Abstract: Metabarcoding and Metagenomics 7: e94298 DOI : 10.3897/mbmg.7.94298 Authors : Masanori Okanishi, Hisanori Kohtsuka, Qianqian Wu, Junpei Shinji, Naoki Shibata, Takashi Tamada, Tomoyuki Nakano, Toshifumi Minamoto : 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. HTML XML PDF PubDate: Tue, 28 Feb 2023 02:59:00 +020
Abstract: Metabarcoding and Metagenomics 7: e98539 DOI : 10.3897/mbmg.7.98539 Authors : Audrey Bourret, Claude Nozères, Eric Parent, Geneviève J. Parent : 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. HTML XML PDF PubDate: Thu, 23 Feb 2023 18:11:22 +020
Abstract: Metabarcoding and Metagenomics 7: e96733 DOI : 10.3897/mbmg.7.96733 Authors : Hailong Dou, Mi Wang, Xuwang Yin, Limin Feng, Haitao Yang : 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. HTML XML PDF PubDate: Tue, 21 Feb 2023 19:19:29 +020
Abstract: Metabarcoding and Metagenomics 7: e95650 DOI : 10.3897/mbmg.7.95650 Authors : Francesco Martoni, Reannon L. Smith, Alexander M. Piper, Narelle Nancarrow, Mohammad Aftab, Piotr Trebicki, Rohan B. E. Kimber, Brendan C. Rodoni, Mark J. Blacket : 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. HTML XML PDF PubDate: Thu, 2 Feb 2023 14:38:16 +0200
Abstract: Metabarcoding and Metagenomics 6: e85844 DOI : 10.3897/mbmg.6.85844 Authors : Ana Baricevic, Cécile Chardon, Maria Kahlert, Satu Maaria Karjalainen, Daniela Maric Pfannkuchen, Martin Pfannkuchen, Frédéric Rimet, Mirta Smodlaka Tankovic, Rosa Trobajo, Valentin Vasselon, Jonas Zimmermann, Agnès Bouchez : 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. HTML XML PDF PubDate: Thu, 24 Nov 2022 18:40:59 +020
Abstract: Metabarcoding and Metagenomics 6: e89857 DOI : 10.3897/mbmg.6.89857 Authors : S. Elizabeth Alter, Jairo Arroyave : 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. HTML XML PDF PubDate: Mon, 24 Oct 2022 11:16:02 +030
Abstract: Metabarcoding and Metagenomics 6: e85199 DOI : 10.3897/mbmg.6.85199 Authors : Omneya Ahmed Osman, Johan Andersson, Pedro M. Martin-Sanchez, Alexander Eiler : 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. HTML XML PDF PubDate: Fri, 7 Oct 2022 17:58:45 +0300
Abstract: Metabarcoding and Metagenomics 6: e87497 DOI : 10.3897/mbmg.6.87497 Authors : Tibor Bíró, Mónika Duleba, Angéla Földi, Keve T. Kiss, Péter Orgoványi, Zsuzsa Trábert, Edit Vadkerti, Carlos E. Wetzel, Éva Ács : 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. HTML XML PDF PubDate: Fri, 7 Oct 2022 12:13:13 +0300
Abstract: Metabarcoding and Metagenomics 6: e89753 DOI : 10.3897/mbmg.6.89753 Authors : Madison A. Moore, Melissa K.R. Scheible, James B. Robertson, Kelly A. Meiklejohn : 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. HTML XML PDF PubDate: Fri, 23 Sep 2022 11:01:27 +030
Abstract: Metabarcoding and Metagenomics 6: e87415 DOI : 10.3897/mbmg.6.87415 Authors : Nariaki Inoue, Masaaki Sato, Naoki Furuichi, Tomohito Imaizumi, Masayuki Ushio : 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. HTML XML PDF PubDate: Fri, 23 Sep 2022 11:01:13 +030
Abstract: Metabarcoding and Metagenomics 6: e79471 DOI : 10.3897/mbmg.6.79471 Authors : Sirje Sildever, Noriko Nishi, Nobuharu Inaba, Taiga Asakura, Jun Kikuchi, Yasuhito Asano, Takanori Kobayashi, Takashi Gojobori, Satoshi Nagai : 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. HTML XML PDF PubDate: Tue, 23 Aug 2022 18:50:32 +030
