results. The “BOLD_NCBI_Merger” bash script is introduced, which combines sequence data obtained from the National Centre for Biotechnology Information (NCBI) GenBank and the Barcode of Life Database (BOLD) and prepares it for taxonomic assignment with the software MEGAN. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2017 Software Description text/html info:doi:10.3897/mbmg.1.22262 https://doi.org/10.3897/mbmg.1.22262 https://mbmg.pensoft.net/article/22262/ https://mbmg.pensoft.net/article/22262/download/pdf/ en

10.3897/mbmg.2.21519 2018-01-02 mbmg

Geographic distance and mountain ranges structure freshwater protist communities on a European scalе Boenigk,Jens Wodniok,Sabina Bock,Christina Beisser,Daniela Hempel,Christopher Grossmann,Lars Lange,Anja Jensen,Manfred protist molecular diversity biogeography microbial ecology algae Metabarcoding and Metagenomics 2: e21519 Protists influence ecosystems by modulating microbial population size, diversity, metabolic outputs and gene flow. In this study we used eukaryotic ribosomal amplicon diversity from 218 European freshwater lakes sampled in August 2012 to assess the effect of mountain ranges as biogeographic barriers on spatial patterns and microbial community structure in European freshwaters. The diversity of microbial communities as reflected by amplicon clusters suggested that the eukaryotic microbial inventory of lakes was well-sampled at the European and at the local scale. Our pan-European diversity analysis indicated that biodiversity and richness of high mountain lakes differed from that of lowland lakes. Further, the taxon inventory of high-mountain lakes strongly contributed to beta-diversity despite a low taxon inventory. Even though ecological factors, in general, strongly affect protist community pattern, we show that geographic distance and geographic barriers significantly contribute to community composition particularly for high mountain regions which presumably act as biogeographic islands. However, community composition in lowland lakes was also affected by geographic distance but less pronounced as in high mountain regions. In consequence protist populations are locally structured into distinct biogeographic provinces and community analyses revealed biogeographic patterns also for lowland lakes whereby European mountain ranges act as dispersal barriers in particular for short to intermediate distances whereas the effect of mountain ranges levels off on larger scale. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Research Article text/html info:doi:10.3897/mbmg.2.21519 https://doi.org/10.3897/mbmg.2.21519 https://mbmg.pensoft.net/article/21519/ https://mbmg.pensoft.net/article/21519/download/pdf/ en

10.3897/mbmg.2.21060 2018-01-15 mbmg

Using DNA metabarcoding for assessing chironomid diversity and community change in mosquito controlled temporary wetlands Theissinger,Kathrin Kästel,Anna Elbrecht,Vasco Makkonen,Jenny Michiels,Susanne Schmidt,Susanne Allgeier,Stefanie Leese,Florian Brühl,Carsten Bacillus thuringensis var. israelensis (Bti) Barcode of Life Database (BOLD) chironomid saprobic index cytochrome oxidase subunit I (COI) ecotoxicology macrozoobenthos emergence operational taxonomic units (OTU) Metabarcoding and Metagenomics 2: e21060 The biocide Bacillus thuringiensis var. israelensis (Bti) is widely applied for mosquito control in temporary wetlands of the German Upper Rhine Valley. Even though Bti is considered environmentally friendly, several studies have shown non-target effects on chironomids, a key food resource in wetland ecosystems. Chironomids have been proposed as important indicators for monitoring freshwater ecosystems, however, morphological determination is very challenging. In this study, we investigated the effectiveness of metabarcoding for chironomid diversity assessment and tested the retrieved chironomid operational taxonomic units (OTUs) for possible changes in relative abundance and species diversity in relation to mosquito control actions in four temporary wetlands. Three of these wetlands were, for the first year after 20 years of Bti treatment, partly left Bti-untreated in a split field design, and one wetland has never been treated with Bti. Our metabarcoding approach detected 54 chironomid OTUs across all study sites, of which almost 70% could be identified to species level comparisons against the BOLD database. We showed that metabarcoding increased chironomid species determination by 70%. However, we found only minor significant effects of Bti on the chironomid community composition, even though Bti reduced chironomid emergence by 65%. This could be due to a time lag of chironomid recolonization, since the study year was the first year of Bti intermittence after about 20 years of Bti application in the study area. Subsequent studies will have to address if and how the chironomid community composition will recover further in the now Bti-untreated temporary wetlands to assess effects of Bti. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Applied Study text/html info:doi:10.3897/mbmg.2.21060 https://doi.org/10.3897/mbmg.2.21060 https://mbmg.pensoft.net/article/21060/ https://mbmg.pensoft.net/article/21060/download/pdf/ en

10.3897/mbmg.2.23297 2018-03-14 mbmg

Quantitative monitoring of multispecies fish environmental DNA using high-throughput sequencing Ushio,Masayuki Murakami,Hiroaki Masuda,Reiji Sado,Tetsuya Miya,Masaki Sakurai,Sho Yamanaka,Hiroki Minamoto,Toshifumi Kondoh,Michio biodiversity monitoring environmental DNA DNA quantification internal standard DNA qPCR metabarcoding Metabarcoding and Metagenomics 2: e23297 Effective ecosystem conservation and resource management require quantitative monitoring of biodiversity, including accurate descriptions of species composition and temporal variations of species abundance. Accordingly, quantitative monitoring of biodiversity has been performed for many ecosystems, but it is often time- and effort-consuming and costly. Recent studies have shown that environmental DNA (eDNA), which is released to the environment from macro-organisms living in a habitat, contains information about species identity and abundance. Thus, analysing eDNA would be a promising approach for more efficient biodiversity monitoring. In the present study, internal standard DNAs (i.e. known amounts of short DNA fragments from fish species that have never been observed in a sampling area) were added to eDNA samples, which were collected weekly from a coastal marine ecosystem in Maizuru Bay, Japan (from April 2015 to March 2016) and metabarcoding analysis was performed using Illumina MiSeq to simultaneously identify fish species and quantify fish eDNA copy numbers. A correction equation was obtained for each sample using the relationship between the number of sequence reads and the added amount of the standard DNAs and this equation was used to estimate the copy numbers from the sequence reads of non-standard fish eDNA. The calculated copy numbers showed significant positive correlations with those determined by quantitative PCR, suggesting that eDNA metabarcoding with standard DNA enabled useful quantification of eDNA. Furthermore, for samples that show a high level of PCR inhibition, this method might allow more accurate quantification than qPCR because the correction equations generated using internal standard DNAs would include the effect of PCR inhibition. A single run of Illumina MiSeq produced >70 quantitative fish eDNA time series in this study, showing that this method could contribute to more efficient and quantitative monitoring of biodiversity. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Research Article text/html info:doi:10.3897/mbmg.2.23297 https://doi.org/10.3897/mbmg.2.23297 https://mbmg.pensoft.net/article/23297/ https://mbmg.pensoft.net/article/23297/download/pdf/ en

10.3897/mbmg.2.22467 2018-03-14 mbmg

Dropping Hints: Estimating the diets of livestock in rangelands using DNA metabarcoding of faeces Lee,Timothy Alemseged,Yohannes Mitchell,Andrew Metabarcoding rbcL ITS2 Ovis aries diet metabarcoding barcode references Metabarcoding and Metagenomics 2: e22467 The introduction of domesticated animals into new environments can lead to considerable ecological disruption, and it can be difficult to predict their impact on the new ecosystem. In this study, we use faecal metabarcoding to characterize the diets of three ruminant taxa in the rangelands of south-western New South Wales, Australia. Our study organisms included goats (Capra aegagrus hircus) and two breeds of sheep (Ovis aries): Merinos, which have been present in Australia for over two hundred years, and Dorpers, which were introduced in the 1990s. We used High-Throughput Sequencing methods to sequence the rbcL and ITS2 genes of plants in the faecal samples, and identified the samples using the GenBank and BOLD online databases, as well as a reference collection of sequences from plants collected in the study area. We found that the diets of all three taxa were dominated by the family Malvaceae, and that the Dorper diet was more diverse than the Merino diet at both the family and the species level. We conclude that Dorpers, like Merinos, are potentially a threat to some vulnerable species in the rangelands of New South Wales. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Research Article text/html info:doi:10.3897/mbmg.2.22467 https://doi.org/10.3897/mbmg.2.22467 https://mbmg.pensoft.net/article/22467/ https://mbmg.pensoft.net/article/22467/download/pdf/ en

10.3897/mbmg.2.24262 2018-05-14 mbmg

Estimating fish alpha- and beta-diversity along a small stream with environmental DNA metabarcoding Li,Yiyuan Evans,Nathan T. Renshaw,Mark A. Jerde,Christopher L. Olds,Brett P. Shogren,Arial J. Deiner,Kristy Lodge,David M. Lamberti,Gary A. Pfrender,Michael eDNA biodiversity assessment biomonitoring α-diversity β-diversity high throughput sequencing Metabarcoding and Metagenomics 2: e24262 Environmental DNA (eDNA) metabarcoding has been increasingly applied to biodiversity surveys in stream ecosystems. In stream networks, the accuracy of eDNA-based biodiversity assessment depends on whether the upstream eDNA influx affects downstream detection. Biodiversity assessment in low-discharge streams should be less influenced by eDNA transport than in high-discharge streams. We estimated α- and β-diversity of the fish community from eDNA samples collected in a small Michigan (USA) stream from its headwaters to its confluence with a larger river. We found that α-diversity increased from upstream to downstream and, as predicted, we found a significant positive correlation between β-diversity and physical distance (stream length) between locations indicating species turnover along the longitudinal stream gradient. Sample replicates and different genetic markers showed similar species composition, supporting the consistency of the eDNA metabarcoding approach to estimate α- and β-diversity of fishes in low-discharge streams. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Research Article text/html info:doi:10.3897/mbmg.2.24262 https://doi.org/10.3897/mbmg.2.24262 https://mbmg.pensoft.net/article/24262/ https://mbmg.pensoft.net/article/24262/download/pdf/ en

10.3897/mbmg.2.25136 2018-05-17 mbmg

Characterising planktonic dinoflagellate diversity in Singapore using DNA metabarcoding Sze,Yue Miranda,Lilibeth N Sin,Tsai Min Huang,Danwei high-throughput sequencing Johor Strait molecular operational taxonomic unit phytoplankton Singapore Strait Metabarcoding and Metagenomics 2: e25136 Dinoflagellates are traditionally identified morphologically using microscopy, which is a time-consuming and labour-intensive process. Hence, we explored DNA metabarcoding using high-throughput sequencing as a more efficient way to study planktonic dinoflagellate diversity in Singapore’s waters. From 29 minimally pre-sorted water samples collected at four locations in western Singapore, DNA was extracted, amplified and sequenced for a 313-bp fragment of the V4–V5 region in the 18S ribosomal RNA gene. Two sequencing runs generated 2,847,170 assembled paired-end reads, corresponding to 573,176 unique sequences. Sequences were clustered at 97% similarity and analysed with stringent thresholds (≥150 bp, ≥20 reads, ≥95% match to dinoflagellates), recovering 28 dinoflagellate taxa. Dinoflagellate diversity captured includes parasitic and symbiotic groups which are difficult to identify morphologically. Richness is similar between the inner and outer West Johor Strait, but variations in community structure are apparent, likely driven by environmental differences. None of the taxa detected in a recent phytoplankton bloom along the West Johor Strait have been recovered in our samples, suggesting that background communities are distinct from bloom communities. The voluminous data obtained in this study contribute baseline information for Singapore’s phytoplankton communities and prompt future research and monitoring to adopt the approach established here. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Research Article text/html info:doi:10.3897/mbmg.2.25136 https://doi.org/10.3897/mbmg.2.25136 https://mbmg.pensoft.net/article/25136/ https://mbmg.pensoft.net/article/25136/download/pdf/ en

10.3897/mbmg.2.24556 2018-07-05 mbmg

Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types Sellers,Graham S. Di Muri,Cristina Gómez,Africa Hänfling,Bernd modular adaptability spin column high-throughput cost effective metabarcoding metagenomics eDNA Metabarcoding and Metagenomics 2: e24556 Efficient DNA extraction is fundamental to molecular studies. However, commercial kits are expensive when a large number of samples need to be processed. Here we present a simple, modular and adaptable DNA extraction ‘toolkit’ for the isolation of high purity DNA from multiple sample types (modular universal DNA extraction method or Mu-DNA). We compare the performance of our method to that of widely used commercial kits across a range of soil, stool, tissue and water samples. Mu-DNA produced DNA extractions of similar or higher yield and purity to that of the commercial kits. As a proof of principle, we carried out replicate fish metabarcoding of aquatic eDNA extractions, which confirmed that the species detection efficiency of our method is similar to that of the most frequently used commercial kit. Our results demonstrate the reliability of Mu-DNA along with its modular adaptability to challenging sample types and sample collection methods. Mu-DNA can substantially reduce the costs and increase the scope of experiments in molecular studies. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Methods text/html info:doi:10.3897/mbmg.2.24556 https://doi.org/10.3897/mbmg.2.24556 https://mbmg.pensoft.net/article/24556/ https://mbmg.pensoft.net/article/24556/download/pdf/ en

10.3897/mbmg.2.25649 2018-07-05 mbmg

BBI: an R package for the computation of Benthic Biotic Indices from composition data Cordier,Tristan Pawlowski,Jan Biotic indices biomonitoring R package benthos composition data Metabarcoding and Metagenomics 2: e25649 The monitoring of impacts of anthropic activities in marine environments, such as aquaculture, oil-drilling platforms or deep-sea mining, relies on Benthic Biotic Indices (BBI). Several indices have been formalised to reduce the multivariate composition data into a single continuous value that is ascribed to a discrete ecological quality status. Such composition data is traditionally obtained from macrofaunal inventories, which is time-consuming and expertise-demanding. Important efforts are ongoing towards using High-Throughput Sequencing of environmental DNA (eDNA metabarcoding) to replace or complement morpho-taxonomic surveys for routine biomonitoring. The computation of BBI from such composition data is usually being undertaken by practitioners with excel spreadsheets or through custom script. Hence, the updating of reference morpho-taxonomic tables and cross studies comparison could be hampered. Here we introduce the R package BBI for the computation of BBI from composition data, either obtained from traditional morpho-taxonomic inventories or from metabarcoding data. Its aim is to provide an open-source, transparent and centralised method to compute BBI for routine biomonitoring. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 R Package text/html info:doi:10.3897/mbmg.2.25649 https://doi.org/10.3897/mbmg.2.25649 https://mbmg.pensoft.net/article/25649/ https://mbmg.pensoft.net/article/25649/download/pdf/ en

10.3897/mbmg.2.26869 2018-08-23 mbmg

A DNA metabarcoding protocol for hyporheic freshwater meiofauna: Evaluating highly degenerate COI primers and replication strategy Weigand,Alexander Macher,Jan-Niklas Hyporheos DNA Metabarcoding Streambed Health Assessment Biological Quality Element Meiofaunal Enrichment Metabarcoding and Metagenomics 2: e26869 The hyporheic zone, i.e. the ecotone between surface water and the groundwater, is a rarely studied freshwater ecosystem. Hyporheic taxa are often meiofaunal (<1 mm) in size and difficult to identify based on morphology. Metabarcoding approaches are promising for the study of these environments and taxa, but it is yet unclear if commonly applied metabarcoding primers and replication strategies can be used. In this study, we took sediment cores from two near natural upstream (NNU) and two ecologically improved downstream (EID) sites in the Boye catchment (Emscher River, Germany), metabarcoding their meiofaunal communities. We evaluated the usability of a commonly used, highly degenerate COI primer pair (BF2/BR2) and tested how sequencing three PCR replicates per sample and removing MOTUs present in only one out of three replicates impacts the inferred community composition. A total of 22,514 MOTUs were detected, of which only 263 were identified as Metazoa. Our results highlight the gaps in reference databases for meiofaunal taxa and the potential problems of using highly degenerate primers for studying samples containing a high number of non-metazoan taxa. Alpha diversity was higher in EID sites and showed higher community similarity when compared to NNU sites. Beta diversity analyses showed that removing MOTUs detected in only one out of three replicates per site greatly increased community similarity in samples. Sequencing three sample replicates and removing rare MOTUs is seen as a good compromise between retaining too many false-positives and introducing too many false-negatives. We conclude that metabarcoding hyporheic communities using highly degenerate COI primers can provide valuable first insights into the diversity of these ecosystems and highlight some potential application scenarios. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Methods text/html info:doi:10.3897/mbmg.2.26869 https://doi.org/10.3897/mbmg.2.26869 https://mbmg.pensoft.net/article/26869/ https://mbmg.pensoft.net/article/26869/download/pdf/ en

10.3897/mbmg.2.26664 2018-08-23 mbmg

Choice of DNA extraction method affects DNA metabarcoding of unsorted invertebrate bulk samples Majaneva,Markus Diserud,Ola H. Eagle,Shannon Hajibabaei,Mehrdad Ekrem,Torbjørn Macroinvertebrates Biomonitoring Freshwater Metabarcoding and Metagenomics 2: e26664 Characterisation of freshwater benthic biodiversity using DNA metabarcoding may allow more cost-effective environmental assessments than the current morphological-based assessment methods. DNA metabarcoding methods where sorting or pre-sorting of samples are avoided altogether are especially interesting, since the time between sampling and taxonomic identification is reduced. Due to the presence of non-target material like plants and sediments in crude samples, DNA extraction protocols become important for maximising DNA recovery and sample replicability. We sampled freshwater invertebrates from six river and lake sites and extracted DNA from homogenised bulk samples in quadruplicate subsamples, using a published method and two commercially available kits: HotSHOT approach, Qiagen DNeasy Blood & Tissue Kit and Qiagen DNeasy PowerPlant Pro Kit. The performance of the selected extraction methods was evaluated by measuring DNA yield and applying DNA metabarcoding to see if the choice of DNA extraction method affects DNA yield and metazoan diversity results. The PowerPlant Kit extractions resulted in the highest DNA yield and a strong significant correlation between sample weight and DNA yield, while the DNA yields of the Blood & Tissue Kit and HotSHOT method did not correlate with the sample weights. Metazoan diversity measures were more repeatable in samples extracted with the PowerPlant Kit compared to those extracted with the HotSHOT method or the Blood & Tissue Kit. Subsampling using Blood & Tissue Kit and HotSHOT extraction failed to describe the same community in the lake samples. Our study exemplifies that the choice of DNA extraction protocol influences the DNA yield as well as the subsequent community analysis. Based on our results, low specimen abundance samples will likely provide more stable results if specimens are sorted prior to DNA extraction and DNA metabarcoding, but the repeatability of the DNA extraction and DNA metabarcoding results was close to ideal in high specimen abundance samples. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Research Article text/html info:doi:10.3897/mbmg.2.26664 https://doi.org/10.3897/mbmg.2.26664 https://mbmg.pensoft.net/article/26664/ https://mbmg.pensoft.net/article/26664/download/pdf/ en

10.3897/mbmg.2.26939 2018-10-10 mbmg

A meta-barcoding census of freshwater planktonic protists in Appalachia – Natural Tunnel State Park, Virginia, USA Cahoon,Bruce Huffman,Ashley Krager,Megan Crowell,Roseanna Metabarcoding microbial eukaryotes algae Bioblitz Metabarcoding and Metagenomics 2: e26939 The purpose of our study was to survey the freshwater planktonic protists within an inland natural preserve in the Ridge and Valley physiographic province of the Appalachian Region using metabarcoding. Microbial eukaryotes are essential primary producers and predators in small freshwater ecosystems, yet they are often overlooked due to the difficulty of identification. This has been remedied, in part, by the cost reduction of high throughput DNA sequencing and the growth of barcode databases, making the identification and analysis of microorganisms by way of metabarcoding surveys in complex ecosystems increasingly feasible. Water samples were collected from five sites at the Natural Tunnel State Park in Scott County, VA (USA), representing three common bodies of water found in this region. Samples were initially collected during a Bioblitz event in April 2016 and then seven and fourteen weeks afterwards. Metabarcode analysis of the 23S and 18S genes identified 3663 OTUs representing 213 family level and 332 genus level taxa. This study provides an initial barcode census within a region that has a reputation as a temperate biodiversity “hotspot”. The overall protist diversity was comparably high to other temperate systems, but not unusually high; the microalgal diversity, however, was higher than that reported for other temperate regions. The three types of water bodies had their own distinctive protist biomes despite close proximity. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Research Article text/html info:doi:10.3897/mbmg.2.26939 https://doi.org/10.3897/mbmg.2.26939 https://mbmg.pensoft.net/article/26939/ https://mbmg.pensoft.net/article/26939/download/pdf/ en

10.3897/mbmg.2.26744 2018-10-12 mbmg

The influence of macroinvertebrate abundance on the assessment of freshwater quality in The Netherlands Beentjes,Kevin Speksnijder,Arjen Schilthuizen,Menno Schaub,Bart van der Hoorn,Berry Water Framework Directive biological quality elements abundance indicator species macrofauna lakes streams Metabarcoding and Metagenomics 2: e26744 The use of molecular tools for the detection and identification of invertebrate species enables the development of more easily standardisable inventories of biological elements for water quality assessments, as it circumvents human-based bias and errors in species identifications. Current Ecological Quality Ratio (EQR) assessments methods, however, often rely on abundance data. Translating metabarcoding sequence data into biomass or specimen abundances has proven difficult, as PCR amplification bias due to primer mismatching often provides skewed proportions of read abundances. While some potential solutions have been proposed in previous research, we instead looked at the necessity of abundance data in EQR assessments. In this study, we used historical monitoring data from natural (lakes, rivers and streams) and artificial (ditches and canals) water bodies to assess the impact of species abundances on the EQR scores for macroinvertebrates in the Water Framework Directive (WFD) monitoring programme of The Netherlands. By removing all the abundance data from the taxon observations, we simulated presence/absence-based monitoring, for which EQRs were calculated according to traditional methods. Our results showed a strong correlation between abundance-based and presence/absence-based EQRs. EQR scores were generally higher without abundances (75.8% of all samples), which resulted in 9.1% of samples being assigned to a higher quality class. The majority of the samples (89.7%) were assigned to the same quality class in both cases. These results are valuable for the incorporation of presence/absence metabarcoding data into water quality assessment methodology, potentially eliminating the need to translate metabarcoding data into biomass or absolute specimen counts for EQR assessments. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Research Article text/html info:doi:10.3897/mbmg.2.26744 https://doi.org/10.3897/mbmg.2.26744 https://mbmg.pensoft.net/article/26744/ https://mbmg.pensoft.net/article/26744/download/pdf/ en

