Friday, May 1, 2020

Weekend reads -- Week 18/2020

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I am revitalizing an older tradition of this blog. A weekly (very subjective) collection of papers relating to DNA barcoding, metabarcoding and everything related:

Insects form an established part of the diet in many parts of the world and insect food products are emerging into the European and North American marketplaces. Consumer confidence in product is key in developing this market, and accurate labelling of content identity is an important component of this. We used DNA barcoding to assess the accuracy of insect food products sold in the UK. We purchased insects sold for human consumption from online retailers in the UK and compared the identity of the material ascertained from DNA barcoding to that stated on the product packaging. To this end, the COI sequence of mitochondrial DNA was amplified and sequenced, and compared the sequences produced to reference sequences in NCBI and the Barcode of Life Data System (BOLD). The barcode identity of all insects that were farmed was consistent with the packaging label. In contrast, disparity between barcode identity and package contents was revealed in two cases of foraged material (mopane worm and winged termites). One case of very broad family-level description was also highlighted, where material described as grasshopper was identified as Locusta migratoria from DNA barcode. Overall these data indicate the need to establish tight protocols to validate product identity in this developing market. Maintaining biosafety and consumer confidence rely on accurate and consistent product labelling that provides a clear chain of information from producer to consumer.

Walnut (Juglans regia L.) is one of the most widely cultivated nuts. Walnut milk beverage is very popular in China due to its nutritional value. However, adulterated walnut milk ingredients have been detected in the Chinese market. Peanut and soybean are sold at much lower prices than walnut and are reported to be commonly used for adulteration in the industrial chain of walnut milk production. The purpose of this study is therefore to develop an accurate and efficient method for detecting the authenticity of the raw materials used in walnut milk beverage. DNA barcoding and high‐resolution melting (HRM) analyses were used to identify common adulterated raw ingredients such as peanut and soybean in commercial walnut milk beverage samples. The chloroplast psbA‐trnH gene was used for sequencing, and HRM analysis was performed. We also prepared experimental mixtures, in the laboratory, with different quantities of walnut, peanut, and soybean. High‐resolution melting analysis of the experimental mixtures clearly distinguished all of them. The results revealed that most of the walnut milk beverage samples fell in the same cluster of walnut species. Several samples fell in the peanut cluster, confirming that they were adulterated products. The results revealed that HRM analysis based on the psbA‐trnH barcode sequence can be used to identify raw ingredients in walnut milk beverages. 

Accurate and cost-effective methods for tracking changes in arthropod communities are needed to develop integrative environmental monitoring programs in the Arctic. To date, even baseline data on their species composition at established ecological monitoring sites are severely lacking. We present the results of a pilot assessment of non-marine arthropod diversity in a middle arctic tundra area near Ikaluktutiak (Cambridge Bay), Victoria Island, Nunavut, undertaken in 2018 using DNA barcodes. A total of 1264 Barcode Index Number (BIN) clusters, used as a proxy for species, were recorded. The efficacy of widely used sampling methods was assessed. Yellow pan traps captured 62% of the entire BIN diversity at the study sites. When complemented with soil and leaf litter sifting, the coverage rose up to 74.6%. Combining community-based data collection with high-throughput DNA barcoding has the potential to overcome many of the logistic, financial, and taxonomic obstacles for large-scale monitoring of the Arctic arthropod fauna.

Improved taxonomic methods are needed to quantify declining populations of insect pollinators. This study devises a high‐throughput DNA barcoding protocol for a regional fauna (United Kingdom) of bees (Apiformes), consisting of reference library construction, a proof‐of‐concept monitoring scheme, and the deep barcoding of individuals to assess potential artefacts and organismal associations. A reference database of cytochrome oxidase c subunit 1 (cox1) sequences including 92.4% of 278 bee species known from the UK showed high congruence with morphological taxon concepts, but molecular species delimitations resulted in numerous split and (fewer) lumped entities within the Linnaean species. Double tagging permitted deep Illumina sequencing of 762 separate individuals of bees from a UK‐wide survey. Extracting the target barcode from the amplicon mix required a new protocol employing read abundance and phylogenetic position, which revealed 180 molecular entities of Apiformes identifiable to species. An additional 72 entities were ascribed to nuclear pseudogenes based on patterns of read abundance and phylogenetic relatedness to the reference set. Clustering of reads revealed a range of secondary operational taxonomic units (OTUs) in almost all samples, resulting from traces of insect species caught in the same traps, organisms associated with the insects including a known mite parasite of bees, and the common detection of human DNA, besides evidence for low‐level cross‐contamination in pan traps and laboratory procedures. Custom scripts were generated to conduct critical steps of the bioinformatics protocol. The resources built here will greatly aid DNA‐based monitoring to inform management and conservation policies for the protection of pollinators.