Abstract: Metabarcoding and Metagenomics 6: e85794 DOI : 10.3897/mbmg.6.85794 Authors : Jon Lapeyra Martin, Ioulia Santi, Paraskevi Pitta, Uwe John, Nathalie Gypens : 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. HTML XML PDF PubDate: Mon, 15 Aug 2022 10:36:12 +030
Abstract: Metabarcoding and Metagenomics 6: e84960 DOI : 10.3897/mbmg.6.84960 Authors : R. Henrik Nilsson, Anders F. Andersson, Andrew Bissett, Anders G. Finstad, Frode Fossøy, Marie Grosjean, Michael Hope, Thomas S. Jeppesen, Urmas Kõljalg, Daniel Lundin, Maria Prager, Saara Suominen, Cecilie S. Svenningsen, Dmitry Schigel : 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. HTML XML PDF PubDate: Tue, 2 Aug 2022 10:58:49 +0300
Abstract: Metabarcoding and Metagenomics 6: e85213 DOI : 10.3897/mbmg.6.85213 Authors : Stephanie J. Swenson, Lisa Eichler, Thomas Hörren, Andreas Kolter, Sebastian Köthe, Gerlind U. C. Lehmann, Gotthard Meinel, Roland Mühlethaler, Martin Sorg, Birgit Gemeinholzer : 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. HTML XML PDF PubDate: Fri, 22 Jul 2022 17:57:26 +030
Abstract: Metabarcoding and Metagenomics 6: e85652 DOI : 10.3897/mbmg.6.85652 Authors : Philippe Blancher, Estelle Lefrançois, Frédéric Rimet, Valentin Vasselon, Christine Argillier, Jens Arle, Pedro Beja, Pieter Boets, Jeanne Boughaba, Christian Chauvin, Michael Deacon, Willie Duncan, Gunilla Ejdung, Stefania Erba, Benoit Ferrari, Helmut Fischer, Bernd Hänfling, Michael Haldin, Daniel Hering, Nicolas Hette-Tronquart, Alice Hiley, Marko Järvinen, Benjamin Jeannot, Maria Kahlert, Martyn Kelly, Julia Kleinteich, Serdar Koyuncuoğlu, Sascha Krenek, Sidsel Langhein-Winther, Florian Leese, David Mann, Rémy Marcel, Stefania Marcheggiani, Kristian Meissner, Patricia Mergen, Olivier Monnier, Frank Narendja, Diane Neu, Veronica Onofre Pinto, Alina Pawlowska, Jan Pawlowski, Martin Petersen, Sandra Poikane, Didier Pont, Marie-Sophie Renevier, Steinar Sandoy, Jonas Svensson, Rosa Trobajo, Andrea Tünde Zagyva, Iakovos Tziortzis, Berry van der Hoorn, Marlen Ines Vasquez, Kerry Walsh, Alexander Weigand, Agnès Bouchez : 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. HTML XML PDF PubDate: Wed, 20 Jul 2022 13:22:09 +030
Abstract: Metabarcoding and Metagenomics 6: e84331 DOI : 10.3897/mbmg.6.84331 Authors : Nuria Jiménez Elvira, Masayuki Ushio, Shoko Sakai : 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. HTML XML PDF PubDate: Wed, 20 Jul 2022 13:21:21 +030
Abstract: Metabarcoding and Metagenomics 6: e78871 DOI : 10.3897/mbmg.6.78871 Authors : Daniel Marquina, Tomas Roslin, Piotr Łukasik, Fredrik Ronquist : 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. HTML XML PDF PubDate: Thu, 16 Jun 2022 19:07:36 +030
Abstract: Metabarcoding and Metagenomics 6: e80139 DOI : 10.3897/mbmg.6.80139 Authors : Eugenia Naro-Maciel, Melissa R. Ingala, Irena E. Werner, Brendan N. Reid, Allison M. Fitzgerald : 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. HTML XML PDF PubDate: Fri, 10 Jun 2022 13:00:48 +030
Abstract: Metabarcoding and Metagenomics 6: e68575 DOI : 10.3897/mbmg.6.e68575 Authors : Gert-Jan Jeunen, Tatsiana Lipinskaya, Helen Gajduchenko, Viktoriya Golovenchik, Michail Moroz, Viktor Rizevsky, Vitaliy Semenchenko, Neil J. Gemmell : 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. HTML XML PDF PubDate: Fri, 10 Jun 2022 08:46:58 +030