10.3897/mbmg.2.28963 2018-11-02 mbmg

Optimising the detection of marine taxonomic richness using environmental DNA metabarcoding: the effects of filter material, pore size and extraction method Deiner,Kristy Lopez,Jacqueline Bourne,Steve Holman,Luke Seymour,Mathew Grey,Erin Lacoursière,Anaïs Li,Yiyuan Renshaw,Mark A. Pfrender,Michael Rius,Marc Bernatchez,Louis Lodge,David M. eDNA 18S ribosomal seawater high-throughput-sequencing metazoan eukaryotes non-indigenous species Metabarcoding and Metagenomics 2: e28963 The analysis of environmental DNA (eDNA) using metabarcoding has increased in use as a method for tracking biodiversity of ecosystems. Little is known about eDNA in marine human-modified environments, such as commercial ports, which are key sites to monitor for anthropogenic impacts on coastal ecosystems. To optimise an eDNA metabarcoding protocol in these environments, seawater samples were collected in a commercial port and methodologies for concentrating and purifying eDNA were tested for their effect on eukaryotic DNA yield and subsequent richness of Operational Taxonomic Units (OTUs). Different filter materials [Cellulose Nitrate (CN) and Glass Fibre (GF)], with different pore sizes (0.5 µm, 0.7 µm and 1.2 µm) and three previously published liquid phase extraction methods were tested. The number of eukaryotic OTUs detected differed by a factor of three amongst the method combinations. The combination of CN filters with phenol-chloroform-isoamyl alcohol extractions recovered a higher amount of eukaryotic DNA and OTUs compared to GF filters and the chloroform-isoamyl alcohol extraction method. Pore size was not independent of filter material but did affect the yield of eukaryotic DNA. For the OTUs assigned to a highly successful non-indigenous species, Styela clava, the two extraction methods with phenol significantly outperformed the extraction method without phenol; other experimental treatments did not contribute significantly to detection. These results highlight that careful consideration of methods is warranted because choice of filter material and extraction method create false negative detections of marine eukaryotic OTUs and underestimate taxonomic richness from environmental samples. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC0 Pensoft Publishers 2018 Research Article text/html info:doi:10.3897/mbmg.2.28963 https://doi.org/10.3897/mbmg.2.28963 https://mbmg.pensoft.net/article/28963/ https://mbmg.pensoft.net/article/28963/download/pdf/ en

10.3897/mbmg.2.30457 2018-12-31 mbmg

Monitoring riverine fish communities through eDNA metabarcoding: determining optimal sampling strategies along an altitudinal and biodiversity gradient Bylemans,Jonas Gleeson,Dianne Lintermans,Mark Hardy,Christopher M Beitzel,Matthew Gilligan,Dean M Furlan,Elise Environmental DNA eDNA Metabarcoding Sampling Fish Metabarcoding and Metagenomics 2: e30457 Monitoring aquatic biodiversity through DNA extracted from environmental samples (eDNA) combined with high-throughput sequencing, commonly referred to as eDNA metabarcoding, is increasing in popularity within the scientific community. However, sampling strategies, laboratory protocols and analytical pipelines can influence the results of eDNA metabarcoding surveys. While the impact of laboratory protocols and analytical pipelines have been extensively studied, the importance of sampling strategies on eDNA metabarcoding surveys has not received the same attention. To avoid underestimating local biodiversity, adequate sampling strategies (i.e. sampling intensity and spatial sampling replication) need to be implemented. This study evaluated the impact of sampling strategies along an altitudinal and biodiversity gradient in the upper section of the Murrumbidgee River (Murray-Darling Basin, Australia). An eDNA metabarcoding survey was used to determine the local fish biodiversity and evaluate the influence of sampling intensity and spatial sampling replication on the biodiversity estimates. The results show that optimal eDNA sampling strategies varied between sites and indicate that river morphology, species richness and species abundance affect the optimal sampling intensity and spatial sampling replication needed to accurately assess the fish biodiversity. While the generality of the patterns will need to be confirmed through future studies, these findings provide a basis to guide future eDNA metabarcoding surveys in river systems. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2018 Research Article text/html info:doi:10.3897/mbmg.2.30457 https://doi.org/10.3897/mbmg.2.30457 https://mbmg.pensoft.net/article/30457/ https://mbmg.pensoft.net/article/30457/download/pdf/ en

10.3897/mbmg.3.33835 2019-04-24 mbmg

Development of a new set of PCR primers for eDNA metabarcoding decapod crustaceans Komai,Tomoyuki Gotoh,Ryo Sado,Tetsuya Miya,Masaki mitochondrial 16S rRNA gene biodiversity monitoring MiDeca natural sea water Metabarcoding and Metagenomics 3: e33835 The Decapoda is one of the largest orders within the class Malacostraca, comprising approximately 14,000 extant species and including many commercially important species. For biodiversity monitoring in a non-invasive manner, a new set of PCR primers was developed for metabarcoding environmental DNA (eDNA) from decapod crustaceans. The new primers (herein named “MiDeca”) were designed for two conservative regions of the mitochondrial 16S rRNA gene, which amplify a short, hyper-variable region (153–184 bp, 164 bp on average) with sufficient interspecific variations. With the use of MiDeca primers and tissue-derived DNA extracts, we successfully determined those sequences (154–189 bp) from 250 species, placed in 186 genera and 65 families across the suborder Dendrobranchiata and 10 of the 11 infraorders of the suborder Pleocyemata. We also preliminarily attempted eDNA metabarcoding from natural seawater collected at Banda, Tateyama, the Pacific coast of central Japan and detected 42 decapod species including 34 and 8 species with sequence identities of > 98% and 80–98%, respectively. The results suggest the usefulness of eDNA metabarcoding with MiDeca primers for biodiversity monitoring of the decapod species. It appears, however, that further optimisation of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2019 Primer Validation text/html info:doi:10.3897/mbmg.3.33835 https://doi.org/10.3897/mbmg.3.33835 https://mbmg.pensoft.net/article/33835/ https://mbmg.pensoft.net/article/33835/download/pdf/ en

10.3897/mbmg.3.34002 2019-06-12 mbmg

Molecular versus morphological data for benthic diatoms biomonitoring in Northern Europe freshwater and consequences for ecological status Bailet,Bonnie Bouchez,Agnès Franc,Alain Frigerio,Jean-marc Keck,Francois Karjalainen,Satu Maaria Rimet,Frederic Schneider,Susanne Kahlert,Maria Metabarcoding environmental assessment 18S-V4 rbcL Bacillariophyta water quality Metabarcoding and Metagenomics 3: e34002 Diatoms are known to be efficient bioindicators for water quality assessment because of their rapid response to environmental pressures and their omnipresence in water bodies. The identification of benthic diatoms communities in the biofilm, coupled with quality indices such as the Indice de polluosensibilité spécifique (IPS) can be used for biomonitoring purposes in freshwater. However, the morphological identification and counting of diatoms species under the microscope is time-consuming and requires extensive expertise to deal with a constantly evolving taxonomy. In response, a molecular-based and potentially more cost-effective method has been developed, coupling high-throughput sequencing and DNA metabarcoding. The method has already been tested for water quality assessment with diatoms in Central Europe. In this study, we applied both the traditional and molecular methods on 180 biofilms samples from Northern Europe (rivers and lakes of Fennoscandia and Iceland). The DNA metabarcoding data were obtained on two different DNA markers, the 18S-V4 and rbcL barcodes, with the NucleoSpin Soil kit for DNA extraction and sequenced on an Ion Torrent PGM platform. We assessed the ability of the molecular method to produce species inventories, IPS scores and ecological status class comparable to the ones generated by the traditional morphology-based approach. The two methods generated correlated but significantly different IPS scores and ecological status assessment. The observed deviations are explained by presence/absence and abundance discrepancies in the species inventories, mainly due to the incompleteness of the barcodes reference databases, primer bias and strictness of the bioinformatic pipeline. Abundance discrepancies are less common than presence/absence discrepancies but have a greater effect on the ecological assessment. Missing species in the reference databases are mostly acidophilic benthic diatoms species, typical of the low pH waters of Northern Europe. The two different DNA markers also generated significantly different ecological status assessments. The use of the 18S-V4 marker generates more species inventories discrepancies, but achieves an ecological assessment more similar to the traditional morphology-based method. Further development of the metabarcoding method is needed for its use in environmental assessment. For its application in Northern Europe, completion and curation of reference databases are necessary, as well as evaluation of the currently available bioinformatics pipelines. New indices, fitted for environmental biomonitoring, should also be developed directly from molecular data. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2019 Research Article text/html info:doi:10.3897/mbmg.3.34002 https://doi.org/10.3897/mbmg.3.34002 https://mbmg.pensoft.net/article/34002/ https://mbmg.pensoft.net/article/34002/download/pdf/ en

10.3897/mbmg.3.36335 2019-07-10 mbmg

Spatial structure of fungal DNA assemblages revealed with eDNA metabarcoding in a forest river network in western Japan Matsuoka,Shunsuke Sugiyama,Yoriko Sato,Hirotoshi Katano,Izumi Harada,Ken Doi,Hideyuki diversity environmental DNA forest river fresh water fungi ITS metabarcoding spatial structure Metabarcoding and Metagenomics 3: e36335 Growing evidence has revealed high diversity and spatial heterogeneity of fungal communities in local habitats of terrestrial ecosystems. Recently, the analysis of environmental DNA has been undertaken to study the biodiversity of organisms, such as animals and plants, in both aquatic and terrestrial habitats. In the present study, we investigated fungal DNA assemblages and their spatial structure using environmental DNA metabarcoding targeting the internal transcribed spacer 1 (ITS1) region of the rRNA gene cluster in habitats across different branches of rivers in forest landscapes. A total of 1,956 operational taxonomic units (OTUs) were detected. Of these, 770 were assigned as Ascomycota, 177 as Basidiomycota, and 38 as Chytridiomycota. The river water was found to contain functionally diverse OTUs of both aquatic and terrestrial fungi, such as plant decomposers and mycorrhizal fungi. These fungal DNA assemblages were more similar within, rather than between, river branches. In addition, the assemblages were more similar between spatially closer branches. This spatial structuring was significantly associated with geographic distances but not with vegetation of the catchment area and the elevation at the sampling points. Our results imply that information on the terrestrial and aquatic fungal compositions of watersheds, and therefore their spatial structure, can be obtained by investigating the fungal DNA assemblages in river water. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2019 Research Article text/html info:doi:10.3897/mbmg.3.36335 https://doi.org/10.3897/mbmg.3.36335 https://mbmg.pensoft.net/article/36335/ https://mbmg.pensoft.net/article/36335/download/pdf/ en

10.3897/mbmg.3.34735 2019-07-11 mbmg

Advancing the use of molecular methods for routine freshwater macroinvertebrate biomonitoring – the need for calibration experiments Blackman,Rosetta Mächler,Elvira Altermatt,Florian Arnold,Amanda Beja,Pedro Boets,Pieter Egeter,Bastian Elbrecht,Vasco Filipe,Ana Filipa Jones,John Macher,Jan-Niklas Majaneva,Markus Martins,Filipa MS Múrria,Cesc Meissner,Kristian Pawlowski,Jan Schmidt Yáñez,Paul Zizka,Vera Leese,Florian Price,Ben Deiner,Kristy Bulk DNA community DNAqua-Net environmental DNA experimental methods fixative DNA monitoring Water Framework Directive Metabarcoding and Metagenomics 3: e34735 Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2019 Forum Paper text/html info:doi:10.3897/mbmg.3.34735 https://doi.org/10.3897/mbmg.3.34735 https://mbmg.pensoft.net/article/34735/ https://mbmg.pensoft.net/article/34735/download/pdf/ en

10.3897/mbmg.3.36236 2019-09-04 mbmg

Mumame: a software tool for quantifying gene-specific point-mutations in shotgun metagenomic data Magesh,Shruthi Jonsson,Viktor Bengtsson-Palme,Johan Antibiotic resistance bioinformatic tools metagenomics mutation frequencies mutation detection statistical methods Metabarcoding and Metagenomics 3: e36236 Metagenomics has emerged as a central technique for studying the structure and function of microbial communities. Often the functional analysis is restricted to classification into broad functional categories. However, important phenotypic differences, such as resistance to antibiotics, are often the result of just one or a few point mutations in otherwise identical sequences. Bioinformatic methods for metagenomic analysis have generally been poor at accounting for this fact, resulting in a somewhat limited picture of important aspects of microbial communities. Here, we address this problem by providing a software tool called Mumame, which can distinguish between wildtype and mutated sequences in shotgun metagenomic data and quantify their relative abundances. We demonstrate the utility of the tool by quantifying antibiotic resistance mutations in several publicly available metagenomic data sets. We also identified that sequencing depth is a key factor to detect rare mutations. Therefore, much larger numbers of sequences may be required for reliable detection of mutations than for most other applications of shotgun metagenomics. Mumame is freely available online (http://microbiology.se/software/mumame). info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2019 Software Description text/html info:doi:10.3897/mbmg.3.36236 https://doi.org/10.3897/mbmg.3.36236 https://mbmg.pensoft.net/article/36236/ https://mbmg.pensoft.net/article/36236/download/pdf/ en

10.3897/mbmg.3.35060 2019-09-05 mbmg

DNA metabarcoding of Neotropical ichthyoplankton: Enabling high accuracy with lower cost Batista Nobile,André Freitas-Souza,Diogo Ruiz-Ruano,Francisco Nobile,Maria Lígia da Costa,Gabriela Lima,Felipe Camacho,Juan Pedro M. Foresti,Fausto Oliveira,Claudio DNA Barcoding Fisheries management Ichthyofauna Upper Paraná River Metabarcoding and Metagenomics 3: e35060 Knowledge of ichthyoplankton dynamics is extremely important for conservation management as it can provide information about preferential spawning sites, reproductive period, migratory routes and recruitment success, which can be used to guide management and conservation efforts. However, identification of the eggs and larvae of Neotropical freshwater fish is a difficult task. DNA barcodes have emerged as an alternative and highly accurate approach for species identification, but DNA barcoding can be time-consuming and costly. To solve this problem, we aimed to develop a simple protocol based on DNA metabarcoding, to investigate whether it is possible to detect and quantify all species present in a pool of organisms. To do this, 230 larvae were cut in half, one half was sequenced by the Sanger technique and the other half was used to compose six arrays with a pool of larvae that were sequenced using a next-generation technique (NGS). The results of the Sanger sequencing allowed the identification of almost all larvae at species level, and the results from NGS showed high accuracy in species detection, ranging from 83% to 100%, with an average of 95% in all samples. No false positives were detected. The frequency of organisms in the two methods was positively correlated (Pearson), with low variation among species. In conclusion, this protocol represents a considerable advance in ichthyoplankton studies, allowing a rapid, cost-effective, quali-quantitative approach that improves the accuracy of identification. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2019 Research Article text/html info:doi:10.3897/mbmg.3.35060 https://doi.org/10.3897/mbmg.3.35060 https://mbmg.pensoft.net/article/35060/ https://mbmg.pensoft.net/article/35060/download/pdf/ en

10.3897/mbmg.3.36408 2019-11-06 mbmg

Metabarcoding of soil nematodes: the importance of taxonomic coverage and availability of reference sequences in choosing suitable marker(s) Ahmed,Mohammed Back,Matthew Prior,Thomas Karssen,Gerrit Lawson,Rebecca Adams,Ian Sapp,Melanie barcoding taxonomy DNA marker nuclear mitochondrial reference database primer Metabarcoding and Metagenomics 3: e36408 For many organisms, there is agreement on the specific genomic region used for developing barcode markers. With nematodes, however, it has been found that the COI region designated for most animals lacks the taxonomic coverage (ability to amplify a diverse group of taxa) required of a metabarcoding marker. For that reason, studies on metabarcoding of nematodes thus far have utilized primarily regions within the highly conserved 18S ribosomal DNA. Two popular markers within this region are the ones flanked by the primer pairs NF1-18Sr2b and SSUF04-SSUR22. The NF1-18Sr2b primer pair, especially, has been critiqued as not being specific enough for nematodes leading to suggestions for other candidate markers while the SSUF04-SSUR22 region has hardly been tested on soil nematodes. The current study aimed to evaluate these two markers against other alternative ones within the 28S rDNA and the COI region for their suitability for nematode metabarcoding. The results showed that the NF1-18Sr2b marker could offer wide coverage and good resolution for characterizing soil nematodes. Sufficient availability of reference sequences for this region was found to be a significant factor that resulted in this marker outperforming the other markers, particularly the 18S-based SSUFO4-SSUR22 marker. None of the other tested regions compared with this marker in terms of the proportion of the taxa recovered. The COI-based marker had the lowest number of taxa recovered, and this was due to the poor performance of its primers and the insufficient number of reference sequences in public databases. In summary, this study highlights how dependent the success of metabarcoding is on the availability of a good reference sequence collection for the marker of choice as well as its taxonomic coverage. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2019 Research Article text/html info:doi:10.3897/mbmg.3.36408 https://doi.org/10.3897/mbmg.3.36408 https://mbmg.pensoft.net/article/36408/ https://mbmg.pensoft.net/article/36408/download/pdf/ en

10.3897/mbmg.3.39646 2019-11-06 mbmg

Assessing pollution of aquatic environments with diatoms’ DNA metabarcoding: experience and developments from France Water Framework Directive networks Vasselon,Valentin Rimet,Frederic Domaizon,Isabelle Monnier,Olivier Reyjol,Yorick Bouchez,Agnès metabarcoding DNA High-Throughput Sequencing diatoms biomonitoring ecological status Water Framework Directive Metabarcoding and Metagenomics 3: e39646 Ecological status assessment of watercourses is based on the calculation of quality indices using pollution sensitivity of targeted biological groups, including diatoms. The determination and quantification of diatom species is generally based on microscopic morphological identification, which requires expertise and is time-consuming and costly. In Europe, this morphological approach is legally imposed by standards and regulatory decrees by the Water Framework Directive (WFD). Over the past decade, a DNA-based molecular biology approach has newly been developed to identify species based on genetic criteria rather than morphological ones (i.e. DNA metabarcoding). In combination with high throughput sequencing technologies, metabarcoding makes it possible both to identify all species present in an environmental sample and to process several hundred samples in parallel. This article presents the results of two recent studies carried out on the WFD networks of rivers of Mayotte (2013–2018) and metropolitan France (2016–2018). These studies aimed at testing the potential application of metabarcoding for biomonitoring in the context of the WFD. We discuss the various methodological developments and optimisations that have been made to make the taxonomic inventories of diatoms produced by metabarcoding more reliable, particularly in terms of species quantification. We present the results of the application of this DNA approach on more than 500 river sites, comparing them with those obtained using the standardised morphological method. Finally, we discuss the potential of metabarcoding for routine application, its limits of application and propose some recommendations for future implementation in WFD. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2019 Research Article text/html info:doi:10.3897/mbmg.3.39646 https://doi.org/10.3897/mbmg.3.39646 https://mbmg.pensoft.net/article/39646/ https://mbmg.pensoft.net/article/39646/download/pdf/ en

10.3897/mbmg.3.46704 2019-12-13 mbmg

Metabarcoding data allow for reliable biomass estimates in the most abundant animals on earth Schenk,Janina Geisen,Stefan Kleinboelting,Nils Traunspurger,Walter Nematodes quantification community analysis Illumina MiSeq species diversity Metabarcoding and Metagenomics 3: e46704 Microscopic organisms are the dominant and most diverse organisms on Earth. Nematodes, as part of this microscopic diversity, are by far the most abundant animals and their diversity is equally high. Molecular metabarcoding is often applied to study the diversity of microorganisms, but has yet to become the standard to determine nematode communities. As such, the information metabarcoding provides, such as in terms of species coverage, taxonomic resolution and especially if sequence reads can be linked to the abundance or biomass of nematodes in a sample, has yet to be determined. Here, we applied metabarcoding using three primer sets located within ribosomal rRNA gene regions to target assembled mock-communities consisting of 18 different nematode species that we established in 9 different compositions. We determined abundances and biomass of all species added to examine if relative sequence abundance or biomass can be linked to relative sequence reads. We found that nematode communities are not equally represented by the three different primer sets and we found that relative read abundances almost perfectly correlated positively with relative species biomass for two of the primer sets. This strong biomass-read number correlation suggests that metabarcoding reads can reveal biomass information even amongst more complex nematode communities as present in the environment and possibly can be transferred to better study other groups of organisms. This biomass-read link is of particular importance for more reliably assessing nutrient flow through food-webs, as well as adjusting biogeochemical models through user-friendly and easily obtainable metabarcoding data. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2019 Research Article text/html info:doi:10.3897/mbmg.3.46704 https://doi.org/10.3897/mbmg.3.46704 https://mbmg.pensoft.net/article/46704/ https://mbmg.pensoft.net/article/46704/download/pdf/ en

10.3897/mbmg.3.39041 2019-12-18 mbmg

Adapting the (fast-moving) world of molecular ecology to the (slow-moving) world of environmental regulation: lessons from the UK diatom metabarcoding exercise Kelly,Martyn diatoms implementation metabarcoding phytobenthos Water Framework Directive Metabarcoding and Metagenomics 3: e39041 Development of effective metabarcoding-based tools for ecological assessment requires more than just detailed knowledge of ecology and molecular genetics. There is also a need to understand the context within which they will be used, and for the organisation that uses it to understand the techniques involved and, more especially, how the data that are produced differs from that generated by traditional ecological methods. Lessons learnt during the development of a metabarcoding tool for phytobenthos in the UK are set out in this paper. This attempted to develop a molecular “mirror” of the existing light microscopy-based approach to ecological assessment. Although this conservative approach does not exploit the full potential of metabarcoding data, it does mean that benchmarks exist against which performance and data can be judged. However, the pace of developments within molecular ecology means that regulators will need to find ways of incorporating new scientific insights whilst, at the same time, ensuring a stable regulatory regime. Installation of a metabarcoding technique within a regulatory organisation, in other words, is more than a transaction in which one approach is switched for another. A deeper transformation of the organisation is required. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2019 Forum Paper text/html info:doi:10.3897/mbmg.3.39041 https://doi.org/10.3897/mbmg.3.39041 https://mbmg.pensoft.net/article/39041/ https://mbmg.pensoft.net/article/39041/download/pdf/ en

10.3897/mbmg.4.48281 2020-02-07 mbmg

Estimation of the relative abundance of species in artificial mixtures of insects using low-coverage shotgun metagenomics Garrido Sanz,Lidia Senar Rosell,Miquel Àngel Piñol,Josep Eukaryotes Metazoa genome skimming PCR-free mock sample sequenced genomes Metabarcoding and Metagenomics 4: e48281 Amplicon metabarcoding is an established technique to analyse the taxonomic composition of communities of organisms using high-throughput DNA sequencing, but there are doubts about its ability to quantify the relative proportions of the species, as opposed to the species list. Here, we bypass the enrichment step and avoid the PCR-bias, by directly sequencing the extracted DNA using shotgun metagenomics. This approach is common practice in prokaryotes, but not in eukaryotes, because of the low number of sequenced genomes of eukaryotic species. We tested the metagenomics approach using insect species whose genome is already sequenced and assembled to an advanced degree. We shotgun-sequenced, at low-coverage, 18 species of insects in 22 single-species and 6 mixed-species libraries and mapped the reads against 110 reference genomes of insects. We used the single-species libraries to calibrate the process of assignation of reads to species and the libraries created from species mixtures to evaluate the ability of the method to quantify the relative species abundance. Our results showed that the shotgun metagenomic method is easily able to set apart closely-related insect species, like four species of Drosophila included in the artificial libraries. However, to avoid the counting of rare misclassified reads in samples, it was necessary to use a rather stringent detection limit of 0.001, so species with a lower relative abundance are ignored. We also identified that approximately half the raw reads were informative for taxonomic purposes. Finally, using the mixed-species libraries, we showed that it was feasible to quantify with confidence the relative abundance of individual species in the mixtures. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Research Article text/html info:doi:10.3897/mbmg.4.48281 https://doi.org/10.3897/mbmg.4.48281 https://mbmg.pensoft.net/article/48281/ https://mbmg.pensoft.net/article/48281/download/pdf/ en