Freshwaters face some of the highest rates of species loss, caused by strong human impact. To decrease or even revert this strong impact, ecological restorations are increasingly applied to restore and maintain the natural ecological status of freshwaters. Their ecological status can be determined by assessing the presence of indicator species (e.g., certain fish species), which is called biomonitoring. However, traditional biomonitoring of fish, such as electrofishing, is often challenging and invasive. To augment traditional biomonitoring of fish, the analysis of environmental DNA (eDNA) has recently been proposed as an alternative, sensitive approach. The present study employed this modern approach to monitor the Rhine sculpin (Cottus rhenanus), a fish species that has been reintroduced into a recently restored stream within the Emscher catchment in Germany, in order to validate the success of the applied restorations and to monitor the species’ dispersal. We monitored the dispersal of the Rhine sculpin using replicated 12S end-point nested PCR eDNA surveillance at a fine spatial and temporal scale. In that way, we investigated if eDNA analysis can be applied for freshwater assessments. We also performed traditional electrofishing in one instance to validate our eDNA-based approach. We could track the dispersal of the Rhine sculpin and showed a higher dispersal potential of the species than we assumed. eDNA detection indicated the species’ dispersal across a potential dispersal barrier and showed a steep increase of positive detections once the reintroduced population had established. In contrast to that, false negative eDNA results occurred at early reintroduction stages. Our results show that eDNA detection can be used to confirm and monitor reintroductions and to contribute to the assessment and modeling of the ecological status of streams.

Environmental DNA (eDNA) is usually defined as genetic material obtained directly from environmental samples, such as soil, water, or ice. Coupled to DNA metabarcoding, eDNA is a powerful tool in biodiversity assessments. Results from eDNA approach provided valuable insights to the studies of past and contemporary biodiversity in terrestrial and aquatic environments. However, the state and fate of eDNA are still investigated and the knowledge about the form of eDNA (i.e., extracellular vs. intracellular) or the DNA degradation under different environmental conditions is limited. Here, we tackle this issue by analyzing foraminiferal sedimentary DNA (sedDNA) from different size fractions of marine sediments: >500 µm, 500-100 µm, 100-63 µm, and < 63 µm. Surface sediment samples were collected at 15 sampling stations located in the Svalbard archipelago. Sequences of the foraminifera-specific 37f region were generated using Illumina technology. The presented data may be used as a reference for a wide range of eDNA-based studies, including biomonitoring and biodiversity assessments across time and space.


Environmental DNA (eDNA) analysis utilises trace DNA released by organisms into their environment for species detection and is revolutionising non-invasive species monitoring. The use of this technology requires rigorous validation - from field sampling to interpretation of PCR-based results - for meaningful application and interpretation. Assays targeting eDNA released by individual species are typically validated with no predefined criteria to answer specific research questions in one ecosystem. Their general applicability, uncertainties and limitations often remain undetermined. The absence of clear guidelines prevents targeted eDNA assays from being incorporated into species monitoring and policy, thus their establishment will be key for the future implementation of eDNA-based surveys. We describe the measures and tests necessary for successful validation of targeted eDNA assays and the associated pitfalls to form the basis of guidelines. A list of 122 variables was compiled and consolidated into a scale to assess the validation status of individual assays. These variables were evaluated for 546 published single-species assays. The resulting dataset was used to provide an overview of current validation practices and test the applicability of the validation scale for future assay rating. The 122 variables representing assay validation status were classified into 14 thematic blocks, such as "in silico analysis", and arranged on a 5-level validation scale from "incomplete" to "operational". Additionally, minimum validation criteria were defined for each level. The majority (30%) of investigated assays were classified as Level 1 (incomplete), and 15% did not achieve this first level. These assays were characterised by minimal in silico and in vitro testing, but their share in annually published eDNA assays has declined since 2014. The total number of reported variables ranged from 20% to 76% and deviated both between and within levels. The meta-analysis demonstrates the suitability of the 5-level validation scale for assessing targeted eDNA assays. It is a user-friendly tool to evaluate previously published assays for future research and routine monitoring, while also enabling appropriate interpretation of results. Finally, it provides guidance on validation and reporting standards for newly developed assays.

We used two large-scale metabarcoding datasets to evaluate phylogenetic signals at global marine and regional terrestrial scales using co-occurrence and co-exclusion networks. Phylogenetic relatedness was estimated using either global pairwise sequence distance or phylogenetic distance and the significance of observed patterns relating networks and phylogenies were evaluated against two null models. In all datasets, we found that phylogenetically close OTUs significantly co-occurred more often, and OTUs with intermediate phylogenetic relatedness co-occurred less often, than expected by chance. Phylogenetically close OTUs co-excluded less often than expected by chance in the marine datasets only. Simultaneous excess of co-occurrences and co-exclusions were observed in the inversion zone between close and intermediate phylogenetic distance classes in marine surface. Similar patterns were observed by using either pairwise sequence or phylogenetic distances, and by using both null models. These results suggest that environmental filtering and dispersal limitation are the preponderant forces driving co-occurrence of protists in both environments, while signal of competitive exclusion was only detected in the marine surface environment. The discrepancy in the co-exclusion pattern is potentially linked to the individual environments: water bodies are more homogeneous while tropical forest soils contain a myriad of nutrient rich micro-environment reducing the strength of mutual exclusion.

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