Abstract: Metabarcoding and Metagenomics 6: e80416 DOI : 10.3897/mbmg.6.80416 Authors : Sanni Hintikka, Jeanette E.L. Carlsson, Jens Carlsson : 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. HTML XML PDF PubDate: Tue, 31 May 2022 18:57:07 +030
Abstract: Metabarcoding and Metagenomics 6: e80444 DOI : 10.3897/mbmg.6.80444 Authors : Masaki Miya, Tetsuya Sado, Shin-ichiro Oka, Takehiko Fukuchi : 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. HTML XML PDF PubDate: Mon, 23 May 2022 12:09:02 +030
Abstract: Metabarcoding and Metagenomics 6: e79351 DOI : 10.3897/mbmg.6.79351 Authors : François Keck, Samuel Hürlemann, Nadine Locher, Christian Stamm, Kristy Deiner, Florian Altermatt : 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. HTML XML PDF PubDate: Tue, 10 May 2022 17:31:17 +030
Abstract: Metabarcoding and Metagenomics 6: e79208 DOI : 10.3897/mbmg.6.79208 Authors : John K. Pearman, Laura Casas, Craig Michell, Naroa Aldanondo, Nazia Mojib, Karie Holtermann, Ioannis Georgakakis, Joao Curdia, Susana Carvalho, Amr Gusti, Xabier Irigoien : 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. HTML XML PDF PubDate: Tue, 12 Apr 2022 13:23:41 +030
Abstract: Metabarcoding and Metagenomics 6: e78762 DOI : 10.3897/mbmg.6.78762 Authors : Brendan N. Reid, Jennifer A. Servis, Molly Timmers, Forest Rohwer, Eugenia Naro-Maciel : 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. HTML XML PDF PubDate: Tue, 12 Apr 2022 13:23:05 +030
Abstract: Metabarcoding and Metagenomics 6: e78756 DOI : 10.3897/mbmg.6.78756 Authors : Physilia Y. S. Chua, Christian Carøe, Alex Crampton-Platt, Claudia S. Reyes-Avila, Gareth Jones, Daniel G. Streicker, Kristine Bohmann : 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. HTML XML PDF PubDate: Thu, 7 Apr 2022 10:00:13 +0300
Abstract: Metabarcoding and Metagenomics 6: e79265 DOI : 10.3897/mbmg.6.79265 Authors : Laura Biessy, John K. Pearman, Sean Waters, Marcus J. Vandergoes, Susanna A. Wood : 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. HTML XML PDF PubDate: Fri, 25 Mar 2022 14:57:33 +020
Abstract: Metabarcoding and Metagenomics 6: e78181 DOI : 10.3897/mbmg.6.78181 Authors : Sayaka Sogawa, Kenji Tsuchiya, Satoshi Nagai, Shinji Shimode, Victor S. Kuwahara : 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. HTML XML PDF PubDate: Mon, 28 Feb 2022 10:05:31 +020
Abstract: Metabarcoding and Metagenomics 6: e77704 DOI : 10.3897/mbmg.6.77704 Authors : Satoshi Nagai, Sirje Sildever, Noriko Nishi, Satoshi Tazawa, Leila Basti, Takanori Kobayashi, Yoshizumi Ishino : 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. HTML XML PDF PubDate: Mon, 21 Feb 2022 11:33:29 +020
Abstract: Metabarcoding and Metagenomics 6: e76534 DOI : 10.3897/mbmg.6.76534 Authors : Masayuki K. Sakata, Mone U. Kawata, Atsushi Kurabayashi, Takaki Kurita, Masatoshi Nakamura, Tomoyasu Shirako, Ryosuke Kakehashi, Kanto Nishikawa, Mohamad Yazid Hossman, Takashi Nishijima, Junichi Kabamoto, Masaki Miya, Toshifumi Minamoto : 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. HTML XML PDF PubDate: Mon, 21 Feb 2022 08:59:01 +020
Abstract: Metabarcoding and Metagenomics 6: e78128 DOI : 10.3897/mbmg.6.78128 Authors : Katie A. Brasell, Xavier Pochon, Jamie Howarth, John K. Pearman, Anastasija Zaiko, Lucy Thompson, Marcus J. Vandergoes, Kevin S. Simon, Susanna A. Wood : 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. HTML XML PDF PubDate: Tue, 1 Feb 2022 10:01:36 +0200
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