10.3897/mbmg.4.53535 2020-06-03 mbmg

BOLDigger – a Python package to identify and organise sequences with the Barcode of Life Data systems Buchner,Dominik Leese,Florian metabarcoding species identification BOLD OTUs taxonomic assignment database Metabarcoding and Metagenomics 4: e53535 DNA metabarcoding workflows produce hundreds to ten-thousands of Operational Taxonomic Units (OTUs) or Exact Sequence Variants (ESVs) per analysis. In most workflows, a taxonomic assignment to these generated sequences is needed. This is typically done using publicly available databases. Especially, yet not exclusively, for Eumetazoan metabarcoding, the Barcode of Life Data system (BOLD) is the most comprehensive and curated reference barcode database and, therefore, typically the first choice for taxonomic assignment. While an application programme interface (API) exists to query data in large batches, no information on the many and important unpublished data are obtained through the API. The alternative approach using the BOLD identification engine on the website provides full access, yet it is restricted to 100 sequences at once. We developed a small platform-independent and graphical user interface (GUI) software package, BOLDigger, which aims to solve this problem by automating the process of sending successive requests of up to 100 sequences without surpassing the capacities of BOLD. BOLDigger can be used to download the results of the identification engine, as well as metadata for the obtained hits. For the selection of the best fitting hit, three different methods are implemented. A new approach, combining a threshold-based approach with the metadata information, was implemented to make use of the metadata. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Software Description text/html info:doi:10.3897/mbmg.4.53535 https://doi.org/10.3897/mbmg.4.53535 https://mbmg.pensoft.net/article/53535/ https://mbmg.pensoft.net/article/53535/download/pdf/ en

10.3897/mbmg.4.51925 2020-07-24 mbmg

Can metabarcoding resolve intraspecific genetic diversity changes to environmental stressors? A test case using river macrozoobenthos Zizka,Vera Weiss,Martina Leese,Florian denoising environmental impact genetic erosion haplotypes pollution Metabarcoding and Metagenomics 4: e51925 Genetic diversity is the most basal level of biodiversity and determines the evolutionary capacity of species to adapt to changing environments, yet it is typically neglected in routine biomonitoring and stressor impact assessment. For a comprehensive analysis of stressor impacts on genetic diversity, it is necessary to assess genetic variants simultaneously in many individuals and species. Such an assessment is not as straightforward and usually limited to one or few focal species. However, nowadays species diversity can be assessed by analysing thousands of individuals of a community simultaneously with DNA metabarcoding. Recent bioinformatic advances also allow for the extraction of exact sequence variants (ESVs or haplotypes) in addition to Operational Taxonomic Units (OTUs). By using this new capability, we here evaluated if the analysis of intraspecific mitochondrial diversity in addition to species diversity can provide insights into responses of stream macrozoobenthic communities to environmental stressors. For this purpose, we analysed macroinvertebrate bulk samples of three German river systems with different stressor levels using DNA metabarcoding. While OTU and haplotype number were negatively correlated with stressor impact, this association was not as clear when studying haplotype diversity across all taxa. However, stressor responses were found for sensitive EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa and those exceedingly resistant to organic stress. An increase in haplotype number per OTU and haplotype diversity of sensitive taxa was observed with an increase in ecosystem quality and stability, while the opposite pattern was detected for pollution resistant taxa. However, this pattern was less prominent than expected based on the strong differences in stressor intensity between sites. To compare genetic diversity among communities in river systems, we focussed on OTUs, which were present in all systems. As OTU composition differed strongly between rivers, this led to the exclusion of a high number of OTUs, especially in diverse river systems of good quality, which potentially diminished the increase in intraspecific diversity. To better understand responses of intraspecific genetic diversity to environmental stressors, for example in river ecosystems, it would be important to increase OTU overlap between compared sites, e.g. by sampling a narrower stressor gradient, and to perform calibrated studies controlling for the number of individuals and their haplotypes. However, this pioneer study shows that the extraction of haplotypes from DNA metabarcoding datasets is a promising source of information to simultaneously assess intraspecific diversity changes in response to environmental impacts for a metacommunity. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Research Article text/html info:doi:10.3897/mbmg.4.51925 https://doi.org/10.3897/mbmg.4.51925 https://mbmg.pensoft.net/article/51925/ https://mbmg.pensoft.net/article/51925/download/pdf/ en

10.3897/mbmg.4.55162 2020-08-12 mbmg

Assessment of species gaps in DNA barcode libraries of non-indigenous species (NIS) occurring in European coastal regions Duarte,Sofia Vieira,Pedro E. Costa,Filipe BOLD DNA barcode markers gap-analysis GenBank marine and coastal ecosystems non-indigenous species Metabarcoding and Metagenomics 4: e55162 DNA metabarcoding has the capacity to bolster current biodiversity assessment techniques, including the early detection and monitoring of non-indigenous species (NIS). However, the success of this approach is greatly dependent on the availability, taxonomic coverage and reliability of reference sequences in genetic databases, whose deficiencies can potentially compromise species identifications at the taxonomic assignment step. In this study we assessed lacunae in availability of DNA sequence data from four barcodes (COI, 18S, rbcL and matK) for NIS occurring in European marine and coastal environments. NIS checklists were based on EASIN and AquaNIS databases. The highest coverage was found for COI for Animalia and rbcL for Plantae (up to 63%, for both) and 18S for Chromista (up to 51%), that greatly increased when only high impact species were taken into account (up to 82 to 89%). Results show that different markers have unbalanced representations in genetic databases, implying that the parallel use of more than one marker can act complimentarily and may greatly increase NIS identification rates through DNA-based tools. Furthermore, based on the COI marker, data for approximately 30% of the species had maximum intra-specific distances higher than 3%, suggesting that many NIS may have undescribed or cryptic diversity. Although completing the gaps in reference libraries is essential to make the most of the potential of the DNA-based tools, a careful compilation, verification and annotation of available sequences is fundamental to assemble large curated and reliable reference libraries that provide support for rigorous species identifications. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Research Article text/html info:doi:10.3897/mbmg.4.55162 https://doi.org/10.3897/mbmg.4.55162 https://mbmg.pensoft.net/article/55162/ https://mbmg.pensoft.net/article/55162/download/pdf/ en

10.3897/mbmg.4.53455 2020-08-26 mbmg

Increasing confidence for discerning species and population compositions from metabarcoding assays of environmental samples: case studies of fishes in the Laurentian Great Lakes and Wabash River Snyder,Matthew Stepien,Carol community composition cytochrome b eDNA Great Lakes population variation species detection species diversity 12S RNA Metabarcoding and Metagenomics 4: e53455 Community composition data are essential for conservation management, facilitating identification of rare native and invasive species, along with abundant ones. However, traditional capture-based morphological surveys require considerable taxonomic expertise, are time consuming and expensive, can kill rare taxa and damage habitats, and often are prone to false negatives. Alternatively, metabarcoding assays can be used to assess the genetic identity and compositions of entire communities from environmental samples, comprising a more sensitive, less damaging, and relatively time- and cost-efficient approach. However, there is a trade-off between the stringency of bioinformatic filtering needed to remove false positives and the potential for false negatives. The present investigation thus evaluated use of four mitochondrial (mt) DNA metabarcoding assays and a customized bioinformatic Bioinformatic pipeline to increase confidence in species identifications by removing false positives, while achieving high detection probability. Positive controls were used to calculate sequencing error, and results that fell below those cutoff values were removed, unless found with multiple assays. The performance of this approach was tested to discern and identify North American freshwater fishes using lab experiments (mock communities and aquarium experiments) and processing of a bulk ichthyoplankton sample. The method then was applied to field environmental (e) DNA water samples taken concomitant with electrofishing surveys and morphological identifications. This protocol detected 100% of species present in concomitant electrofishing surveys in the Wabash River and an additional 21 that were absent from traditional sampling. Using single 1 L water samples collected from just four locations, the metabarcoding assays discerned 73% of the total fish species that were discerned during four months of an extensive electrofishing river survey in the Maumee River, along with an additional nine species. In both rivers, total fish species diversity was best resolved when all four metabarcoding assays were used together, which identified 35 additional species missed by electrofishing. Ecological distinction and diversity levels among the fish communities also were better resolved with the metabarcoding assays than with morphological sampling and identifications, especially using all four assays together. At the population-level, metabarcoding analyses targeting the invasive round goby Neogobius melanostomus and the silver carp Hypophthalmichthys molitrix identified all population haplotype variants found using Sanger sequencing of morphologically sampled fish, along with additional intra-specific diversity, meriting further investigation. Overall findings demonstrated that the use of multiple metabarcoding assays and custom bioinformatics that filter potential error from true positive detections improves confidence in evaluating biodiversity. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC0 Pensoft Publishers 2020 Research Article text/html info:doi:10.3897/mbmg.4.53455 https://doi.org/10.3897/mbmg.4.53455 https://mbmg.pensoft.net/article/53455/ https://mbmg.pensoft.net/article/53455/download/pdf/ en

10.3897/mbmg.4.51433 2020-08-28 mbmg

Exploring the potential of metabarcoding to disentangle macroinvertebrate community dynamics in intermittent streams Laini,Alex Beermann,Arne Bolpagni,Rossano Burgazzi,Gemma Elbrecht,Vasco Zizka,Vera Leese,Florian Viaroli,Pierluigi beta diversity biodiversity exact sequence variants IRES nestedness taxonomic sufficiency turnover Metabarcoding and Metagenomics 4: e51433 Taxonomic sufficiency represents the level of taxonomic detail needed to detect ecological patterns to a level that match the requirement of a study. Most bioassessments apply the taxonomic sufficiency concept and assign specimens to the family or genus level given time constraints and the difficulty to correctly identify species. This holds particularly true for stream invertebrates because small and morphologically similar larvae are hard to distinguish. Low taxonomic resolution may hinder detecting true community dynamics, which thus leads to incorrect inferences about community assembly processes. DNA metabarcoding is a new, affordable and cost-effective tool for the identification of multiple species from bulk samples of organisms. As it provides high taxonomic resolution, it can be used to compare results obtained from different identification levels. Measuring the effect of taxonomic resolution on the detection of community dynamics is especially interesting in extreme ecosystems like intermittent streams to test if species at intermittent sites are subsets of those from perennial sources or if independently recruiting taxa exist. Here we aimed to compare the performance of morphological identification and metabarcoding to detect macroinvertebrate community dynamics in the Trebbia River (Italy). Macroinvertebrates were collected from four perennial and two intermittent sites two months after flow resumption and before the next dry phase. The identification level ranged from family to haplotype. Metabarcoding and morphological identifications found similar alpha diversity patterns when looking at family and mixed taxonomic levels. Increasing taxonomic resolution with metabarcoding revealed a strong partitioning of beta diversity in nestedness and turnover components. At flow resumption, beta diversity at intermittent sites was dominated by nestedness when family-level information was employed, while turnover was evidenced as the most important component when using Operational Taxonomic Units (OTUs) or haplotypes. The increased taxonomic resolution with metabarcoding allowed us to detect species adapted to deal with intermittency, like the chironomid Cricotopus bicinctus and the ephemeropteran Cloeon dipterum. Our study thus shows that family and mixed taxonomic level are not sufficient to detect all aspects of macroinvertebrate community dynamics. High taxonomic resolution is especially important for intermittent streams where accurate information about species-specific habitat preference is needed to interpret diversity patterns induced by drying and the nestedness/turnover components of beta diversity are of interest to understand community assembly processes. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Research Article text/html info:doi:10.3897/mbmg.4.51433 https://doi.org/10.3897/mbmg.4.51433 https://mbmg.pensoft.net/article/51433/ https://mbmg.pensoft.net/article/51433/download/pdf/ en

10.3897/mbmg.4.55815 2020-09-10 mbmg

Alignment-free classification of COI DNA barcode data with the Python package Alfie Nugent,Cameron Adamowicz,Sarah COI DNA barcoding eDNA environmental DNA machine learning metabarcoding neural network Metabarcoding and Metagenomics 4: e55815 Characterization of biodiversity from environmental DNA samples and bulk metabarcoding data is hampered by off-target sequences that can confound conclusions about a taxonomic group of interest. Existing methods for isolation of target sequences rely on alignment to existing reference barcodes, but this can bias results against novel genetic variants. Effectively parsing targeted DNA barcode data from off-target noise improves the quality of biodiversity estimates and biological conclusions by limiting subsequent analyses to a relevant subset of available data. Here, we present Alfie, a Python package for the alignment-free classification of cytochrome c oxidase subunit I (COI) DNA barcode sequences to taxonomic kingdoms. The package determines k-mer frequencies of DNA sequences, and the frequencies serve as input for a neural network classifier that was trained and tested using ~58,000 publicly available COI sequences. The classifier was designed and optimized through a series of tests that allowed for the optimal set of DNA k-mer features and optimal machine learning algorithm to be selected. The neural network classifier rapidly assigns COI sequences of varying lengths to kingdoms with greater than 99% accuracy and is shown to generalize effectively and make accurate predictions about data from previously unseen taxonomic classes. The package contains an application programming interface that allows the Alfie package’s functionality to be extended to different DNA sequence classification tasks to suit a user’s need, including classification of different genes and barcodes, and classification to different taxonomic levels. Alfie is free and publicly available through GitHub (https://github.com/CNuge/alfie) and the Python package index (https://pypi.org/project/alfie/). info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Software Description text/html info:doi:10.3897/mbmg.4.55815 https://doi.org/10.3897/mbmg.4.55815 https://mbmg.pensoft.net/article/55815/ https://mbmg.pensoft.net/article/55815/download/pdf/ en

10.3897/mbmg.4.58259 2020-09-21 mbmg

The mitochondrial genomes of 11 aquatic macroinvertebrate species from Cyprus Macher,Jan-Niklas Drakou,Katerina Papatheodoulou,Athina van der Hoorn,Berry Vasquez,Marlen Ines freshwater biodiversity Mediterranean insects Biodiversity Hotspot Metabarcoding and Metagenomics 4: e58259 Aquatic macroinvertebrates are often identified, based on morphology, but molecular approaches like DNA barcoding, metabarcoding and metagenomics are increasingly used for species identification. These approaches require the availability of DNA references deposited in public databases. Here we report the mitochondrial genomes of 11 aquatic macroinvertebrate species from Cyprus, a European Union island country in the Mediterranean. Only three species could be provisionally assigned to a binomial species name, highlighting the current lack of molecular references for aquatic macroinvertebrates from Cyprus. Graphical Abstract info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Data Paper text/html info:doi:10.3897/mbmg.4.58259 https://doi.org/10.3897/mbmg.4.58259 https://mbmg.pensoft.net/article/58259/ https://mbmg.pensoft.net/article/58259/download/pdf/ en

10.3897/mbmg.4.56959 2020-10-22 mbmg

Read counts from environmental DNA (eDNA) metabarcoding reflect fish abundance and biomass in drained ponds Di Muri,Cristina Lawson-Handley,Lori Bean,Colin Li,Jianlong Peirson,Graeme Sellers,Graham S. Walsh,Kerry Watson,Hayley Winfield,Ian Hänfling,Bernd biodiversity eDNA monitoring freshwater lake lentic Metabarcoding and Metagenomics 4: e56959 The sampling of environmental DNA (eDNA) coupled with cost-efficient and ever-advancing sequencing technology is propelling changes in biodiversity monitoring within aquatic ecosystems. Despite the increasing number of eDNA metabarcoding approaches, the ability to quantify species biomass and abundance in natural systems is still not fully understood. Previous studies have shown positive but sometimes weak correlations between abundance estimates from eDNA metabarcoding data and from conventional capture methods. As both methods have independent biases a lack of concordance is difficult to interpret. Here we tested whether read counts from eDNA metabarcoding provide accurate quantitative estimates of the absolute abundance of fish in holding ponds with known fish biomass and number of individuals. Environmental DNA samples were collected from two fishery ponds with high fish density and broad species diversity. In one pond, two different DNA capture strategies (on-site filtration with enclosed filters and three different preservation buffers versus lab filtration using open filters) were used to evaluate their performance in relation to fish community composition and biomass/abundance estimates. Fish species read counts were significantly correlated with both biomass and abundance, and this result, together with information on fish diversity, was repeatable when open or enclosed filters with different preservation buffers were used. This research demonstrates that eDNA metabarcoding provides accurate qualitative and quantitative information on fish communities in small ponds, and results are consistent between different methods of DNA capture. This method flexibility will be beneficial for future eDNA-based fish monitoring and their integration into fisheries management. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Research Article text/html info:doi:10.3897/mbmg.4.56959 https://doi.org/10.3897/mbmg.4.56959 https://mbmg.pensoft.net/article/56959/ https://mbmg.pensoft.net/article/56959/download/pdf/ en

10.3897/mbmg.4.56087 2020-11-26 mbmg

Using DNA metabarcoding to investigate diet and niche partitioning in the native European otter (Lutra lutra) and invasive American mink (Neovison vison) Harper,Lynsey Watson,Hayley Donnelly,Robert Hampshire,Richard Sayer,Carl Breithaupt,Thomas Hänfling,Bernd carnivore faecal DNA high-throughput sequencing mustelid predator-prey interactions scats spraints trophic ecology Metabarcoding and Metagenomics 4: e56087 In the UK, the native European otter (Lutra lutra) and invasive American mink (Neovison vison) have experienced concurrent declines and expansions. Currently, the otter is recovering from persecution and waterway pollution, whereas the mink is in decline due to population control and probable interspecific interaction with the otter. We explored the potential of DNA metabarcoding for investigating diet and niche partitioning between these mustelids. Otter spraints (n = 171) and mink scats (n = 19) collected from three sites (Malham Tarn, River Hull and River Glaven) in northern and eastern England were screened for vertebrates using high-throughput sequencing. Otter diet mainly comprised aquatic fishes (81.0%) and amphibians (12.7%), whereas mink diet predominantly consisted of terrestrial birds (55.9%) and mammals (39.6%). The mink used a lower proportion (20%) of available prey (n = 40 taxa) than the otter and low niche overlap (0.267) was observed between these mustelids. Prey taxon richness of mink scats was lower than otter spraints and beta diversity of prey communities was driven by taxon turnover (i.e. the otter and mink consumed different prey taxa). Considering otter diet only, prey taxon richness was higher in spraints from the River Hull catchment and beta diversity of prey communities was driven by taxon turnover (i.e. the otter consumed different prey taxa at each site). Studies using morphological faecal analysis may misidentify the predator as well as prey items. Faecal DNA metabarcoding can resolve these issues and provide more accurate and detailed dietary information. When scaled up across multiple habitat types, DNA metabarcoding should greatly improve future understanding of resource use and niche overlap between the otter and mink. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Research Article text/html info:doi:10.3897/mbmg.4.56087 https://doi.org/10.3897/mbmg.4.56087 https://mbmg.pensoft.net/article/56087/ https://mbmg.pensoft.net/article/56087/download/pdf/ en

10.3897/mbmg.4.58365 2020-11-26 mbmg

Soil stabilisation for DNA metabarcoding of plants and fungi. Implications for sampling at remote locations or via third-parties Avila Clasen,Lina Detheridge,Andrew Scullion,John Griffith,Gareth chitinolytic fungi freeze-drying freeze-thaw sample preservation Metabarcoding and Metagenomics 4: e58365 Storage of soil samples prior to metagenomic analysis presents a problem. If field sites are remote or if samples are collected by third parties, transport to analytical laboratories may take several days or even weeks. The bulk of such samples and requirement for later homogenisation precludes the convenient use of a stabilisation buffer, so samples are usually cooled or frozen during transit. There has been limited testing of the most appropriate storage methods for later study of soil organisms by eDNA approaches. Here we tested a range of storage methods on two contrasting soils, comparing these methods to the control of freezing at -80 °C, followed by freeze-drying. To our knowledge, this is the first study to examine the effect of storage conditions on eukaryote DNA in soil, including both viable organisms (fungi) and DNA contained within dying/dead tissues (plants). For fungi, the best storage regimes (closest to the control) were storage at 4 °C (for up to 14 d) or active air-drying at room temperature. The worst treatments involved initial freezing, followed by thawing which led to significant later spoilage. The key spoilage organisms were identified as Metarhizium carneum and Mortierella spp., with a general increase in saprotrophic fungi and reduced abundances of mycorrhizal/biotrophic fungi. Plant data showed a similar pattern, but with greater variability in community structure, especially in the freeze-thaw treatments, probably due to stochastic variation in substrates for fungal decomposition, algal proliferation and some seed germination. In the absence of freeze drying facilities, samples should be shipped refrigerated, but not frozen if there is any risk of thawing. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Methods text/html info:doi:10.3897/mbmg.4.58365 https://doi.org/10.3897/mbmg.4.58365 https://mbmg.pensoft.net/article/58365/ https://mbmg.pensoft.net/article/58365/download/pdf/ en

10.3897/mbmg.4.60854 2020-12-11 mbmg

Corrigendum: Using DNA metabarcoding for assessing chironomid diversity and community change in mosquito controlled temporary wetlands. MBMG 2: e21060. https://doi.org/10.3897/mbmg.2.21060 Theissinger,Kathrin Kästel,Anna Elbrecht,Vasco Makkonen,Jenny Michiels,Susanne Schmidt,Susanne Isabel Allgeier,Stefanie Leese,Florian Brühl,Carsten Bacillus thuringensis var. israelensis (Bti) Barcode of Life Datasystems (BOLD) chironomid saprobic index cytochrome oxidase subunit I (COI) ecotoxicology macrozoobenthos emergence operational taxonomic units (OTU) Metabarcoding and Metagenomics 4: e60854 Chironomids have been proposed as important indicators for monitoring freshwater ecosystems, however, morphological determination is very challenging. In this study, we investigated the effectiveness of metabarcoding for chironomid diversity assessment and tested the retrieved chironomid operational taxonomic units (OTUs) for possible changes in relative abundance and species diversity in relation to mosquito control actions in four temporary wetlands. The biocide Bacillus thuringiensis var. israelensis (Bti) is widely applied for mosquito control in temporary wetlands of the German Upper Rhine Valley. Even though Bti is considered environmentally friendly, several studies have shown non-target effects on chironomids, a key food resource in wetland ecosystems Three of the studied wetlands were, for the first year after 20 years of Bti treatment, partly left Bti-untreated in a split field design, and one wetland has never been treated with Bti. Our metabarcoding approach detected 54 chironomid OTUs across all study sites, of which 70% could be identified to species level comparisons against the BOLD database. We showed that metabarcoding increased chironomid species determination by 70%. However, we found only minor significant effects of Bti on the chironomid community composition. Subsequent studies will have to address if and how the chironomid community composition might change in the now Bti-untreated temporary wetlands to assess effects of Bti. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Corrigendum text/html info:doi:10.3897/mbmg.4.60854 https://doi.org/10.3897/mbmg.4.60854 https://mbmg.pensoft.net/article/60854/ https://mbmg.pensoft.net/article/60854/download/pdf/ en

10.3897/mbmg.4.58572 2020-12-31 mbmg

Methodological considerations for monitoring soil/litter arthropods in tropical rainforests using DNA metabarcoding, with a special emphasis on ants, springtails and termites Basset,Yves Donoso,David Hajibabaei,Mehrdad Wright,Michael T.G. Perez,Kate Lamarre,Greg De León,Luis F. Palacios-Vargas,José G. Castaño-Meneses,Gabriela Rivera,Marleny Perez,Filonila Bobadilla,Ricardo Lopez,Yacksecari Ramirez,José Alejandro Barrios,Héctor Barro Colorado Island Berlese-Tullgren Collembola Formicidae Isoptera light trap Malaise trap Metabarcoding and Metagenomics 4: e58572 Robust data to refute or support claims of global insect decline are currently lacking, particularly for the soil fauna in the tropics. DNA metabarcoding represents a powerful approach for rigorous spatial and temporal monitoring of the taxonomically challenging soil fauna. Here, we provide a detailed field protocol, which was successfully applied in Barro Colorado Island (BCI) in Panama, to collect soil samples and arthropods in a tropical rainforest, to be later processed with metabarcoding. We also estimate the proportion of soil/litter ant, springtail and termite species from the local fauna that can be detected by metabarcoding samples obtained either from Berlese-Tullgren (soil samples), Malaise or light traps. Each collecting method detected a rather distinct fauna. Soil and Malaise trap samples detected 213 species (73%) of all target species. Malaise trap samples detected many ant species, whereas soil samples were more efficient at detecting springtail and termite species. With respect to long-term monitoring of soil-dwelling and common species (more amenable to statistical trends), the best combination of two methods were soil and light trap samples, detecting 94% of the total of common species. A protocol including 100 soil, 40 Malaise and 80 light trap samples annually processed by metabarcoding would allow the long-term monitoring of at least 11%, 18% and 16% of species of soil/litter ants, springtails and termites, respectively, present on BCI, and a high proportion of the total abundance (up to 80% of all individuals) represented by these taxa. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2020 Monitoring Schema text/html info:doi:10.3897/mbmg.4.58572 https://doi.org/10.3897/mbmg.4.58572 https://mbmg.pensoft.net/article/58572/ https://mbmg.pensoft.net/article/58572/download/pdf/ en

10.3897/mbmg.5.57278 2021-01-04 mbmg

Application of propylene glycol in DNA-based studies of invertebrates Weigand,Alexander Desquiotz,Noah Weigand,Hannah Szucsich,Nikolaus alternative fixative bioassessment DNA integrity DNA preservation environmental monitoring Metabarcoding and Metagenomics 5: e57278 High-throughput sequencing (HTS) studies on invertebrates commonly use ethanol as the main sample fixative (upon collection) and preservative (for storage and curation). However, alternative agents exists, which should not be automatically neglected when studies are newly designed. This review provides an overview of the application of propylene glycol (PG) in DNA-based studies of invertebrates, thus to stimulate an evidence-based discussion. The use of PG in DNA-based studies of invertebrates is still limited (n = 79), but a steady increase has been visible since 2011. Most studies used PG as a fixative for passive trapping (73%) and performed Sanger sequencing (66%; e.g. DNA barcoding). More recently, HTS setups joined the field (11%). Terrestrial Coleoptera (30%) and Diptera (20%) were the most studied groups. Very often, information on the grade of PG used (75%) or storage conditions (duration, temperature) were lacking. This rendered direct comparisons of study results difficult, and highlight the need for further systematic studies on these subjects. When compared to absolute ethanol, PG can be more widely and cheaply acquired (e.g. as an antifreeze, 13% of studies). It also enables longer trapping intervals, being especially relevant at remote or hard-to-reach places. Shipping of PG-conserved samples is regarded as risk-free and is authorised, pinpointing its potential for larger trapping programs or citizen science projects. Its property to retain flexibility of morphological characters as well as to lead to a reduced shrinkage effect was especially appraised by integrative study designs. Finally, the so far limited application of PG in the context of HTS showed promising results for short read amplicon sequencing and reduced representation methods. Knowledge of the influence of PG fixation and storage for long(er) read HTS setups is currently unavailable. Given our review results and taking difficulties of direct methodological comparisons into account, future DNA-based studies of invertebrates should on a case-by-case basis critically scrutinise if the application of PG in their anticipated study design can be of benefit. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Review Article text/html info:doi:10.3897/mbmg.5.57278 https://doi.org/10.3897/mbmg.5.57278 https://mbmg.pensoft.net/article/57278/ https://mbmg.pensoft.net/article/57278/download/pdf/ en

10.3897/mbmg.5.58056 2021-02-26 mbmg

Metadata standards and practical guidelines for specimen and DNA curation when building barcode reference libraries for aquatic life Rimet,Frederic Aylagas,Eva Borja,Ángel Bouchez,Agnès Canino,Alexis Chauvin,Christian Chonova,Teofana Čiampor Jr,Fedor Costa,Filipe Ferrari,Benoit Gastineau,Romain Goulon,Chloé Gugger,Muriel Holzman,Maria Jahn,Regine Kahlert,Maria Kusber,Wolf-Henning Laplace-Treyture,Christophe Leese,Florian Leliaert,Frederik Mann,David G. Marchand,Frédéric Méléder,Vona Pawlowski,Jan Rasconi,Serena Rivera,Sinziana Rougerie,Rodolphe Schweizer,Magali Trobajo,Rosa Vasselon,Valentin Vivien,Régis Weigand,Alexander Witkowski,Andrzej Zimmermann,Jonas Ekrem,Torbjørn aquatic organisms barcode DNA metadata quality reference library traceability Metabarcoding and Metagenomics 5: e58056 DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Methods text/html info:doi:10.3897/mbmg.5.58056 https://doi.org/10.3897/mbmg.5.58056 https://mbmg.pensoft.net/article/58056/ https://mbmg.pensoft.net/article/58056/download/pdf/ en

10.3897/mbmg.5.62823 2021-04-09 mbmg

Metabarcoding of environmental samples suggest wide distribution of eelgrass (Zostera marina) pathogens in the north Pacific Menning,Damian Gravley,Hunter Cady,Melissa Pepin,Daniel Wyllie-Echeverria,Sandy Ward,David Talbot,Sandra Alaska eDNA disease Japan seagrass Metabarcoding and Metagenomics 5: e62823 Seagrass meadows provide important ecological services to the marine environment but are declining worldwide. Although eelgrass meadows in the north Pacific are thought to be relatively healthy, few studies have assessed the presence of known disease pathogens in these meadows. In a pilot study to test the efficacy of the methods and to provide foundational disease biodiversity data in the north Pacific, we leveraged metabarcoding of environmental DNA extracted from water, sediment, and eelgrass tissue samples collected from five widely distributed eelgrass meadows in Alaska and one in Japan and uncovered wide prevalence of two classes of pathogenic organisms – Labyrinthula zosterae and other associated strains of Labyrinthula, and the Phytophthora/Halophytophthora blight species complex – known to have caused decline in eelgrass (Zostera marina) elsewhere in the species’ global distribution. Although the distribution of these disease organisms is not well understood in the north Pacific, we uncovered the presence of at least one eelgrass pathogen at every locality sampled. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC0 Pensoft Publishers 2021 Research Article text/html info:doi:10.3897/mbmg.5.62823 https://doi.org/10.3897/mbmg.5.62823 https://mbmg.pensoft.net/article/62823/ https://mbmg.pensoft.net/article/62823/download/pdf/ en

10.3897/mbmg.5.59201 2021-04-09 mbmg

Effects of Malaise trap spacing on species richness and composition of terrestrial arthropod bulk samples Steinke,Dirk Braukmann,Thomas Manerus,Laura Woodhouse,Allan Elbrecht,Vasco Biodiversity biomonitoring experimental design insects metabarcoding Metabarcoding and Metagenomics 5: e59201 The Malaise trap is a popular device for assessing diverse terrestrial arthropod communities because it collects large samples with modest effort. A number of factors influence its collection efficiency, placement being one of them. For instance, when designing larger biotic surveys using arrays of Malaise traps we need to know the optimal distance between individual traps that maximises observable species richness and community composition. We examined the influence of spacing between Malaise traps by metabarcoding samples from two field experiments at a site in Waterloo, Ontario, Canada. For one experiment, we used two trap pairs deployed at weekly increasing distances (3 m increments from 3 to 30 m). The second experiment involved a total of 10 traps set up in a row at 3 m distance intervals for three consecutive weeks. Results show that community similarity of samples decreases over distance between traps. The amount of species shared between trap pairs drops considerably at about 18 m trap-to-trap distance. This change can be observed across all major taxonomic groups and for two different habitat types (grassland and forest). Large numbers of OTUs found only once within samples cause rather large dissimilarity between distance pairs even at close proximity. This could be caused by a large number of transient species from adjacent habitats which arrive at the trap through passive transport, as well as capture of rare taxa, which end up in different traps by chance. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Research Article text/html info:doi:10.3897/mbmg.5.59201 https://doi.org/10.3897/mbmg.5.59201 https://mbmg.pensoft.net/article/59201/ https://mbmg.pensoft.net/article/59201/download/pdf/ en

10.3897/mbmg.5.66503 2021-05-17 mbmg

Potential for cross-contamination of diatom DNA samples when using toothbrushes Kelly,Martyn Jones,Tim Walsh,Kerry diatoms metabarcoding ecological assessment Water Framework Directive Metabarcoding and Metagenomics 5: e66503 The use of toothbrushes and similar devices for sampling diatoms from hard surfaces is a well-established approach. Toothbrushes are routinely cleaned and reused when sampling for analysis by light microscopy. This paper looks at the scale of contamination encountered when this technique is used to sample diatoms for metabarcoding analyses, as well as at the scale of contamination to be expected if stream, rather than distilled water, is used to wash diatoms from stones. Although some contamination attributable to toothbrushes was detected, read numbers were low and had no effect on index calculation or ecological status estimates. However, if the primary focus of a study is to thoroughly document diversity in a sample, then even this small level of contamination may be unacceptable and more stringent measures may be required. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Methods text/html info:doi:10.3897/mbmg.5.66503 https://doi.org/10.3897/mbmg.5.66503 https://mbmg.pensoft.net/article/66503/ https://mbmg.pensoft.net/article/66503/download/pdf/ en

10.3897/mbmg.5.66557 2021-07-13 mbmg

Beyond fish eDNA metabarcoding: Field replicates disproportionately improve the detection of stream associated vertebrate species Macher,Till-Hendrik Schütz,Robin Arle,Jens Beermann,Arne Koschorreck,Jan Leese,Florian biomonitoring birds bycatch conservation environmental DNA mammals Metabarcoding and Metagenomics 5: e66557 Fast, reliable, and comprehensive biodiversity monitoring data are needed for environmental decision making and management. Recent work on fish environmental DNA (eDNA) metabarcoding shows that aquatic diversity can be captured fast, reliably, and non-invasively at moderate costs. Because water in a catchment flows to the lowest point in the landscape, often a stream, it can collect traces of terrestrial species via surface or subsurface runoff along its way or when specimens come into direct contact with water (e.g., when drinking). Thus, fish eDNA metabarcoding data can provide information on fish but also on other vertebrate species that live in riparian habitats. This additional data may offer a much more comprehensive approach for assessing vertebrate diversity at no additional costs. Studies on how the sampling strategy affects species detection especially of stream-associated communities, however, are scarce. We therefore performed an analysis on the effects of biological replication on both fish as well as (semi-)terrestrial species detection. Along a 2 km stretch of the river Mulde (Germany), we collected 18 1-L water samples and analyzed the relation of detected species richness and quantity of biological replicates taken. We detected 58 vertebrate species, of which 25 were fish and lamprey, 18 mammals, and 15 birds, which account for 50%, 22.2%, and 7.4% of all native species to the German federal state of Saxony-Anhalt. However, while increasing the number of biological replicates resulted in only 24.8% more detected fish and lamprey species, mammal, and bird species richness increased disproportionately by 68.9% and 77.3%, respectively. Contrary, PCR replicates showed little stochasticity. We thus emphasize to increase the number of biological replicates when the aim is to improve general species detections. This holds especially true when the focus is on rare aquatic taxa or on (semi-)terrestrial species, the so-called ‘bycatch’. As a clear advantage, this information can be obtained without any additional sampling or laboratory effort when the sampling strategy is chosen carefully. With the increased use of eDNA metabarcoding as part of national fish bioassessment and monitoring programs, the complimentary information provided on bycatch can be used for biodiversity monitoring and conservation on a much broader scale. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Research Article text/html info:doi:10.3897/mbmg.5.66557 https://doi.org/10.3897/mbmg.5.66557 https://mbmg.pensoft.net/article/66557/ https://mbmg.pensoft.net/article/66557/download/pdf/ en

10.3897/mbmg.5.67533 2021-07-20 mbmg

Wet grinding of invertebrate bulk samples – a scalable and cost-efficient protocol for metabarcoding and metagenomics Buchner,Dominik Haase,Peter Leese,Florian bioassessment biodiversity soup bulk sample community metabarcoding DNA isolation LTER Metabarcoding and Metagenomics 5: e67533 Most metabarcoding protocols for invertebrate bulk samples start with sample homogenisation, followed by DNA extraction, amplification of a specific marker region, and sequencing. Many of the above-mentioned laboratory steps have been verified thoroughly and best practice strategies exist, yet, no clear recommendation for the basis of almost all metabarcoding studies exists: the homogenisation of samples itself. Two different categories of devices are typically used for homogenisation: bead mills or blenders. Both have upsides and downsides. Bead mills rely on single-use plastics and therefore produce a lot of waste and are expensive. In addition to that, processing times can go up to 30 minutes making them unsuitable for large-scale studies. Blenders can handle larger sample volumes in a shorter time, and be cleaned – yet suffer from an increased risk of cross-contamination. We aimed to develop a fast, robust, cheap, and reliable sample homogenisation protocol that overcomes limitations of both approaches, i.e. does not produce difficult to discard waste and avoid single-use plastics while reducing overall costs. We tested the performance of the new protocol using six size-sorted Malaise trap samples and six unsorted stream macroinvertebrate kick-net samples. We used 14 replicates per sample and included many negative controls at different steps of the protocol to quantify the impacts of i) insufficient homogenisation and ii) cross-contamination. Our results show that 3-min homogenisation is sufficient to recover about 80% of OTUs per sample in each replicate and that a non-hazardous DIY cleaning solution provides an effective and efficient way of cleaning. The improvements of the protocol in terms of speed, ease of handling, an overall reduction of costs as well as the documented reliability and robustness make it an important candidate for sample homogenisation after sampling in particular for large-scale and regulatory metabarcoding but also metagenomics biodiversity assessments and monitoring. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Methods text/html info:doi:10.3897/mbmg.5.67533 https://doi.org/10.3897/mbmg.5.67533 https://mbmg.pensoft.net/article/67533/ https://mbmg.pensoft.net/article/67533/download/pdf/ en

10.3897/mbmg.5.64735 2021-07-21 mbmg

Evaluating the performance of DNA metabarcoding for assessment of zooplankton communities in Western Lake Superior using multiple markers Meredith,Christy Hoffman,Joel Trebitz,Anett Pilgrim,Erik Okum,Sara Martinson,John Cameron,Ellen zooplankton Great Lakes Lake Superior identification metabarcoding Metabarcoding and Metagenomics 5: e64735 For DNA metabarcoding to attain its potential as a community assessment tool, we need to better understand its performance versus traditional morphological identification and work to address any remaining performance gaps in incorporating DNA metabarcoding into community assessments. Using fragments of the 18S nuclear and 16S mitochondrial rRNA genes and two fragments of the mitochondrial COI marker, we examined the use of DNA metabarcoding and traditional morphological identification for understanding the diversity and composition of crustacean zooplankton at 42 sites across western Lake Superior. We identified 51 zooplankton taxa (genus or species, depending on the finest resolution of the taxon across all identification methods), of which 17 were identified using only morphological traits, 13 using only DNA and 21 using both methods. The taxa found using only DNA metabarcoding included four species and one genus-level identification not previously known to occur in Lake Superior, the presence of which still needs to be confirmed. A substantial portion of taxa that were identified to genus or species by morphological identification, but not identified using DNA metabarcoding, had zero (“no record”) or < 2 (“underrepresented records”) reference barcodes in the BOLD or NCBI databases (63% for COI, 80% for 16S, 74% for 18S). The two COI marker fragments identified the most genus- and species-level taxa, whereas 18S was the only marker whose family-level percent sequence abundance patterns showed high correlation to composition patterns from morphological identification, based on a NMDS analysis of Bray-Curtis similarities. Multiple replicates were collected at a subset of sites and an occupancy analysis was performed, which indicated that rare taxa were more likely to be detected using DNA metabarcoding than traditional morphology. Our results support that DNA metabarcoding can augment morphological identification for estimating zooplankton diversity and composition of zooplankton over space and time, but may require use of multiple markers. Further addition of taxa to reference DNA databases will improve our ability to use DNA metabarcoding to identify zooplankton and other invertebrates in aquatic surveys. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC0 Pensoft Publishers 2021 Research Article text/html info:doi:10.3897/mbmg.5.64735 https://doi.org/10.3897/mbmg.5.64735 https://mbmg.pensoft.net/article/64735/ https://mbmg.pensoft.net/article/64735/download/pdf/ en

10.3897/mbmg.5.68938 2021-07-28 mbmg

The power of metabarcoding: Can we improve bioassessment and biodiversity surveys of stream macroinvertebrate communities? Turunen,Jarno Mykrä,Heikki Elbrecht,Vasco Steinke,Dirk Braukmann,Thomas Aroviita,Jukka biomonitoring Fennoscandia mining molecular data species identification taxonomic resolution Metabarcoding and Metagenomics 5: e68938 Most stream bioassessment and biodiversity surveys are currently based on morphological identification of communities. However, DNA metabarcoding is emerging as a fast and cost-effective alternative for species identification. We compared both methods in a survey of benthic macroinvertebrate communities across 36 stream sites in northern Finland. We identified 291 taxa of which 62% were identified only by DNA metabarcoding. DNA metabarcoding produced extensive species level inventories for groups (Oligochaeta, Chironomidae, Simuliidae, Limoniidae and Limnephilidae), for which morphological identification was not feasible due to the high level of expertise needed. Metabarcoding also provided more insightful taxonomic information on the occurrence of three red-listed vulnerable or data deficient species, the discovery of two likely cryptic and potentially new species to Finland and species information of insect genera at an early larval stage that could not be separated morphologically. However, it systematically failed to reliably detect the occurrence of gastropods that were easily identified morphologically. The impact of mining on community structure could only be shown using DNA metabarcoding data which suggests that the finer taxonomic detail can improve detection of subtle impacts. Both methods generally exhibited similar strength of community-environment relationships, but DNA metabarcoding showed better performance with presence/absence data than with relative DNA sequence abundances. Our results suggest that DNA metabarcoding holds a promise for future anthropogenic impact assessments, although, in our case, the performance did not improve much from the morphological species identification. The key advantage of DNA metabarcoding lies in efficient biodiversity surveys, taxonomical studies and applications in conservation biology. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Research Article text/html info:doi:10.3897/mbmg.5.68938 https://doi.org/10.3897/mbmg.5.68938 https://mbmg.pensoft.net/article/68938/ https://mbmg.pensoft.net/article/68938/download/pdf/ en

10.3897/mbmg.5.68438 2021-08-25 mbmg

How can integrated morphotaxonomy- and metabarcoding-based diatom assemblage analyses best contribute to the ecological assessment of streams? Pissaridou,Panayiota Cantonati,Marco Bouchez,Agnès Tziortzis,Iakovos Dörflinger,Gerald Vasquez,Marlen Ines DNA metabarcoding morpho-taxonomy river biomonitoring Cyprus rivers Metabarcoding and Metagenomics 5: e68438 Environmental conditions, such as nutrient concentrations, salinity, elevation etc., shape diatom assemblages of periphytic biofilms. These assemblages respond rapidly to environmental changes, a fact which makes diatoms valuable bioindicators. Hence, freshwater biomonitoring programmes currently use diatom indices (e.g. EU Water Framework Directive - WFD). To date, microscopy-based assessments require high taxonomic expertise for diatom identification at the species level. High-throughput technologies now provide cost-effective identification approaches that are promising, complementary or alternative tools for bioassessment. The suitability of the metabarcoding method is evaluated for the first time in the Cyprus streams WFD monitoring network, an eastern Mediterranean country with many endemic species and results are compared to the results acquired from the morphotaxonomic analysis. Morphotaxonomic identification was conducted microscopically, using the most updated taxonomic concepts, literature and online resources. At the same time, DNA metabarcoding involved the use of the rbcL 312 bp barcode, high-throughput sequencing and bioinformatic analysis. The ecological status was calculated using the IPS Index. Results show a positive correlation between morpho-taxonomic and molecular IPS scores. Discrepancies between the two methodologies are related to the limitations of both techniques. This study confirmed that Fistulifera saprophila can have a crucial role in key differences observed, as it negatively influences IPS scores and microscopy methods frequently overlook it. Importantly, gaps in the DNA barcoding reference databases lead to a positive overestimation in IPS scores. Overall, we conclude that DNA metabarcoding offsets the morphotaxonomic methodology for the ecological quality assessment of freshwaters. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Research Article text/html info:doi:10.3897/mbmg.5.68438 https://doi.org/10.3897/mbmg.5.68438 https://mbmg.pensoft.net/article/68438/ https://mbmg.pensoft.net/article/68438/download/pdf/ en

10.3897/mbmg.5.69602 2021-08-30 mbmg

Ciliate SSU-rDNA reference alignments and trees for phylogenetic placements of metabarcoding data Rajter,Lubomir Dunthorn,Micah Ciliophora diversity environmental sequencing HTS protists 18S Metabarcoding and Metagenomics 5: e69602 Although ciliates are one of the most dominant microbial eukaryotic groups in many environments, there is a lack of updated global ciliate alignments and reference trees that can be used for phylogenetic placement methods to analyze environmental metabarcoding data. Here we fill this gap by providing reference alignments and trees for those ciliates taxa with available SSU-rDNA sequences derived from identified species. Each alignment contains 478 ciliate and six outgroup taxa, and they were made using different masking strategies for alignment positions (unmasked, masked and masked except the hypervariable V4 region). We constrained the monophyly of the major ciliate groups based on the recently updated classification of protists and based on phylogenomic data. Taxa of uncertain phylogenetic position were kept unconstrained, except for Mesodinium species that we constrained to form a clade with the Litostomatea. These ciliate reference alignments and trees can be used to perform taxonomic assignments of metabarcoding data, discover novel ciliate clades, estimate species richness, and overlay measured ecological parameters onto the phylogenetic placements. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Data Paper text/html info:doi:10.3897/mbmg.5.69602 https://doi.org/10.3897/mbmg.5.69602 https://mbmg.pensoft.net/article/69602/ https://mbmg.pensoft.net/article/69602/download/pdf/ en

10.3897/mbmg.5.68155 2021-09-02 mbmg

Internal transcribed spacer primer evaluation for vascular plant metabarcoding Kolter,Andreas Gemeinholzer,Birgit barcoding DMSO internal transcribed spacer Spermatophyta mock community PCR Tracheophyta Metabarcoding and Metagenomics 5: e68155 The unprecedented ongoing biodiversity decline necessitates scalable means of monitoring in order to fully understand the underlying causes. DNA metabarcoding has the potential to provide a powerful tool for accurate and rapid biodiversity monitoring. Unfortunately, in many cases, a lack of universal standards undermines the widespread application of metabarcoding. One of the most important considerations in metabarcoding of plants, aside from selecting a potent barcode marker, is primer choice. Our study evaluates published ITS primers in silico and in vitro, through mock communities and presents newly designed primers. We were able to show that a large proportion of previously available ITS primers have unfavourable attributes. Our combined results support the recommendation of the introduced primers ITS-3p62plF1 and ITS-4unR1 as the best current universal plant specific ITS2 primer combination. We also found that PCR optimisation, such as the addition of 5% DMSO, is essential to obtain meaningful results in ITS2 metabarcoding. Finally, we conclude that continuous quality assurance is indispensable for reliable metabarcoding results. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Primer Validation text/html info:doi:10.3897/mbmg.5.68155 https://doi.org/10.3897/mbmg.5.68155 https://mbmg.pensoft.net/article/68155/ https://mbmg.pensoft.net/article/68155/download/pdf/ en

10.3897/mbmg.5.69691 2021-09-07 mbmg

18S rRNA amplicon sequence data (V1–V3) of the Bronx river estuary, New York Ingala,Melissa Werner,Irena Fitzgerald,Allison Naro-Maciel,Eugenia (Genia) eDNA metabarcoding New York City next-generation sequencing QIIME2 urban ecology Metabarcoding and Metagenomics 5: e69691 Characterising and monitoring biological diversity to foster sustainable ecosystems is highly recommended as urban centres rapidly expand. However, much of New York City’s biodiversity remains undescribed, including in the historically degraded, but recovering Bronx River Estuary. In a pilot study to identify organisms and characterise biodiversity patterns there, 18S rRNA gene amplicons (V1–V3 region), obtained from river sediments and surface waters of Hunts Point Riverside and Soundview Parks, were sequenced. Across 48 environmental samples collected over three seasons in 2015 and 2016, following quality control and contaminant removal, 2,763 Amplicon Sequence Variants (ASVs) were identified from 1,918,463 sequences. Rarefaction analysis showed sufficient sampling depth, and community composition varied over time and by substrate at the study sites over the sampling period. Protists, plants, fungi and animals, including organisms of management concern, such as Eastern oysters (Crassostrea virginica), wildlife pathogens and groups related to Harmful Algal Blooms, were detected. The most common taxa identified in river sediments were annelid worms, nematodes and diatoms. In the water column, the most commonly observed organisms were diatoms, algae of the phylum Cryptophyceae, ciliates and dinoflagellates. The presented dataset demonstrates the reach of 18S rRNA metabarcoding for characterising biodiversity in an urban estuary. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Data Paper text/html info:doi:10.3897/mbmg.5.69691 https://doi.org/10.3897/mbmg.5.69691 https://mbmg.pensoft.net/article/69691/ https://mbmg.pensoft.net/article/69691/download/pdf/ en

10.3897/mbmg.5.72371 2021-11-03 mbmg

Plankton biodiversity and species co-occurrence based on environmental DNA – a multiple marker study Sildever,Sirje Laas,Peeter Kolesova,Natalja Lips,Inga Lips,Urmas Nagai,Satoshi bacteria Baltic Sea metabarcoding phytoplankton rRNA genes seasons zooplankton Metabarcoding and Metagenomics 5: e72371 Metabarcoding in combination with high-throughput sequencing (HTS) allows simultaneous detection of multiple taxa by targeting single or several taxonomically informative gene regions from environmental DNA samples. In this study, a multiple-marker HTS approach was applied to investigate the plankton diversity and seasonal succession in the Baltic Sea from winter to autumn. Four different markers targeting the 16S, 18S, and 28S ribosomal RNA genes were employed, including a marker for more efficient dinoflagellate detection. Typical seasonal changes were observed in phyto- and bacterioplankton communities. In phytoplankton, the appearance patterns of selected common, dominant, or harmful species followed the patterns also confirmed based on 20 years of phytoplankton monitoring data. In the case of zooplankton, both macro- and microzooplankton species were detected. However, no seasonal patterns were detected in their appearance. In total, 15 and 2 new zoo- and phytoplankton species were detected from the Baltic Sea. HTS approach was especially useful for detecting microzooplankton species as well as for investigating the co-occurrence and potential interactions of different taxa. The results of this study further exemplify the efficiency of metabarcoding for biodiversity monitoring and the advantage of employing multiple markers through the detection of species not identifiable based on a single marker survey and/or by traditional morphology-based methods. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Research Article text/html info:doi:10.3897/mbmg.5.72371 https://doi.org/10.3897/mbmg.5.72371 https://mbmg.pensoft.net/article/72371/ https://mbmg.pensoft.net/article/72371/download/pdf/ en

10.3897/mbmg.5.69657 2021-11-03 mbmg

The Dark mAtteR iNvestigator (DARN) tool: getting to know the known unknowns in COI amplicon data Zafeiropoulos,Haris Gargan,Laura Hintikka,Sanni Pavloudi,Christina Carlsson,Jens Docker environmental DNA (eDNA) metabarcoding mitochondrial cytochrome C oxidase subunit I software tool tree of life (tol) unassigned sequences Metabarcoding and Metagenomics 5: e69657 The mitochondrial cytochrome C oxidase subunit I gene (COI) is commonly used in environmental DNA (eDNA) metabarcoding studies, especially for assessing metazoan diversity. Yet, a great number of COI operational taxonomic units (OTUs) or/and amplicon sequence variants (ASVs) retrieved from such studies do not get a taxonomic assignment with a reference sequence. To assess and investigate such sequences, we have developed the Dark mAtteR iNvestigator (DARN) software tool. For this purpose, a reference COI-oriented phylogenetic tree was built from 1,593 consensus sequences covering all the three domains of life. With respect to eukaryotes, consensus sequences at the family level were constructed from 183,330 sequences retrieved from the Midori reference 2 database, which represented 70% of the initial number of reference sequences. Similarly, sequences from 431 bacterial and 15 archaeal taxa at the family level (29% and 1% of the initial number of reference sequences respectively) were retrieved from the BOLD and the PFam databases. DARN makes use of this phylogenetic tree to investigate COI pre-processed sequences of amplicon samples to provide both a tabular and a graphical overview of their phylogenetic assignments. To evaluate DARN, both environmental and bulk metabarcoding samples from different aquatic environments using various primer sets were analysed. We demonstrate that a large proportion of non-target prokaryotic organisms, such as bacteria and archaea, are also amplified in eDNA samples and we suggest prokaryotic COI sequences to be included in the reference databases used for the taxonomy assignment to allow for further analyses of dark matter. DARN source code is available on GitHub at https://github.com/hariszaf/darn and as a Docker image at https://hub.docker.com/r/hariszaf/darn. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Software Description text/html info:doi:10.3897/mbmg.5.69657 https://doi.org/10.3897/mbmg.5.69657 https://mbmg.pensoft.net/article/69657/ https://mbmg.pensoft.net/article/69657/download/pdf/ en

10.3897/mbmg.5.74096 2021-12-15 mbmg

Phytool, a ShinyApp to homogenise taxonomy of freshwater microalgae from DNA barcodes and microscopic observations Canino,Alexis Bouchez,Agnès Laplace-Treyture,Christophe Domaizon,Isabelle Rimet,Frederic barcoding data bioinformatics biomonitoring freshwater microalgae metabarcoding microscopy phytoplankton taxonomy web-based application Metabarcoding and Metagenomics 5: e74096 Methods for biomonitoring of freshwater phytoplankton are evolving rapidly with eDNA-based methods, offering great complementarity with microscopy. Metabarcoding approaches have been more commonly used over the last years, with a continuous increase in the amount of data generated. Depending on the researchers and the way they assigned barcodes to species (bioinformatic pipelines and molecular reference databases), the taxonomic assignment obtained for HTS DNA reads might vary. This is also true for traditional taxonomic studies by microscopy with regular adjustments of the classification and taxonomy. For those reasons (leading to non-homogeneous taxonomies), gap-analyses and comparisons between studies become even more challenging and the curation processes to find potential consensus names are time-consuming. Here, we present a web-based application (Phytool), developed with ShinyApp (Rstudio), that aims to make the harmonisation of taxonomy easier and in a more efficient way, using a complete and up-to-date taxonomy reference database for freshwater microalgae. Phytool allows users to homogenise and update freshwater phytoplankton taxonomical names from sequence files and data tables directly uploaded in the application. It also gathers barcodes from curated references in a user-friendly way in which it is possible to search for specific organisms. All the data provided are downloadable with the possibility to apply filters in order to select only the required taxa and fields (e.g. specific taxonomic ranks). The main goal is to make accessible to a broad range of users the connection between microscopy and molecular biology and taxonomy through different ready-to-use functions. This study estimates that only 25% of species of freshwater phytoplankton in Phytobs are associated with a barcode. We plead for an increased effort to enrich reference databases by coupling taxonomy and molecular methods. Phytool should make this crucial work more efficient. The application is available at https://caninuzzo.shinyapps.io/phytool_v1/ info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Software Description text/html info:doi:10.3897/mbmg.5.74096 https://doi.org/10.3897/mbmg.5.74096 https://mbmg.pensoft.net/article/74096/ https://mbmg.pensoft.net/article/74096/download/pdf/ en

10.3897/mbmg.5.67862 2021-12-16 mbmg

Revision and annotation of DNA barcode records for marine invertebrates: report of the 8th iBOL conference hackathon Radulovici,Adriana Vieira,Pedro E. Duarte,Sofia Teixeira,Marcos Borges,Luisa Deagle,Bruce Majaneva,Sanna Redmond,Niamh Schultz,Jessica Costa,Filipe annotation data curation DNA barcoding marine invertebrates metabarcoding reference libraries Metabarcoding and Metagenomics 5: e67862 The accuracy of specimen identification through DNA barcoding and metabarcoding relies on reference libraries containing records with reliable taxonomy and sequence quality. The considerable growth in barcode data requires stringent data curation, especially in taxonomically difficult groups such as marine invertebrates. A major effort in curating marine barcode data in the Barcode of Life Data Systems (BOLD) was undertaken during the 8th International Barcode of Life Conference (Trondheim, Norway, 2019). Major taxonomic groups (crustaceans, echinoderms, molluscs, and polychaetes) were reviewed to identify those which had disagreement between Linnaean names and Barcode Index Numbers (BINs). The records with disagreement were annotated with four tags: a) MIS-ID (misidentified, mislabeled, or contaminated records), b) AMBIG (ambiguous records unresolved with the existing data), c) COMPLEX (species names occurring in multiple BINs), and d) SHARE (barcodes shared between species). A total of 83,712 specimen records corresponding to 7,576 species were reviewed and 39% of the species were tagged (7% MIS-ID, 17% AMBIG, 14% COMPLEX, and 1% SHARE). High percentages (>50%) of AMBIG tags were recorded in gastropods, whereas COMPLEX tags dominated in crustaceans and polychaetes. The high proportion of tagged species reflects either flaws in the barcoding workflow (e.g., misidentification, cross-contamination) or taxonomic difficulties (e.g., synonyms, undescribed species). Although data curation is essential for barcode applications, such manual attempts to examine large datasets are unsustainable and automated solutions are extremely desirable. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Data Paper text/html info:doi:10.3897/mbmg.5.67862 https://doi.org/10.3897/mbmg.5.67862 https://mbmg.pensoft.net/article/67862/ https://mbmg.pensoft.net/article/67862/download/pdf/ en

10.3897/mbmg.5.70107 2021-12-20 mbmg

Using DNA metabarcoding to decipher the diet plant component of mammals from the Eastern Mediterranean region BOUKHDOUD,Liliane Saliba,Carole PARKER,Lillian D McINERNEY,Nancy Rotzel Kahale,Rhea Saliba,Issam Maldonado,Jesus Bou Dagher Kharrat,Magda conservation diet DNA metabarcoding reforestation seed dispersal wildlife Metabarcoding and Metagenomics 5: e70107 Longevity of species populations depends largely on interactions among animals and plants in an ecosystem. Predation and seed dispersal are among the most important interactions necessary for species conservation and persistence, and diet analysis is a prerequisite tool to evaluate these interactions. Understanding these processes is crucial for identifying conservation targets and for executing efficient reforestation and ecological restoration. In this study, we applied a scat DNA metabarcoding technique using the P6-loop of the trnL (UAA) chloroplastic marker to describe the seasonal plant diet composition of 15 mammal species from a highly biodiverse Lebanese forest in the Eastern Mediterranean. We also recovered plant seeds, when present, from the scats for identification. The mammal species belong to 10 families from 5 different orders. More than 133 plant species from 54 plant families were detected and identified. Species from the Rosaceae, Poaceae, Apiaceae, Fabaceae, Fagaceae and Berberidaceae families were consumed by the majority of the mammals and should be taken into consideration in future reforestation and conservation projects. Our results showed that the DNA metabarcoding approach provides a promising method for tracking the dietary plant components of a wide diversity of mammals, yielding key insights into plant-animal interactions inside Lebanon’s forests. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC0 Pensoft Publishers 2021 Research Article text/html info:doi:10.3897/mbmg.5.70107 https://doi.org/10.3897/mbmg.5.70107 https://mbmg.pensoft.net/article/70107/ https://mbmg.pensoft.net/article/70107/download/pdf/ en

10.3897/mbmg.5.71107 2021-12-29 mbmg

Towards harmonization of DNA metabarcoding for monitoring marine macrobenthos: the effect of technical replicates and pooled DNA extractions on species detection Van den Bulcke,Laure De Backer,Annelies Ampe,Bart Maes,Sara Wittoeck,Jan Waegeman,Willem Hostens,Kris Derycke,Sofie biological replicates Bulk DNA diversity DNA replicates Macrobenthos Metabarcoding protocol North Sea PCR replicates pooling technical replicates Metabarcoding and Metagenomics 5: e71107 DNA-based monitoring methods are potentially faster and cheaper compared to traditional morphological benthic identification. DNA metabarcoding involves various methodological choices which can introduce bias leading to a different outcome in biodiversity patterns. Therefore, it is important to harmonize DNA metabarcoding protocols to allow comparison across studies and this requires a good understanding of the effect of methodological choices on diversity estimates. This study investigated the impact of DNA and PCR replicates on the detection of macrobenthos species in locations with high, medium and low diversity. Our results show that two to three DNA replicates were needed in locations with a high and medium diversity to detect at least 80% of the species found in the six DNA replicates, while three to four replicates were needed in the location with low diversity. In contrast to general belief, larger body size or higher abundance of the species in a sample did not increase its detection prevalence among DNA replicates. However, rare species were less consistently detected across all DNA replicates of the location with high diversity compared to locations with less diversity. Our results further show that pooling of DNA replicates did not significantly alter diversity patterns, although a small number of rare species was lost. Finally, our results confirm high variation in species detection between PCR replicates, especially for the detection of rare species. These results contribute to create reliable, time and cost efficient metabarcoding protocols for the characterization of macrobenthos. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Research Article text/html info:doi:10.3897/mbmg.5.71107 https://doi.org/10.3897/mbmg.5.71107 https://mbmg.pensoft.net/article/71107/ https://mbmg.pensoft.net/article/71107/download/pdf/ en

10.3897/mbmg.5.70063 2021-12-29 mbmg

Comparing species detection success between molecular markers in DNA metabarcoding of coastal macroinvertebrates Leite,Barbara Vieira,Pedro E. Troncoso,Jesús Costa,Filipe COI 18S DNA metabarcoding marine macrozoobenthic diversity primer efficiency taxonomic discrimination Metabarcoding and Metagenomics 5: e70063 DNA metabarcoding has great potential to improve marine biomonitoring programs by providing a rapid and accurate assessment of species composition in zoobenthic communities. However, some methodological improvements are still required, especially regarding failed detections, primers efficiency and incompleteness of databases. Here we assessed the efficiency of two different marker loci (COI and 18S) and three primer pairs in marine species detection through DNA metabarcoding of the macrozoobenthic communities colonizing three types of artificial substrates (slate, PVC and granite), sampled between 3 and 15 months of deployment. To accurately compare detection success between markers, we also compared the representativeness of the detected species in public databases and revised the reliability of the taxonomic assignments. Globally, we recorded extensive complementarity in the species detected by each marker, with 69% of the species exclusively detected by either 18S or COI. Individually, each of the three primer pairs recovered, at most, 52% of all species detected on the samples, showing also different abilities to amplify specific taxonomic groups. Most of the detected species have reliable reference sequences in their respective databases (82% for COI and 72% for 18S), meaning that when a species was detected by one marker and not by the other, it was most likely due to faulty amplification, and not by lack of matching sequences in the database. Overall, results showed the impact of marker and primer applied on species detection ability and indicated that, currently, if only a single marker or primer pair is employed in marine zoobenthos metabarcoding, a fair portion of the diversity may be overlooked. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2021 Methods text/html info:doi:10.3897/mbmg.5.70063 https://doi.org/10.3897/mbmg.5.70063 https://mbmg.pensoft.net/article/70063/ https://mbmg.pensoft.net/article/70063/download/pdf/ en

10.3897/mbmg.6.78128 2022-02-01 mbmg

Shifts in DNA yield and biological community composition in stored sediment: implications for paleogenomic studies Brasell,Katie Pochon,Xavier Howarth,Jamie Pearman,John Zaiko,Anastasija Thompson,Lucy Vandergoes,Marcus Simon,Kevin Wood,Susanna chilled storage droplet digital PCR extracellular DNA metabarcoding sedimentary DNA (sedDNA) Metabarcoding and Metagenomics 6: e78128 Lake sediments hold a wealth of information from past environments that is highly valuable for paleolimnological reconstructions. These studies increasingly apply modern molecular tools targeting sedimentary DNA (sedDNA). However, sediment core sampling can be logistically difficult, making immediate subsampling for sedDNA challenging. Sediment cores are often refrigerated (4 °C) for weeks or months before subsampling. We investigated the impact of storage time on changes in DNA (purified or as cell lysate) concentrations and shifts in biological communities following storage of lake surface sediment at 4 °C for up to 24 weeks. Sediment samples (~ 0.22 g, in triplicate per time point) were spiked with purified DNA (100 or 200 ng) or lysate from a brackish water cyanobacterium that produces the cyanotoxin nodularin or non-spiked. Samples were analysed every 1–4 weeks over a 24-week period. Droplet digital PCR showed no significant decrease in the target gene (nodularin synthetase – subunit F; ndaF) over the 24-week period for samples spiked with purified DNA, while copy number decreased by more than half in cell lysate-spiked samples. There was significant change over time in bacteria and eukaryotic community composition assessed using metabarcoding. Amongst bacteria, the cyanobacterial signal became negligible after 5 weeks while Proteobacteria increased. In the eukaryotic community, Cercozoa became dominant after 6 weeks. These data demonstrate that DNA yields and community composition data shift significantly when sediments are stored chilled for more than 5 weeks. This highlights the need for rapid subsampling and appropriate storage of sediment core samples for paleogenomic studies. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.78128 https://doi.org/10.3897/mbmg.6.78128 https://mbmg.pensoft.net/article/78128/ https://mbmg.pensoft.net/article/78128/download/pdf/ en

10.3897/mbmg.6.76534 2022-02-21 mbmg

Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia Sakata,Masayuki Kawata,Mone U. Kurabayashi,Atsuki Kurita,Takaki Nakamura,Masatoshi Shirako,Tomoyasu Kakehashi,Ryosuke Nishikawa,Kanto Hossman,Mohamad Yazid Nishijima,Takashi Kabamoto,Junichi Miya,Masaki Minamoto,Toshifumi Аgricultural ecosystem Amphibia biodiversity monitoring environmental DNA metabarcoding universal primer Metabarcoding and Metagenomics 6: e76534 Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians’ ecological traits (e.g. nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding – analysis of extra-organismal DNA released into the environment – allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160–311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set “Amph16S” had the highest resolution amongst the tested sets. Finally, we applied Amph16S to the water samples collected in the field and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.76534 https://doi.org/10.3897/mbmg.6.76534 https://mbmg.pensoft.net/article/76534/ https://mbmg.pensoft.net/article/76534/download/pdf/ en

10.3897/mbmg.6.77704 2022-02-21 mbmg

Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding Nagai,Satoshi Sildever,Sirje Nishi,Noriko Tazawa,Satoshi Basti,Leila Kobayashi,Takanori Ishino,Yoshizumi chimeric sequences DNA polymerase high throughput sequencing metabarcoding PCR-generated artifacts 18S rRNA gene Metabarcoding and Metagenomics 6: e77704 Accuracy of PCR amplification is vital for obtaining reliable amplicon-sequencing results by metabarcoding. Here, we performed a comparative analysis of error profiles in the PCR products by 14 different PCR kits using a mock eukaryotic community DNA sample mimicking metabarcoding analysis. To prepare a mock eukaryotic community from the marine environment, equal amounts of plasmid DNA from 40 microalgal species were mixed and used for amplicon-sequencing by a high-throughput sequencing approach. To compare the differences in PCR kits used for this experiment, we focused on the following seven parameters: 1) Quality, 2) Chimera, 3) Blast top hit accuracy, 4) Deletion, 5) Insertion, 6) Base substitution and 7) Amplification bias amongst species. The results showed statistically significant differences (p < 0.05) for all of the seven parameters depending on the PCR kits used. These differences may result from the different DNA polymerases included in each kit, although the result can also be influenced by PCR reaction conditions. Simultaneous analysis of several parameters suggested that kits containing KOD plus Neo (TOYOBO) and HotStart Taq DNA polymerase (BiONEER, CA, US) at the annealing temperature of 65 °C displayed better results in terms of parameters associated with chimeras, top hit similarity and deletions. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.77704 https://doi.org/10.3897/mbmg.6.77704 https://mbmg.pensoft.net/article/77704/ https://mbmg.pensoft.net/article/77704/download/pdf/ en

10.3897/mbmg.6.78181 2022-02-28 mbmg

Annual dynamics of eukaryotic and bacterial communities revealed by 18S and 16S rRNA metabarcoding in the coastal ecosystem of Sagami Bay, Japan Sogawa,Sayaka Tsuchiya,Kenji Nagai,Satoshi Shimode,Shinji Kuwahara,Victor S. bacteria bloom coastal marine ecosystem deep seawater environmental factor microbial plankton phytoplankton zooplankton Metabarcoding and Metagenomics 6: e78181 Sagami Bay, Japan is influenced by both the warm Kuroshio Current and the cold Oyashio Current and rich nutrients are supplied from multiple river sources and the deep-sea, forming a dynamic ecosystem. The aim of the present study was to investigate eukaryotic and bacterial communities in the coastal waters of Sagami Bay, using 16S rRNA and 18S rRNA sequencing and to assess the seasonal and vertical dynamics in relation to physicochemical and biological conditions. Eukaryotic and bacterial communities showed synchronous seasonal and vertical changes along with environmental variability. Diversity of plankton community suspended in the surface was lower than those at the subsurface layers in both the eukaryotes and bacteria communities; however, community diversity showed different characteristics in the subsurface where the eukaryotic community at the deeper layer (100 m) was as low as the surface and highest in intermediate depth layers (10–50 m), while that of bacterial community was highest in the deeper layer (100 m). The annual variability of the coastal microbial communities was driven, not only by the seasonal changes of abiotic and biotic factors and short-term rapid changes by river water inflow and phytoplankton blooms, but also largely influenced by deep-seawater upwellings due to the unique seafloor topography. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.78181 https://doi.org/10.3897/mbmg.6.78181 https://mbmg.pensoft.net/article/78181/ https://mbmg.pensoft.net/article/78181/download/pdf/ en

10.3897/mbmg.6.79265 2022-03-25 mbmg

Metagenomic insights to the functional potential of sediment microbial communities in freshwater lakes Biessy,Laura Pearman,John Waters,Sean Vandergoes,Marcus Wood,Susanna bacteria cyanobacteria New Zealand nutrients surface sediments water quality Metabarcoding and Metagenomics 6: e79265 Molecular-based techniques offer considerable potential to provide new insights into the impact of anthropogenic stressors on lake ecosystems. Microbial communities are involved in many geochemical cycling processes in lakes and a greater understanding of their functions could assist in guiding more targeted remedial actions. Recent advances in metagenomics now make it possible to determine the functional potential of entire microbial communities. The present study investigated microbial communities and their functional potential in surface sediments collected from three lakes with differing trophic states and characteristics. Surface sediments were analysed for their nutrient and elemental contents and metagenomics and metabarcoding analysis undertaken. The nutrients content of the surface sediments did not show as distinct a gradient as water chemistry monitoring data, likely reflecting effects of other lake characteristics, in particular, depth. Metabarcoding and metagenomics revealed differing bacterial community composition and functional potential amongst lakes. Amongst the differentially abundant metabolic pathways, the most prominent were clusters in the energy and xenobiotics pathways. Differences in the energy metabolism paths of photosynthesis and oxidative phosphorylation were observed. These were most likely related to changes in the community composition and especially the presence of cyanobacteria in two of the three lakes. Xenobiotic pathways, such as those involving polycyclic aromatic hydrocarbons, were highest in the lakes with the greatest agricultural land-use in their catchment. These results highlight how microbial metagenomics can be used to gain insights into the causes of differences in trophic status amongst lakes. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.79265 https://doi.org/10.3897/mbmg.6.79265 https://mbmg.pensoft.net/article/79265/ https://mbmg.pensoft.net/article/79265/download/pdf/ en

10.3897/mbmg.6.78756 2022-04-07 mbmg

A two-step metagenomics approach for the identification and mitochondrial DNA contig assembly of vertebrate prey from the blood meals of common vampire bats (Desmodus rotundus) Chua,Physilia Carøe,Christian Crampton-Platt,Alex Reyes-Avila,Claudia Jones,Gareth Streicker,Daniel Bohmann,Kristine animal diet Desmodus rotundus high-throughput sequencing molecular ecology mtDNA assembly sanguivory shotgun sequencing Metabarcoding and Metagenomics 6: e78756 The feeding behaviour of the sanguivorous common vampire bat (Desmodus rotundus) facilitates the transmission of pathogens that can impact both human and animal health. To formulate effective strategies in controlling the spread of diseases, there is a need to obtain information on which animals they feed on. One DNA-based approach, shotgun sequencing, can be used to obtain such information. Even though it is costly, shotgun sequencing can be used to simultaneously retrieve prey and vampire bat mitochondrial DNA for population studies within one round of sequencing. However, due to the challenges of analysing shotgun sequenced metagenomic data such as false negatives/positives and typically low proportion of reads mapped to diet items, shotgun sequencing has not been used for the identification of prey from common vampire bat blood meals. To overcome these challenges and generate longer mitochondrial contigs which could be useful for prey population studies, we shotgun sequenced common vampire bat blood meal samples (n = 8) and utilised a two-step metagenomic approach based on combining existing bioinformatic workflows (alignment and mtDNA contig assembly) to identify prey. After validating our results from detections made through metabarcoding, we accurately identified the common vampire bats’ prey in six out of eight samples without any false positives. We also generated prey mitochondrial contig lengths between 138 bp to 3231 bp (median = 770 bp, Q1 = 262 bp, Q3 = 1766 bp). This opens the potential to conduct phylogenetic and phylogeographic monitoring of elusive prey species in future studies, through the analyses of blood meal metagenomic data. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Emerging Technique text/html info:doi:10.3897/mbmg.6.78756 https://doi.org/10.3897/mbmg.6.78756 https://mbmg.pensoft.net/article/78756/ https://mbmg.pensoft.net/article/78756/download/pdf/ en

10.3897/mbmg.6.78762 2022-04-12 mbmg

18S rDNA amplicon sequence data (V1–V3) of the Palmyra Atoll National Wildlife Refuge, Central Pacific Reid,Brendan Servis,Jennifer Timmers,Molly Rohwer,Forest Naro-Maciel,Eugenia (Genia) ARMS eDNA next-generation sequencing PANWR QIIME2 seawater sediment sessile Metabarcoding and Metagenomics 6: e78762 To address the global biodiversity crisis, standardized data that are rapidly obtainable through minimally invasive means are needed for documenting change and informing conservation within threatened and diverse systems, such as coral reefs. In this data paper, we describe 18S rRNA gene amplicon data (V1–V3 region) generated from samples collected to begin characterizing coral reef eukaryotic community composition at the Palmyra Atoll National Wildlife Refuge in the Central Pacific Ocean. Sixteen samples were obtained across four sample types: sediments from two sieved fractions (100–500 μm, n = 3; 500 μm-2 mm, n = 3) and sessile material scrapings (n = 3) from Autonomous Reef Monitoring Structures (ARMS) sampled in 2015, as well as seawater from 2012 (n = 7). After filtering and contaminant removal, 3,861 Amplicon Sequence Variants (ASVs) were produced from 1,062,238 reads. The rarefaction curves demonstrated adequate sampling depth, and communities grouped by sample type. The dominant orders across samples were polychaete worms (Eunicida), demosponges (Poecilosclerida), and bryozoans (Cheilostomatida). The ten most common orders in terms of relative abundance comprised ~60% of all sequences and 23% of ASVs, and included reef-building crustose coralline algae (CCA; Corallinophycidae) and stony corals (Scleractinia), two taxa associated with healthy reefs. Highlighting the need for further study, ~21% of the ASVs were identified as uncultured, incertae sedis, or not assigned to phylum or order. This data paper presents the first 18S rDNA survey at Palmyra Atoll and serves as a baseline for biodiversity assessment, monitoring, and conservation of this remote and pristine ecosystem. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC0 Pensoft Publishers 2022 Data Paper text/html info:doi:10.3897/mbmg.6.78762 https://doi.org/10.3897/mbmg.6.78762 https://mbmg.pensoft.net/article/78762/ https://mbmg.pensoft.net/article/78762/download/pdf/ en

10.3897/mbmg.6.79208 2022-04-12 mbmg

Comparative metagenomics of phytoplankton blooms after nutrient enrichment of oligotrophic marine waters Pearman,John Casas,Laura Michell,Craig Aldanondo,Naroa Mojib,Nazia Holtermann,Karie Georgakakis,Ioannis Curdia,Joao Carvalho,Susana Gusti,Amr Irigoien,Xabier algal blooms eutrophication metagenomics nutrient enrichment Metabarcoding and Metagenomics 6: e79208 Increasing anthropogenic pressures on the coastal marine environments impact these ecosystems via a variety of mechanisms including nutrient loading, leading to eutrophication and increases in algal blooms. Here, we use a metagenomics approach to assess the taxonomic and functional changes of the microbial community throughout a nutrient enriched mesocosm phytoplankton bloom. We tested four different nutrient treatments consisting of either nitrate and phosphate or nitrate, phosphate and silicate, administered on the first day or continuously for the first two weeks of the experiment. Our results show a shift in the taxonomic composition of the community over time that is dependent on the nutrient addition regime. Significant differences in the functional potential of the communities were detected, with an interaction between bloom period (pre-bloom, bloom and post-bloom) and nutrient treatment (p = 0.004). A sharp drop in functional similarity was observed in the first week in all treatments and after 20 days had not returned to pre-bloom levels. Changes within energy metabolism pathways showed a remarkable enrichment of the dissimilatory nitrate reduction pathway in the post-bloom period. Eukaryotic oxidative phosphorylation and photosynthetic antenna proteins were more abundant during the bloom, especially in the continuous treatment with silicate. Our results suggest that continuous (i.e. chronic) nutrient enrichment has a larger effect on the functioning of marine systems compared to a single (i.e acute) addition. A deep understanding of the functional and taxonomic shifts in the community during blooms is essential to reverse or mitigate human impacts on coastal environments. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.79208 https://doi.org/10.3897/mbmg.6.79208 https://mbmg.pensoft.net/article/79208/ https://mbmg.pensoft.net/article/79208/download/pdf/ en

10.3897/mbmg.6.79351 2022-05-10 mbmg

A triad of kicknet sampling, eDNA metabarcoding, and predictive modeling to assess richness of mayflies, stoneflies and caddisflies in rivers Keck,François Hürlemann,Samuel Locher,Nadine Stamm,Christian Deiner,Kristy Altermatt,Florian Ephemeroptera metabarcoding Plecoptera Trichoptera water DNA Metabarcoding and Metagenomics 6: e79351 Monitoring biodiversity is essential to understand the impacts of human activities and for effective management of ecosystems. Thereby, biodiversity can be assessed through direct collection of targeted organisms, through indirect evidence of their presence (e.g. signs, environmental DNA, camera trap, etc.), or through extrapolations from species distribution and species richness models. Differences in approaches used in biodiversity assessment, however, may come with individual challenges and hinder cross-study comparability. In the context of rapidly developing techniques, we compared three different approaches in order to better understand assessments of aquatic macroinvertebrate diversity. Specifically, we compared the community composition and species richness of three orders of aquatic macroinvertebrates (mayflies, stoneflies, and caddisflies, hereafter EPT) obtained via eDNA metabarcoding and via traditional in situ kicknet sampling to catchment-level based predictions of a species richness model. We used kicknet data from 24 sites in Switzerland and compared taxonomic lists to those obtained using eDNA amplified with two different primer sets. Richness detected by these methods was compared to the independent predictions made by a statistical species richness model, that is, a generalized linear model using landscape-level features to estimate EPT diversity. Despite the ability of eDNA to consistently detect some EPT species found by traditional sampling, we found important discrepancies in community composition between the kicknet and eDNA approaches, particularly at a local scale. We found the EPT-specific primer set fwhF2/EPTDr2n, detected a greater number of targeted EPT species compared to the more general primer set mlCOIintF/HCO2198. Moreover, we found that the species richness measured by eDNA from either primer set was poorly correlated to the richness measured by kicknet sampling (Pearson correlation = 0.27) and that the richness estimated by eDNA and kicknet were poorly correlated with the prediction of the species richness model (Pearson correlation = 0.30 and 0.44, respectively). The weak relationships between the traditional kicknet sampling and eDNA with this model indicates inherent limitations in upscaling species richness estimates, and possibly a limited ability of the model to meet real world expectations. It is also possible that the number of replicates was not sufficient to detect ambiguous correlations. Future challenges include improving the accuracy and sensitivity of each approach individually, yet also acknowledging their respective limitations, in order to best meet stakeholder demands and address the biodiversity crisis we are facing. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.79351 https://doi.org/10.3897/mbmg.6.79351 https://mbmg.pensoft.net/article/79351/ https://mbmg.pensoft.net/article/79351/download/pdf/ en

10.3897/mbmg.6.80444 2022-05-23 mbmg

The use of citizen science in fish eDNA metabarcoding for evaluating regional biodiversity in a coastal marine region: A pilot study Miya,Masaki Sado,Tetsuya Oka,Shin-ichiro Fukuchi,Takehiko citizen science conservation eDNA metabarcoding education fish biodiversity outreach Metabarcoding and Metagenomics 6: e80444 To test the feasibility of a citizen science program for fish eDNA metabarcoding in coastal marine environments, we recruited six groups of voluntary citizens for a science education course at a natural history museum. We held a seminar on eDNA and a workshop for seawater sampling and on-site filtration using syringes and filter cartridges for the participants. After that, they selected single survey sites following the guidelines for conducting a safe field trip. They performed seawater sampling and on-site filtration at these sites during their summer holidays. The six selected sites unexpectedly included diverse coastal habitats within a 40 km radius, located at temperate latitudes in central Japan (~35°N). After the field trips, they returned filtered cartridges to the museum, and we extracted eDNA from the filters. We performed fish eDNA metabarcoding, along with data analysis. Consequently, we identified 140 fish species across 66 families and 118 genera from the six samples, with species richness ranging from 14 to 66. Despite its limited sample size, such a diverse taxonomic range of fish species exhibited spatial biodiversity patterns within the region, which are consistent with species distribution. These include north-south and urbanization gradients of species richness, geographic structure of the fish communities, and varying salinity preferences of the component species. This case study demonstrates the potential of fish eDNA metabarcoding as an educational and scientific tool to raise public awareness and perform large-scale citizen science initiatives encompassing regional, national, or global fauna. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Case Study text/html info:doi:10.3897/mbmg.6.80444 https://doi.org/10.3897/mbmg.6.80444 https://mbmg.pensoft.net/article/80444/ https://mbmg.pensoft.net/article/80444/download/pdf/ en

10.3897/mbmg.6.80416 2022-05-31 mbmg

The bacterial hitchhiker’s guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind Hintikka,Sanni Carlsson,Jeanette Carlsson,Jens eDNA magnetic bead capture metabarcoding mtDNA Metabarcoding and Metagenomics 6: e80416 Environmental DNA (eDNA) metabarcoding from water samples has, in recent years, shown great promise for biodiversity monitoring. However, universal primers targeting the cytochrome oxidase I (COI) marker gene popular in metazoan studies have displayed high levels of nontarget amplification. To date, enrichment methods bypassing amplification have not been able to match the detection levels of conventional metabarcoding. This study evaluated the use of universal metabarcoding primers as capture probes to either isolate target DNA or to remove nontarget DNA, prior to amplification, by using biotinylated versions of universal metazoan and bacterial barcoding primers, namely metazoan COI (mlCOIintF) and bacterial 16S (515F). Additionally, each step of the protocol was assessed by amplifying for both metazoan COI (mlCOIintF/jgHCO2198) and bacterial 16S (515F/806R) to investigate the effect on the metazoan and bacterial communities. Bacterial read abundance increased significantly in response to the captures (COI library), while the quality of the captured DNA was also improved. The metazoan-oriented probe captured bacterial DNA in a range that was also amplifiable with the 16S primers, demonstrating the ability of capture probes to isolate fragments of DNA spanning over a longer distance than perhaps expected, from eDNA. Although the use of the tested COI probe cannot be recommended for metazoan enrichment, based on the experimental results, the concept of capturing these longer fragments could be applied to metazoan metabarcoding. By using a truly conserved site without a high-level taxonomic resolution as a target for capture, it may be possible to isolate DNA fragments large enough to span over a nearby barcoding region (e.g., COI), which can then be processed through a conventional metabarcoding-by-amplification protocol. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.80416 https://doi.org/10.3897/mbmg.6.80416 https://mbmg.pensoft.net/article/80416/ https://mbmg.pensoft.net/article/80416/download/pdf/ en

10.3897/mbmg.6.e68575 2022-06-10 mbmg

Environmental DNA (eDNA) metabarcoding surveys show evidence of non-indigenous freshwater species invasion to new parts of Eastern Europe Jeunen,Gert-Jan Lipinskaya,Tatsiana Gajduchenko,Helen Golovenchik,Viktoriya Moroz,Michail Rizevsky,Viktor Semenchenko,Vitaliy Gemmell,Neil aquatic biodiversity assessment Central European invasion corridor fish invasive species detection macroinvertebrate passive eDNA surveillance traditional survey methods Metabarcoding and Metagenomics 6: e68575 Active environmental DNA (eDNA) surveillance through species-specific amplification has shown increased sensitivity in the detection of non-indigenous species (NIS) compared to traditional approaches. When many NIS are of interest, however, active surveillance decreases in cost- and time-efficiency. Passive surveillance through eDNA metabarcoding takes advantage of the complex DNA signal in environmental samples and facilitates the simultaneous detection of multiple species. While passive eDNA surveillance has previously detected NIS, comparative studies are essential to determine the ability of eDNA metabarcoding to accurately describe the range of invasion for multiple NIS versus alternative approaches. Here, we surveyed twelve sites, covering nine rivers across Belarus for NIS with three different techniques, i.e. an ichthyological, hydrobiological and eDNA survey, whereby DNA was extracted from 500 ml surface water samples and amplified with two 16S rDNA primer assays targeting the fish and macroinvertebrate biodiversity. Nine non-indigenous fish and ten non-indigenous benthic macroinvertebrates were detected by traditional surveys, while seven NIS eDNA signals were picked up, including four fish, one aquatic and two benthic macroinvertebrates. Passive eDNA surveillance extended the range of invasion further north for two invasive fish and identified a new NIS for Belarus, the freshwater jellyfish Craspedacusta sowerbii. False-negative detections for the eDNA survey might be attributed to: (i) preferential amplification of aquatic over benthic macroinvertebrates from surface water samples and (ii) an incomplete reference database. The evidence provided in this study recommends the implementation of both molecular-based and traditional approaches to maximise the probability of early detection of non-native organisms. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.e68575 https://doi.org/10.3897/mbmg.6.e68575 https://mbmg.pensoft.net/article/68575/ https://mbmg.pensoft.net/article/68575/download/pdf/ en

10.3897/mbmg.6.80139 2022-06-10 mbmg

COI amplicon sequence data of environmental DNA collected from the Bronx River Estuary, New York City Naro-Maciel,Eugenia (Genia) Ingala,Melissa Werner,Irena Reid,Brendan Fitzgerald,Allison eDNA MEGAN metabarcoding next-generation sequencing QIIME2 river sediment river water urban ecology Metabarcoding and Metagenomics 6: e80139 In this data paper, we describe environmental DNA (eDNA) cytochrome c oxidase (COI) amplicon sequence data from New York City’s Bronx River Estuary. As urban systems continue to expand, describing and monitoring their biodiversity is increasingly important for sustainability. Once polluted and overexploited, New York City’s Bronx River Estuary is undergoing revitalization and restoration. To investigate and characterize the area’s diversity, we collected and sequenced river sediment and surface water samples from Hunts Point Riverside and Soundview Parks (ntotal = 48; nsediment = 25; nwater = 23). COI analysis using universal primers mlCOIintF and jgHCO2198 detected 27,328 Amplicon Sequence Variants (ASVs) from 7,653,541 sequences, and rarefaction curves reached asymptotes indicating sufficient sampling depth. Of these, eukaryotes represented 9,841ASVs from 3,562,254 sequences. At the study sites over the sampling period, community composition varied by substrate (river sediment versus surface water) and with water temperature, but not pH. The three most common phyla were Bacillariophyta (diatoms), Annelida (segmented worms), and Ochrophyta (e.g. brown and golden algae). Of the eukaryotic ASVs, we identified 614 (6.2%) to species level, including several dinoflagellates linked to Harmful Algal Blooms such as Heterocapsa spp., as well as the invasive amphipod Grandidierella japonica. The analysis detected common bivalves including blue (Mytilus edulis) and ribbed (Geukensia demissa) mussels, as well as soft-shell clams (Mya arenaria), in addition to Eastern oysters (Crassostrea virginica) that are being reintroduced to the area. Fish species undergoing restoration such as river herring (Alosa pseudoharengus, A. aestivalis) failed to be identified, although relatively common fish including Atlantic silversides (Menidia menidia), menhaden (Brevoortia tyrannus), striped bass (Morone saxatilis), and mummichogs (Fundulus heteroclitus) were found. The data highlight the utility of eDNA metabarcoding for analyzing urban estuarine biodiversity and provide a baseline for future work in the area. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Data Paper text/html info:doi:10.3897/mbmg.6.80139 https://doi.org/10.3897/mbmg.6.80139 https://mbmg.pensoft.net/article/80139/ https://mbmg.pensoft.net/article/80139/download/pdf/ en

10.3897/mbmg.6.78871 2022-06-16 mbmg

Evaluation of non-destructive DNA extraction protocols for insect metabarcoding: gentler and shorter is better Marquina,Daniel Roslin,Tomas Łukasik,Piotr Ronquist,Fredrik DNA extraction insects metabarcoding non-destructive taxonomy Metabarcoding and Metagenomics 6: e78871 DNA metabarcoding can accelerate research on insect diversity, as it is cheap and fast compared to manual sorting and identification. Most metabarcoding protocols require homogenisation of the sample, preventing further work on the specimens. Mild digestion of the tissue by incubation in a lysis buffer has been proposed as an alternative, and, although some mild lysis protocols have already been presented, they have so far not been evaluated against each other. Here, we analyse how two mild lysis buffers (one more aggressive, one gentler in terms of tissue degradation), two different incubation times, and two DNA purification methods (a manual precipitation and an automated protocol) affect the accuracy of retrieving the true composition of mock communities using two mitochondrial markers (COI and 16S). We found that protocol-specific variation in concentration and purity of the DNA extracts produced had little effect on the recovery of species. However, the two lysis treatments differed in quantification of species abundances. Digestion in the gentler buffer and for a shorter time yielded better representation of original sample composition. Digestion in a more aggressive buffer or longer incubation time yielded lower alpha diversity values and increased differences between metabarcoding results and the true species-abundance distribution. We conclude that the details of non-destructive protocols can have a significant effect on metabarcoding performance. A short and mild lysis treatment appears the best choice for recovering the true composition of the sample. This not only improves accuracy, but also comes with a faster processing time than the other treatments. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.78871 https://doi.org/10.3897/mbmg.6.78871 https://mbmg.pensoft.net/article/78871/ https://mbmg.pensoft.net/article/78871/download/pdf/ en

10.3897/mbmg.6.84331 2022-07-20 mbmg

Are microbes growing on flowers evil? Effects of old flower microbes on fruit set in a wild ginger with one-day flowers, Alpinia japonica (Zingiberaceae) Jiménez Elvira,Nuria Ushio,Masayuki Sakai,Shoko anthosphere flower longevity fruit set microbiome pollination Metabarcoding and Metagenomics 6: e84331 Flowers are colonized and inhabited by diverse microbes. Flowers have various mechanisms to suppress microbial growth, such as flower volatiles, reactive oxygen and secondary compounds. Besides, plants rapidly replace flowers that have a short lifespan, and old flowers senesce. They may contribute to avoiding adverse effects of the microbes. In this study, we investigate if the flower microbial community on old flowers impedes fruit and seed production in a wild ginger with one-day flowers. We focus on microbes on old flowers because they may be composed of microbes that would grow during flowering if the flowers did not have mechanisms to suppress microbial growth. We inoculated newly opened flowers with old flower microbes, and monitored the effects on fruit and seed set. We also assessed prokaryotic communities on the flowers using 16S rRNA amplicon sequencing. We found six bacterial amplicon sequence variants (ASVs) whose proportions were increased on the inoculated flowers. These ASVs were also found on flower buds and flowers that were bagged by net or paper during anthesis, suggesting that they had been present in small numbers prior to flowering. Fruit set was negatively associated with the proportions of these ASVs, while seed set was not. The results suggest that old flowers harbor microbial communities different from those at anthesis, and that the microbes abundant on old flowers negatively affect plant reproduction. Although it has received little attention, antagonistic microbes that rapidly proliferate on the flowers may have affected the evolution of various flower characteristics such as flower volatiles and life span. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.84331 https://doi.org/10.3897/mbmg.6.84331 https://mbmg.pensoft.net/article/84331/ https://mbmg.pensoft.net/article/84331/download/pdf/ en

10.3897/mbmg.6.85652 2022-07-20 mbmg

A strategy for successful integration of DNA-based methods in aquatic monitoring Blancher,Philippe Lefrançois,Estelle Rimet,Frederic Vasselon,Valentin Argillier,Christine Arle,Jens Beja,Pedro Boets,Pieter Boughaba,Jeanne Chauvin,Christian Deacon,Michael Duncan,Willie Ejdung,Gunilla Erba,Stefania Ferrari,Benoit Fischer,Helmut Hänfling,Bernd Haldin,Michael Hering,Daniel Hette-Tronquart,Nicolas Hiley,Alice Järvinen,Marko Jeannot,Benjamin Kahlert,Maria Kelly,Martyn Kleinteich,Julia Koyuncuoğlu,Serdar Krenek,Sascha Langheim-Winther,Sidsel Leese,Florian Mann,David G. Marcel,Rémy Marcheggiani,Stefania Meissner,Kristian Mergen,Patricia Monnier,Olivier Narendja,Frank Neu,Diane Onofre Pinto,Veronica Pawlowska,Alina Pawlowski,Jan Petersen,Martin Poikane,Sandra Pont,Didier Renevier,Marie-Sophie Sandoy,Steinar Svensson,Jonas Trobajo,Rosa Tünde Zagyva,Andrea Tziortzis,Iakovos van der Hoorn,Berry Vasquez,Marlen Ines Walsh,Kerry Weigand,Alexander Bouchez,Agnès Aquatic ecosystems DNAqua-Net Ecological assessment eDNA Water Framework Directive Metabarcoding and Metagenomics 6: e85652 Recent advances in molecular biomonitoring open new horizons for aquatic ecosystem assessment. Rapid and cost-effective methods based on organismal DNA or environmental DNA (eDNA) now offer the opportunity to produce inventories of indicator taxa that can subsequently be used to assess biodiversity and ecological quality. However, the integration of these new DNA-based methods into current monitoring practices is not straightforward, and will require coordinated actions in the coming years at national and international levels. To plan and stimulate such an integration, the European network DNAqua-Net (COST Action CA15219) brought together international experts from academia, as well as key environmental biomonitoring stakeholders from different European countries. Together, this transdisciplinary consortium developed a roadmap for implementing DNA-based methods with a focus on inland waters assessed by the EU Water Framework Directive (2000/60/EC). This was done through a series of online workshops held in April 2020, which included fifty participants, followed by extensive synthesis work. The roadmap is organised around six objectives: 1) to highlight the effectiveness and benefits of DNA-based methods, 2) develop an adaptive approach for the implementation of new methods, 3) provide guidelines and standards for best practice, 4) engage stakeholders and ensure effective knowledge transfer, 5) support the environmental biomonitoring sector to achieve the required changes, 6) steer the process and harmonise efforts at the European level. This paper provides an overview of the forum discussions and the common European views that have emerged from them, while reflecting the diversity of situations in different countries. It highlights important actions required for a successful implementation of DNA-based biomonitoring of aquatic ecosystems by 2030. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Forum Paper text/html info:doi:10.3897/mbmg.6.85652 https://doi.org/10.3897/mbmg.6.85652 https://mbmg.pensoft.net/article/85652/ https://mbmg.pensoft.net/article/85652/download/pdf/ en

10.3897/mbmg.6.85213 2022-07-22 mbmg

The potential of metabarcoding plant components of Malaise trap samples to enhance knowledge of plant-insect interactions Swenson,Stephanie Eichler,Lisa Hörren,Thomas Kolter,Andreas Köthe,Sebastian Lehmann,Gerlind U. C. Meinel,Gotthard Mühlethaler,Roland Sorg,Martin Gemeinholzer,Birgit biomonitoring DNA metabarcoding insect decline landscape change nature conservation plant-insect interactions Metabarcoding and Metagenomics 6: e85213 The worldwide rapid declines in insect and plant abundance and diversity that have occurred in the past decades have gained public attention and demand for political actions to counteract these declines are growing. Rapid large-scale biomonitoring can aid in observing these changes and provide information for decisions for land management and species protection. Malaise traps have long been used for insect sampling and when insects are captured in these traps, they carry traces of plants they have visited on the body surface or as digested food material in the gut contents. Metabarcoding offers a promising method for identifying these plant traces, providing insight into the plants with which insects are directly interacting at a given time. To test the efficacy of DNA metabarcoding with these sample types, 79 samples from 21 sites across Germany were analysed with the ITS2 barcode. This study, to our knowledge, is the first examination of metabarcoding plant DNA traces from Malaise trap samples. Here, we report on the feasibility of sequencing these sample types, analysis of the resulting taxa, the usage of cultivated plants by insects near nature conservancy areas and the detection of rare and neophyte species. Due to the frequency of contamination and false positive reads, isolation and PCR negative controls should be used in every reaction. Metabarcoding has advantages in efficiency and resolution over microscopic identification of pollen and is the only possible identification method for the other plant traces from Malaise traps and could provide a broad utility for future studies of plant-insect interactions. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.85213 https://doi.org/10.3897/mbmg.6.85213 https://mbmg.pensoft.net/article/85213/ https://mbmg.pensoft.net/article/85213/download/pdf/ en

10.3897/mbmg.6.84960 2022-08-02 mbmg

Introducing guidelines for publishing DNA-derived occurrence data through biodiversity data platforms Nilsson,R. Henrik Andersson,Anders Bissett,Andrew Finstad,Anders Gravbrøt Fossøy,Frode Grosjean,Marie Hope,Michael Jeppesen,Thomas Kõljalg,Urmas Lundin,Daniel Prager,Maria Suominen,Saara Svenningsen,Cecilie Schigel,Dmitry biological data management DNA sequences metabarcoding metagenomics occurrence record open data scientific credit scientific reproducibility Metabarcoding and Metagenomics 6: e84960 DNA sequencing efforts of environmental and other biological samples disclose unprecedented and largely untapped opportunities for advances in the taxonomy, ecology, and geographical distributions of our living world. To realise this potential, DNA-derived occurrence data (notably sequences with dates and coordinates) – much like traditional specimens and observations – need to be discoverable and interpretable through biodiversity data platforms. The Global Biodiversity Information Facility (GBIF) recently headed a community effort to assemble a set of guidelines for publishing DNA-derived data. These guidelines target the principles and approaches of exposing DNA-derived occurrence data in the context of broader biodiversity data. They cover a choice of terms using a controlled vocabulary, common pitfalls, and good practices, without going into platform-specific details. Our hope is that they will benefit anyone interested in better exposure of DNA-derived occurrence data through general biodiversity data platforms, including national biodiversity portals. This paper provides a brief rationale and an overview of the guidelines, an up-to-date version of which is maintained at https://doi.org/10.35035/doc-vf1a-nr22. User feedback and interaction are encouraged as new techniques and best practices emerge. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Forum Paper text/html info:doi:10.3897/mbmg.6.84960 https://doi.org/10.3897/mbmg.6.84960 https://mbmg.pensoft.net/article/84960/ https://mbmg.pensoft.net/article/84960/download/pdf/ en

10.3897/mbmg.6.85794 2022-08-15 mbmg

Towards quantitative metabarcoding of eukaryotic plankton: an approach to improve 18S rRNA gene copy number bias Lapeyra Martin,Jon Santi,Ioulia Paraskevi,Pitta John,Uwe Gypens,Nathalie 18S rRNA copy number correction factor plankton protists quantitative metabarcoding Metabarcoding and Metagenomics 6: e85794 Plankton metabarcoding is increasingly implemented in marine ecosystem assessments and is more cost-efficient and less time-consuming than monitoring based on microscopy (morphological). 18S rRNA gene is the most widely used marker for groups’ and species’ detection and classification within marine eukaryotic microorganisms. These datasets have commonly relied on the acquisition of organismal abundances directly from the number of DNA sequences (i.e. reads). Besides the inherent technical biases in metabarcoding, the largely varying 18S rRNA gene copy numbers (GCN) among marine protists (ranging from tens to thousands) is one of the most important biological biases for species quantification. In this work, we present a gene copy number correction factor (CF) for four marine planktonic groups: Bacillariophyta, Dinoflagellata, Ciliophora miscellaneous and flagellated cells. On the basis of the theoretical assumption that ‘1 read’ is equivalent to ‘1 GCN’, we used the GCN median values per plankton group to calculate the corrected cell number and biomass relative abundances. The species-specific absolute GCN per cell were obtained from various studies published in the literature. We contributed to the development of a species-specific 18S rRNA GCN database proposed by previous authors. To assess the efficiency of the correction factor we compared the metabarcoding, morphological and corrected relative abundances (in cell number and biomass) of 15 surface water samples collected in the Belgian Coastal Zone. Results showed that the application of the correction factor over metabarcoding results enables us to significantly improve the estimates of cell abundances for Dinoflagellata, Ciliophora and flagellated cells, but not for Bacillariophyta. This is likely to due to large biovolume plasticity in diatoms not corresponding to genome size and gene copy numbers. C-biomass relative abundance estimations directly from amplicon reads were only improved for Dinoflagellata and Ciliophora. The method is still facing biases related to the low number of species GCN assessed. Nevertheless, the increase of species in the GCN database may lead to the refinement of the proposed correction factor. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.85794 https://doi.org/10.3897/mbmg.6.85794 https://mbmg.pensoft.net/article/85794/ https://mbmg.pensoft.net/article/85794/download/pdf/ en

10.3897/mbmg.6.79471 2022-08-23 mbmg

Monitoring harmful microalgal species and their appearance in Tokyo Bay, Japan, using metabarcoding Sildever,Sirje Nishi,Noriko Inaba,Nobuharu Asakura,Taiga Kikuchi,Jun Asano,Yasuhito Kobayashi,Takanori Gojobori,Takashi Nagai,Satoshi 18S ribosomal RNA gene bacteria bloom-forming species next-generation sequencing phytoplankton toxin-producing species Metabarcoding and Metagenomics 6: e79471 During the recent decade, high-throughput sequencing (HTS) techniques, in particular, DNA metabarcoding, have facilitated increased detection of biodiversity, including harmful algal bloom (HAB) species. In this study, the presence of HAB species and their appearance patterns were investigated by employing molecular and light microscopy-based monitoring in Tokyo Bay, Japan. The potential co-appearance patterns between the HAB species, as well as with other eukaryotes and prokaryotes were investigated using correlation and association rule-based time-series analysis. In total, 40 unique HAB species were detected, including 12 toxin-producing HAB species previously not reported from the area. More than half of the HAB species were present throughout the sampling season (summer to autumn) and no structuring or succession patterns associated with the environmental conditions could be detected. Statistically significant (p < 0.05, rS ranging from −0.88 to 0.90) associations were found amongst the HAB species and other eukaryotic and prokaryotic species, including genera containing growth-limiting bacteria. However, significant correlations between species differed amongst the years, indicating that variability in environmental conditions between the years may have a stronger influence on the microalgal community structure and interspecies interactions than the variability during the sampling season. The association rule-based time-series analysis allowed the detection of a previously reported negative relationship between Synechococcus sp. and Skeletonema sp. in nature. Overall, the results support the applicability of metabarcoding and HTS-based microalgae monitoring, as it facilitates more precise species identification compared to light microscopy, as well as provides input for investigating potential interactions amongst different species/groups through simultaneous detection of multiple species/genera. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.79471 https://doi.org/10.3897/mbmg.6.79471 https://mbmg.pensoft.net/article/79471/ https://mbmg.pensoft.net/article/79471/download/pdf/ en

10.3897/mbmg.6.87415 2022-09-23 mbmg

The relationship between eDNA density distribution and current fields around an artificial reef in the waters of Tateyama Bay, Japan INOUE,NARIAKI Sato,Masaaki Furuichi,Naoki Imaizumi,Tomohito Ushio,Masayuki ADCP artificial reef current environmental DNA fish Metabarcoding and Metagenomics 6: e87415 Monitoring of artificial reefs (ARs) has been conducted through such methods as visual censuses, surveys using fishing gear, and echo sounder. These methods have disadvantages: visual census is not possible at ARs in deeper waters, fishing gear surveys are invasive to fish individuals, and echo sounders have difficulty in species identification. A new AR monitoring method is required to compensate for these disadvantages. While eDNA has become a valid monitoring tool for marine biodiversities, it is influenced by degradation and transport of the molecules that affect information about the spatio-temporal distribution of fish. An understanding of the relationship between current fields and eDNA distribution, particularly in open waters, is critical when using eDNA as an index for fish aggregation at ARs. We investigated the relationship between eDNA distribution and current fields around an AR for four dominant species (Engraulis japonicus, Parapristipoma trilineatum, Scomber spp and Trachurus japonicus) in Tateyama Bay, Japan. The highest density of fish schools is formed directly above or at the upstream side of ARs. If we assume that the center of eDNA originates at these locations at an AR and eDNA is simply transported by currents, a higher density of eDNA would distribute downstream from the AR. However, our results indicate that eDNA distribution is in accord with actual fish distribution, namely eDNA densities are more abundant in the upstream side of ARs. We thus consider that eDNA distribution is more influenced by actual distribution patterns than by the transport processes. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.87415 https://doi.org/10.3897/mbmg.6.87415 https://mbmg.pensoft.net/article/87415/ https://mbmg.pensoft.net/article/87415/download/pdf/ en

10.3897/mbmg.6.89753 2022-09-23 mbmg

Assessing the lysis of diverse pollen from bulk environmental samples for DNA metabarcoding Moore,Madison A Scheible,Melissa KR Robertson,James B Meiklejohn,Kelly A DNA extraction DNA metabarcoding internal transcribed spacer 2 pollen surface soils Metabarcoding and Metagenomics 6: e89753 Pollen is ubiquitous year-round in bulk environmental samples and can provide useful information on previous and current plant communities. Characterization of pollen has traditionally been completed based on morphology, requiring significant time and expertise. DNA metabarcoding is a promising approach for characterizing pollen from bulk environmental samples, but accuracy hinges on successful lysis of pollen grains to free template DNA. In this study, we assessed the lysis of morphologically and taxonomically diverse pollen from one of the most common bulk environmental sample types for DNA metabarcoding, surface soil. To achieve this, a four species artificial pollen mixture was spiked into surface soils collected from Colorado, North Carolina, and Pennsylvania, and subsequently subjected to DNA extraction using both the PowerSoil and PowerSoil Pro Kits (Qiagen) with a heated incubation (either 65 °C or 90 °C). Amplification and Illumina sequencing of the internal transcribed spacer subunit 2 (ITS2) was completed in duplicate for each sample (total n, 76), and the resulting sequencing reads taxonomically identified using GenBank. The PowerSoil Pro Kit statistically outperformed the PowerSoil Kit for total DNA yield. When using either kit, incubation temperature (65 °C or 90 °C) used had no impact on the recovery of DNA, plant amplicon sequence variants (ASVs), or total plant ITS2 reads. This study highlighted that lysis of pollen in bulk environmental samples is feasible using commercially available kits, and downstream DNA metabarcoding can be used to accurately characterize pollen DNA from such sample types. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.89753 https://doi.org/10.3897/mbmg.6.89753 https://mbmg.pensoft.net/article/89753/ https://mbmg.pensoft.net/article/89753/download/pdf/ en

10.3897/mbmg.6.87497 2022-10-07 mbmg

Metabarcoding as an effective complement of microscopic studies in revealing the composition of the diatom community – a case study of an oxbow lake of Tisza River (Hungary) with the description of a new Mayamaea species Bíró,Tibor Duleba,Mónika Földi,Angéla Kiss,Keve Orgoványi,Péter Trábert,Zsuzsa Vadkerti,Edit Wetzel,Carlos Ács,Éva Mayamaea metabarcoding new diatom species oxbow Metabarcoding and Metagenomics 6: e87497 Diatoms are valuable bioindicators and their traditional classification and identification are mainly based on the morphological characteristics of their frustules. However, in recent years, DNA-based methods have been proposed and are rapidly growing in the scientific literature as a complementary tool to assess the ecological status of freshwaters. Diatom-based ecological status assessment uses indices calculated from sensitivity and tolerance values as well as relative abundance of species. Correct assessment requires an accurate identification of species. In the present study, diatom assemblages of an oxbow lake were investigated using light and scanning electron microscopy as well as metabarcoding using rbcL marker, and the identification results were compared, intending to match barcode sequences of species that are currently missing in the diatom reference database. The investigated oxbow is an important wetland for bird conservation, although it is impacted by land use. Taxon lists based on morphology and metabarcoding considerably differed when bioinformatics analysis involved DADA2 pipeline with Diat.barcode database. Previously unknown sequence variants of four pennate species were found with additional BLAST search. Using phylogeny and p-distance calculations sequences could be matched to three small-celled naviculoid species that were found under a microscope. One of them was found to be a new species of the genus Mayamaea and was described as a new species, Mayamaea ectorii. Additionally, spatial distribution maps for several small-celled naviculoid species are provided for the Hungarian territory. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.87497 https://doi.org/10.3897/mbmg.6.87497 https://mbmg.pensoft.net/article/87497/ https://mbmg.pensoft.net/article/87497/download/pdf/ en

10.3897/mbmg.6.85199 2022-10-07 mbmg

National eDNA-based monitoring of Batrachochytrium dendrobatidis and amphibian species in Norway Osman,Omneya Andersson,Johan Martin-Sanchez,Pedro M. Eiler,Alexander environmental monitoring invasive species Metabarcoding qPCR risk assessment Metabarcoding and Metagenomics 6: e85199 Freshwaters represent the most threatened environments with regard to biodiversity loss and, therefore, there is a need for national monitoring programs to effectively document species distribution and evaluate potential risks for vulnerable species. The monitoring of species for effective management practices is, however, challenged by insufficient data acquisition when using traditional methods. Here we present the application of environmental DNA (eDNA) metabarcoding of amphibians in combination with quantitative PCR (qPCR) assays for an invasive pathogenic chytrid species (Batrachochytrium dendrobatidis -Bd), a potential threat to endemic and endangered amphibian species. Statistical comparison of amphibian species detection using either traditional or eDNA-based approaches showed weak correspondence. By tracking the distribution of Bd over three years, we concluded that the risk for amphibian extinction is low since Bd was only detected at five sites where multiple amphibians were present over the sampled years. Our results show that eDNA-based detection can be used for simultaneous monitoring of amphibian diversity and the presence of amphibian pathogens at the national level in order to assess potential species extinction risks and establish effective management practices. As such our study represents suggestions for a national monitoring program based on eDNA. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.85199 https://doi.org/10.3897/mbmg.6.85199 https://mbmg.pensoft.net/article/85199/ https://mbmg.pensoft.net/article/85199/download/pdf/ en

10.3897/mbmg.6.89857 2022-10-24 mbmg

Environmental DNA metabarcoding is a promising method for assaying fish diversity in cenotes of the Yucatán Peninsula, Mexico Alter,S. Elizabeth Arroyave,Jairo eDNA freshwater fishes groundwater karst aquifer Metabarcoding and Metagenomics 6: e89857 The karst aquifer of the Yucatán Peninsula (YP) in southeastern Mexico is a unique ecosystem in which water-filled sinkholes, locally known as cenotes, connect subterranean waters with the surface. This system is home to around 20 species of freshwater fishes, including several that are endemic and/or threatened. Studies on this unique ichthyofauna have been partially hampered by the technical difficulties associated with sampling these habitats, particularly submerged caves. In this proof-of-concept study, we use environmental DNA (eDNA) metabarcoding to survey the diversity of freshwater fishes associated with the YP karst aquifer by sampling six cenotes from across the Ring of Cenotes region in northwestern Yucatán, a 180-km-diameter semicircular band of abundant karst sinkholes. Through a combination of conventional sampling (direct observation, fishing) and eDNA metabarcoding, we detected eight species of freshwater fishes across the six sampled cenotes. Overall, our eDNA metabarcoding approach was effective at detecting the presence of fishes from cenote water samples, including one of the two endemic cave-dwelling fish species restricted to the subterranean section of the aquifer. Although our study was focused on detecting fishes via eDNA, we also recovered DNA from several other vertebrate groups, particularly bats. These results suggest that the eDNA metabarcoding approach represents a promising and largely noninvasive method to assay aquatic biodiversity in these vulnerable habitats, allowing more effective, frequent, and wide-ranging surveys. Our detection of DNA from aerial and terrestrial vertebrate fauna implies that eDNA from cenotes, besides being a means to survey aquatic fauna, may also offer an effective way to quickly survey non-aquatic biodiversity associated with these persistent water bodies. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.89857 https://doi.org/10.3897/mbmg.6.89857 https://mbmg.pensoft.net/article/89857/ https://mbmg.pensoft.net/article/89857/download/pdf/ en

10.3897/mbmg.6.85844 2022-11-24 mbmg

Recommendations for the preservation of environmental samples in diatom metabarcoding studies Baricevic,Ana Chardon,Cécile Kahlert,Maria Karjalainen,Satu Maaria Maric Pfannkuchen,Daniela Pfannkuchen,Martin Rimet,Frederic Smodlaka Tankovic,Mirta Trobajo,Rosa Vasselon,Valentin Zimmermann,Jonas Bouchez,Agnès biomonitoring diatom assemblages DNA metabarcoding European Water Framework Directive methods sample preservation Metabarcoding and Metagenomics 6: e85844 Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa (< 100 reads) differed among preservation methods and storage durations. For biomonitoring purposes, freshwater ecological index values were not affected by the preservation method and storage duration tested (including ethanol preservation), all treatments returning the same ecological status for a site. This study contributes to consolidating diatom metabarcoding. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2022 Research Article text/html info:doi:10.3897/mbmg.6.85844 https://doi.org/10.3897/mbmg.6.85844 https://mbmg.pensoft.net/article/85844/ https://mbmg.pensoft.net/article/85844/download/pdf/ en

10.3897/mbmg.7.95650 2023-02-02 mbmg

Non-destructive insect metabarcoding as a surveillance tool for the Australian grains industry: a first trial for the iMapPESTS smart trap Martoni,Francesco Smith,Reannon Piper,Alexander Nancarrow,Narelle Aftab,Mohammad Trebicki,Piotr Kimber,Rohan Rodoni,Brendan Blacket,Mark aphids biosecurity diversity environmental data morphology Metabarcoding and Metagenomics 7: e95650 Surveillance and long-term monitoring of insect pest populations are of paramount importance to limit dispersal and inform pest management. Molecular methods have been employed in diagnostics, surveillance and monitoring for the past few decades, often paired with more traditional techniques relying on morphological examinations. Within this context, the ‘iMapPESTS: Sentinel Surveillance for Agriculture’ project was conceptualised to enhance on-farm pest management decision-making via development and deployment of smart traps, able to collect insects, as well as recording associated environmental data. Here, we compared an iMapPESTS ‘Sentinel’ smart trap to an alternative suction trap over a 10-week period. We used a non-destructive insect metabarcoding approach complemented by insect morphological diagnostics to assess and compare aphid species presence and diversity across trap samples and time. Furthermore, we paired this with environmental data recorded throughout the sampling period. This methodology recorded a total of 497 different taxa from 70 traps over a 10-week period in the grain-growing region in western Victoria. This included not only the 14 aphid target species, but an additional 12 aphid species, including a new record for Victoria. Ultimately, with more than 450 bycatch species detected, this highlighted the value of insect metabarcoding, not only for pest surveillance, but also at a broader ecosystem level, with potential applications in integrated pest management and biocontrol. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2023 Research Article text/html info:doi:10.3897/mbmg.7.95650 https://doi.org/10.3897/mbmg.7.95650 https://mbmg.pensoft.net/article/95650/ https://mbmg.pensoft.net/article/95650/download/pdf/ en

10.3897/mbmg.7.96733 2023-02-21 mbmg

Can the Eurasian otter (Lutra lutra) be used as an effective sampler of fish diversity? Using molecular assessment of otter diet to survey fish communities Dou,Hailong Wang,Mi Yin,Xuwang Feng,Limin Yang,Haitao 12S rRNA diet diversity sampler DNA barcoding species resolution spraints Metabarcoding and Metagenomics 7: e96733 The Eurasian otter Lutra lutra is a generalist carnivore that is widely distributed in many aquatic ecosystems. Based on its inherent attributes of opportunistic foraging behaviour and broad dietary range, it is naturally considered a potential sampler of the diversity of aquatic vertebrates. To test the ability and efficiency of otters as a diversity sampler, we used DNA metabarcoding to investigate the composition in vertebrates of the diet of otters that inhabit a forest stream area in northeast China. Twenty vertebrate prey taxa were detected in 98 otter spraints. Otter diet mainly comprised aquatic fishes (59.4%) and amphibians (39.0%). We also used traditional approaches to investigate fish communities at 60 sampling sites in the same area to determine the relationship between fish population composition in the environment and otter diet. The comparison revealed that 28 species of fish were distributed in this area, of which five are simultaneously detected in otter spraints. This indicates that molecular analysis of the diet of otters is not an ideal approach for investigating fish diversity, at least when using the 12SV5 primer pair. Based on a review of the available molecular research on otter diet, we conclude that the low species resolution may be due to the presence of many closely-related prey species in native habitats and lack of suitable barcodes. Considering the remarkable power of diet metabarcoding analysis in capturing elusive and rare species, it represents an approach that can compensate for the defects associated with fishing methods and we suggest that it can be used as an auxiliary means of measuring traditional fish diversity. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2023 Research Article text/html info:doi:10.3897/mbmg.7.96733 https://doi.org/10.3897/mbmg.7.96733 https://mbmg.pensoft.net/article/96733/ https://mbmg.pensoft.net/article/96733/download/pdf/ en

10.3897/mbmg.7.98539 2023-02-23 mbmg

Maximizing the reliability and the number of species assignments in metabarcoding studies using a curated regional library and a public repository Bourret,Audrey Nozeres,Claude Eric,Parent Parent,Genevieve J. classifier cytochrome C oxidase I GenBank marine species metagenomics reference sequence library Metabarcoding and Metagenomics 7: e98539 Biodiversity assessments relying on DNA have increased rapidly over the last decade. However, the reliability of taxonomic assignments in metabarcoding studies is variable and affected by the reference databases and the assignment methods used. Species level assignments are usually considered as reliable using regional libraries but unreliable using public repositories. In this study, we aimed to test this assumption for metazoan species detected in the Gulf of St. Lawrence in the Northwest Atlantic. We first created a regional library (GSL-rl) by data mining COI barcode sequences from BOLD, and included a reliability ranking system for species assignments. We then estimated 1) the accuracy and precision of the public repository NCBI-nt for species assignments using sequences from the regional library and 2) compared the detection and reliability of species assignments of a metabarcoding dataset using either NCBI-nt or the regional library and popular assignment methods. With NCBI-nt and sequences from the regional library, the BLAST-LCA (least common ancestor) method was the most precise method for species assignments, but the accuracy was higher with the BLAST-TopHit method (>80% over all taxa, between 70% and 90% amongst taxonomic groups). With the metabarcoding dataset, the reliability of species assignments was greater using GSL-rl compared to NCBI-nt. However, we also observed that the total number of reliable species assignments could be maximized using both GSL-rl and NCBI-nt with different optimized assignment methods. The use of a two-step approach for species assignments, i.e., using a regional library and a public repository, could improve the reliability and the number of detected species in metabarcoding studies. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC0 Pensoft Publishers 2023 Research Article text/html info:doi:10.3897/mbmg.7.98539 https://doi.org/10.3897/mbmg.7.98539 https://mbmg.pensoft.net/article/98539/ https://mbmg.pensoft.net/article/98539/download/pdf/ en

10.3897/mbmg.7.94298 2023-02-28 mbmg

Development of two new sets of PCR primers for eDNA metabarcoding of brittle stars (Echinodermata, Ophiuroidea) Okanishi,Masnaori Kohtsuka,Hisanori Wu,Qianqian Shinji,Jumpei Shibata,Naoki Tamada,Takashi Nakano,Tomoyuki Minamoto,Toshifumi 16SOph1 16SOph2 deep sea waters environmental monitoring mitochondrial 16S rRNA Sagami Bay Metabarcoding and Metagenomics 7: e94298 Brittle stars (class Ophiuroidea) are marine invertebrates comprising approximately 2,100 extant species, and are considered to constitute the most diverse taxon of the phylum Echinodermata. As a non-invasive method for monitoring biodiversity, we developed two new sets of PCR primers for metabarcoding environmental DNA (eDNA) from brittle stars. The new primer sets were designed to amplify 2 short regions of the mitochondrial 16S rRNA gene, comprising a conserved region (111–115 bp, 112 bp on average; named “16SOph1”) and a hyper-variable region (180–195 bp, 185 bp on average; named “16SOph2”) displaying interspecific variation. The performance of the primers was tested using eDNA obtained from two sources: a) rearing water of an 2.5 or 170 L aquarium tanks containing 15 brittle star species and b) from natural seawater collected around Misaki, the Pacific coast of central Japan, at depths ranging from shallow (2 m) to deep (> 200 m) sea. To build a reference library, we obtained 16S rRNA sequences of brittle star specimens collected from around Misaki and from similar depths in Japan, and sequences registered in International Nucleotide Sequence Database Collaboration. As a result of comparison of the obtained eDNA sequences with the reference library 37 (including cryptic species) and 26 brittle star species were detected with certain identities by 16SOph1 and 16SOph2 analyses, respectively. In shallow water, the number of species and reads other than the brittle stars detected with 16SOph1 was less than 10% of the total number. On the other hand, the number of brittle star species and reads detected with 16SOph2 was less than half of the total number, and the number of detected non-brittle star metazoan species ranged from 20 to 46 species across 6 to 8 phyla (only the reads at the “Tank” were less than 0.001%). The number of non-brittle star species and reads at 80 m was less than 10% with both of the primer sets. These findings suggest that 16SOph1 is specific to the brittle star and 16SOph2 is suitable for a variety of marine metazoans. It appears, however, that further optimization of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts. Moreover, a detailed elucidation of the brittle star fauna in the examined area, and the accurate identification of brittle star species in the current DNA databank is required. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2023 Research Article text/html info:doi:10.3897/mbmg.7.94298 https://doi.org/10.3897/mbmg.7.94298 https://mbmg.pensoft.net/article/94298/ https://mbmg.pensoft.net/article/94298/download/pdf/ en

10.3897/mbmg.7.101266 2023-06-13 mbmg

Does phylogeny explain bias in quantitative DNA metabarcoding? Liu,Mingxin Burridge,Christopher Clarke,Laurence Baker,Susan jordan,greg Coleoptera high-throughput sequencing PCR amplification phylogenetic signal species biomass Metabarcoding and Metagenomics 7: e101266 Estimating species biomass or abundance from the number of high-throughput sequencing (HTS) reads is an aspirational goal for DNA metabarcoding, yet studies have found varied correlations. Performance varies depending on the gene marker and taxonomic group and, in part, may be related to primer-template mismatches, which are likely to exhibit phylogenetic signals. In this study, we compared commonly used fragments of two gene markers for beetles, the mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S), which have similar lengths, but different propensity for primer-template mismatches. We tested whether primer-template mismatches influence the relationship between species biomass and HTS read abundance and whether the effect of mismatches was explained by phylogeny. A significant correlation between species biomass and HTS read abundance existed for 16S, but not for COI, which had more primer-template mismatches. Models incorporating the effects of mismatch type or number improved the estimation of species biomass from HTS read abundance for COI and strong phylogenetic signals were identified. Researchers seeking to quantify biomass from metabarcoding studies should consider the effect of primer-template mismatches for the taxonomic group of interest and, for beetles, 16S appears a good candidate. Phylogenetic correction can also improve biomass estimation when using gene markers with higher primer mismatching. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2023 Research Article text/html info:doi:10.3897/mbmg.7.101266 https://doi.org/10.3897/mbmg.7.101266 https://mbmg.pensoft.net/article/101266/ https://mbmg.pensoft.net/article/101266/download/pdf/ en

10.3897/mbmg.7.86883 2023-07-05 mbmg

Pollen metabarcoding of museum specimens and recently collected bumblebees (Bombus) indicates foraging shifts Kolter,Andreas Husemann,Martin Podsiadlowski,Lars Gemeinholzer,Birgit barcoding Bombus bumblebee Cuxhaven Hamburg ITS1 ITS ITS2 natural history collection plant metabarcoding pollen trnL-F P6 Metabarcoding and Metagenomics 7: e86883 Landscape changes, over time, lead to changes of floral resources available to pollinators, which in turn may result in the disappearance of ecologically specialized species. Here, we use pollen metabarcoding to infer historic and recent interactions between plants and bumblebees (Bombus). Bumblebees from Cuxhaven (Germany) were sampled from historical museum collections (1968/69) and in the field (2019). Pollen attached to their bodies was barcoded using multiple plant markers (ITS1, ITS2 and trnL-P6 loop). Our results show shifts in foraging habits between the historic and recent sampling periods, mostly determined by fewer Fabaceae interactions in 2019. The successful implementation of scalable molecular techniques for the analysis of historical pollen samples underscores the value of museum collections as a resource for biodiversity research. This study provides proof of concept of a comparative analysis of recent and historical pollination data. However, to ensure the robustness of our results, it is crucial to consider the broader methodology used. Our study found variation in the efficacy of the three plant barcoding markers. The ITS1 marker exhibited the highest species-level identification success, while the trnL-P6 loop demonstrated utility in amplifying degraded DNA across diverse plant families. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2023 Research Article text/html info:doi:10.3897/mbmg.7.86883 https://doi.org/10.3897/mbmg.7.86883 https://mbmg.pensoft.net/article/86883/ https://mbmg.pensoft.net/article/86883/download/pdf/ en

10.3897/mbmg.7.102455 2023-07-25 mbmg

Direct PCR meets high-throughput sequencing – metabarcoding of chironomid communities without DNA extraction Röder,Nina Schwenk,Klaus cytochrome c oxidase I (COI or COX1) DNA isolation metabarcoding next-generation sequencing (NGS) presence-absence community composition read abundance size-sorting Metabarcoding and Metagenomics 7: e102455 Aquatic emergent insect communities form an important link between aquatic and terrestrial ecosystems, yet studying them is costly and time-consuming as they are usually diverse and superabundant. Metabarcoding is a valuable tool to investigate arthropod community compositions, however high-throughput applications, such as for biomonitoring, require cost-effective and user-friendly procedures. To investigate if the time-consuming and labour-intensive DNA extraction step can be omitted in metabarcoding, we studied the difference in detection rates and individual read abundance using standard DNA extraction versus direct PCR protocols. Metabarcoding with and without DNA extraction was performed with artificially created communities of known composition as well as on natural communities both of the dipteran family Chironomidae to compare detection rates, individual read abundances and presence-absence community composition. We found that the novel approach of direct PCR metabarcoding presented here did not alter detection rates and had a minor effect on individual read abundances in artificially created communities. Furthermore, presence-absence community compositions of natural chironomid communities were highly comparable using both approaches. In conclusion, we showed that direct PCR protocols can be applied in chironomid metabarcoding approaches, with possible application for a wider range of arthropod taxa, enabling us to study communities more efficiently in the future. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2023 Research Article text/html info:doi:10.3897/mbmg.7.102455 https://doi.org/10.3897/mbmg.7.102455 https://mbmg.pensoft.net/article/102455/ https://mbmg.pensoft.net/article/102455/download/pdf/ en

10.3897/mbmg.7.106844 2023-07-25 mbmg

DNA metabarcoding data from faecal samples of the lesser (Myotis blythii) and the greater (Myotis myotis) mouse-eared bats from Bulgaria Hubancheva,Antoniya Bozicevic,Vedran Moriniere,Jerome Goerlitz,Holger R. Bats (Chiroptera) metabarcoding Myotis myotis Myotis blythii parasite-host interactions predator-prey interactions Metabarcoding and Metagenomics 7: e106844 A comprehensive understanding of trophic interactions in terrestrial ecosystems is crucial for ecological research and conservation. Recent advances in non-invasive methods, such as environmental DNA (eDNA) metabarcoding, have enabled researchers to collect vast amounts of data on wild animal diets. However, sharing this data and metadata effectively and transparently presents new challenges. To address this, a new type of scholarly journal publication has emerged that aims to describe datasets rather than report research investigations. In this paper, we present a dataset of consumed prey species and parasites based on the metabarcoding of 113 faecal samples from the greater and lesser mouse-eared bats (Myotis myotis and Myotis blythii), along with a detailed description of the data sampling, laboratory analysis, and bioinformatics pipeline. Our dataset comprises 1018 unique Barcode Index Numbers (BINs) from 12 Classes and 43 Orders. In addition, we provide interactive Krona charts to visually summarize the taxonomic relationships and relative read abundance of the consumed prey species and parasites. This data can be used for meta-analysis, exploring new predator-prey and host-parasite interactions, studying inter- and intraspecific ecological interactions, and informing protected area management, among other applications. By sharing this dataset, we hope to encourage other researchers to use it to answer additional ecological questions and advance our understanding of trophic interactions in terrestrial ecosystems. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2023 Data Paper text/html info:doi:10.3897/mbmg.7.106844 https://doi.org/10.3897/mbmg.7.106844 https://mbmg.pensoft.net/article/106844/ https://mbmg.pensoft.net/article/106844/download/pdf/ en

10.3897/mbmg.7.99979 2023-08-09 mbmg

Ecological signals of arctic plant-microbe associations are consistent across eDNA and vegetation surveys Parisy,Bastien Schmidt,Niels Martin Wirta,Helena Stewart,Laerke Pellissier,Loic Holben,Bill Pannoni,Sam Somervuo,Panu Jones,Mirkka Siren,Jukka Vesterinen,Eero Ovaskainen,Otso Roslin,Tomas eDNA metabarcoding environmental gradients Greenland joint species distribution model observational data plant-soil microbe associations vegetation assessment Metabarcoding and Metagenomics 7: e99979 Understanding how different taxa respond to abiotic characteristics of the environment is of key interest for understanding the assembly of communities. Yet, whether eDNA data will suffice to accurately capture environmental imprints has been the topic of some debate. In this study, we characterised patterns of species occurrences and co-occurrences in Zackenberg in northeast Greenland using environmental DNA. To explore the potential for extracting ecological signals from eDNA data alone, we compared two approaches (visual vegetation surveys and soil eDNA metabarcoding) to describing plant communities and their responses to abiotic conditions. We then examined plant associations with microbes using a joint species distribution model. We found that most (68%) of plant genera were detectable by both vegetation surveys and eDNA signatures. Species-specific occurrence data revealed how plants, bacteria and fungi responded to their abiotic environment – with plants, bacteria and fungi all responding similarly to soil moisture. Nonetheless, a large proportion of fungi decreased in occurrences with increasing soil temperature. Regarding biotic associations, the nature and proportion of the plant-microbe associations detected were consistent between plant data identified via vegetation surveys and eDNA. Of pairs of plants and microbe genera showing statistically supported associations (while accounting for joint responses to the environment), plants and bacteria mainly showed negative associations, whereas plants and fungi mainly showed positive associations. Ample ecological signals detected by both vegetation surveys and by eDNA-based methods and a general correspondence in biotic associations inferred by both methods, suggested that purely eDNA-based approaches constitute a promising and easily applicable tool for studying plant-soil microbial associations in the Arctic and elsewhere. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2023 Research Article text/html info:doi:10.3897/mbmg.7.99979 https://doi.org/10.3897/mbmg.7.99979 https://mbmg.pensoft.net/article/99979/ https://mbmg.pensoft.net/article/99979/download/pdf/ en

10.3897/mbmg.7.103279 2023-09-05 mbmg

Unravelling bird nest arthropod community structure using metabarcoding Levesque-Beaudin,Valerie Steinke,Dirk Böcker,Mieke Thalinger,Bettina Berlese-Tullgren funnel dust environmental DNA Passeriformes PCR inhibition trophic interactions Metabarcoding and Metagenomics 7: e103279 Bird nests are fascinating microcosms harboring a wide range of arthropods parasitizing the nesting birds or feeding on prey remains, feces, and the nest material. Studies of these communities have been entirely based on emergence traps which collect live organisms out of the nests. The analysis of nest contents and environmental DNA (eDNA) via metabarcoding could expand our knowledge and identify prey, exuviae, and other animal remains in bird nests. Here, we investigated the potential of arthropod remains, nest dust, and feathers to better describe arthropod diversity accumulated in 20 bird nests collected in Guelph (Canada). We used subsampling strategies and tested two extraction approaches to investigate the distribution of DNA in nests, account for low-quality DNA, and the presence of inhibitory substances. In total, 103 taxa were detected via metabarcoding. Arthropod remains delivered the highest number of taxa (n = 67), followed by nest dust (n = 29). Extractions with the PowerSoil kit outperformed DNeasy extractions coupled with PowerClean Pro inhibitor removal. Per nest, on average 5.5% taxonomic overlap between arthropod remains of different size classes was detected and subsamples of nest dust extracted with the PowerSoil kit showed 47.3% taxonomic overlap indicating a heterogeneous eDNA distribution in nests. Most detected species were either feeding in the nest, i.e., herbivorous / predatory, or bird food. We also detected molecular traces of 25 bird species, whose feathers were likely used as nest material. Consequently, the metabarcoding of bird nest materials provides a more complete picture of nest communities, which can enable future studies on functional diversity and better comparisons between nesting species. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2023 Research Article text/html info:doi:10.3897/mbmg.7.103279 https://doi.org/10.3897/mbmg.7.103279 https://mbmg.pensoft.net/article/103279/ https://mbmg.pensoft.net/article/103279/download/pdf/ en

10.3897/mbmg.7.103856 2023-09-05 mbmg

Evaluating five primer pairs for environmental DNA metabarcoding of Central European fish species based on mock communities Macher,Till-Hendrik Schütz,Robin Yildiz,Atakan Beermann,Arne Leese,Florian 12S 16S biomonitoring COI eDNA Metabarcoding and Metagenomics 7: e103856 Environmental DNA (eDNA) metabarcoding has become a powerful tool for examining fish communities. Prior to the introduction of eDNA-based assessments into regulatory monitoring contexts (e.g., EU Water Framework Directive), there is a demand for methodological standardization. To ensure methodical accuracy and to meet regulatory standards, various sampling, laboratory and bioinformatic workflows have been established. However, a crucial prerequisite for comprehensive fish monitoring is the choice of suitable primer pairs to accurately identify the fishes present in a given water body. Various fish-specific primer pairs targeting different genetic marker regions were published over the past decade. However, a dedicated study to evaluate the performance of frequently applied fish primer pairs to assess Central European fish species has not yet been conducted. Therefore, we created an artificial 'mock' community composed of DNA from 45 Central European fish species and examined the detection ability and reproducibility of five primer pairs. Our study highlights the effect of primer choice and bioinformatic filtering on the outcome of eDNA metabarcoding results. From the five primer pairs evaluated in our study the tele02 (12S gene) primer pair was the best choice for eDNA metabarcoding of Central European freshwater fish. Also, the MiFish-U (12S) and SeaDNA-mid (COI) primer pairs displayed good detection ability and reproducibility. However, less specific primer pairs (i.e., targeting vertebrates) were found to be less reliable and generated high numbers of false-positive and false-negative detections. Our study illustrates how the careful selection of primer pairs and bioinformatic pipelines can make eDNA metabarcoding a more reliable tool for fish monitoring. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2023 Primer Validation text/html info:doi:10.3897/mbmg.7.103856 https://doi.org/10.3897/mbmg.7.103856 https://mbmg.pensoft.net/article/103856/ https://mbmg.pensoft.net/article/103856/download/pdf/ en

10.3897/mbmg.7.96780 2023-10-18 mbmg

Fish diversity assessed by eDNA detection methods in the Rioni River Beridze,Tamar Japoshvili,Bella Edisherashvili,Tamari Anderson,Cort Mumladze,Levan Caucasus eDNA fish diversity Rioni River Metabarcoding and Metagenomics 7: e96780 Due to anthropogenic influences, habitat degradation and a continuous loss of biodiversity in freshwater ecosystems are occurring on a large scale, while these ecosystems constitute invaluable natural resources. Therefore, it is essential to study and monitor freshwater ecosystems to guide conservation efforts. Freshwater ecosystems are one of the less-studied fields in Georgia. Studies about the species distribution of many taxa and/or regions carried out during the last century have not been updated for decades. Here, we report the results of an environmental DNA (eDNA) metabarcoding exercise, based on samples collected from the Rioni River, a tributary to the Black Sea and a crucial aquatic ecosystem regionally and globally. The only comprehensive review of the fish of the Rioni River dates back to 1956. We compared the eDNA-based taxonomic composition to the known faunal composition within the Rioni River and found that the eDNA-based taxonomic coverage approached 75% of the expected total fish fauna. A number of new species occurrences were also found, including the first detection of three invasive alien species (Carassius gibelio, Pseudorasbora parva, Rhinogobius lindbergi) in the Rionis River Basin and a new country record of the ninespine stickleback (genus Pungitius) for Georgia. In spite of the usefulness of the eDNA metabarcoding approach, the sparsity of the fish DNA barcode reference library for the region emerged as a limitation to this study. However, our findings still represent a great leap forward in updating fish status on the Rioni River and testing the effectiveness of the eDNA sampling for aquatic species. info:eu-repo/semantics/altIdentifier/eissn/2534-9708 info:eu-repo/semantics/openAccess CC BY 4.0 Pensoft Publishers 2023 Applied Study text/html info:doi:10.3897/mbmg.7.96780 https://doi.org/10.3897/mbmg.7.96780 https://mbmg.pensoft.net/article/96780/ https://mbmg.pensoft.net/article/96780/download/pdf/ en cGFnZT0xJnNldD1tYm1nJmZyb209JnVudGlsPSZtZXRhZGF0YV9wcmVmaXg9b2FpX2Rj