Exploring the potential of metabarcoding to disentangle macroinvertebrate community dynamics in intermittent streams

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

Metabarcoding unsorted kick‐samples facilitates macroinvertebrate‐based biomonitoring with increased taxonomic resolution, while outperforming environmental DNA

Pereira‐da‐Conceicoa, L, Elbrecht, V, Hall, A, Briscoe, A, Barber‐James, H, Price, B. Metabarcoding unsorted kick‐samples facilitates macroinvertebrate‐based biomonitoring with increased taxonomic resolution, while outperforming environmental DNA. Environmental DNA. 2020; 00: 1– 19. https://doi.org/10.1002/edn3.116© 2020 The Authors. Environmental DNA published by John Wiley & Sons Ltd This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. The attached file is the published pdf

Connecting high-throughput biodiversity inventories: Opportunities for a site-based genomic framework for global integration and synthesis

High‐throughput sequencing (HTS) is increasingly being used for the characterization and monitoring of biodiversity. If applied in a structured way, across broad geographical scales, it offers the potential for a much deeper understanding of global biodiversity through the integration of massive quantities of molecular inventory data generated independently at local, regional and global scales. The universality, reliability and efficiency of HTS data can potentially facilitate the seamless linking of data among species assemblages from different sites, at different hierarchical levels of diversity, for any taxonomic group and regardless of prior taxonomic knowledge. However, collective international efforts are required to optimally exploit the potential of site‐based HTS data for global integration and synthesis, efforts that at present are limited to the microbial domain. To contribute to the development of an analogous strategy for the nonmicrobial terrestrial domain, an international symposium entitled “Next Generation Biodiversity Monitoring” was held in November 2019 in Nicosia (Cyprus). The symposium brought together evolutionary geneticists, ecologists and biodiversity scientists involved in diverse regional and global initiatives using HTS as a core tool for biodiversity assessment. In this review, we summarize the consensus that emerged from the 3‐day symposium. We converged on the opinion that an effective terrestrial Genomic Observatories network for global biodiversity integration and synthesis should be spatially led and strategically united under the umbrella of the metabarcoding approach. Subsequently, we outline an HTS‐based strategy to collectively build an integrative framework for site‐based biodiversity data generation

Non‐specific amplification compromises environmental DNA metabarcoding with COI

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

The problem of omnivory: a synthesis on omnivory and DNA metabarcoding

Dietary analysis using DNA metabarcoding is a powerful tool that is increasingly being used to further our knowledge of trophic interactions in highly complex food webs but is not without limitations. Omnivores, the most generalist of consumers, pose unique challenges when using such methods. Here, we provide the rationale to understand the problems associated with analysing the complex diets of omnivores. By reviewing existing metabarcoding studies of omnivorous diet, and constructing hypothetical scenarios arising from each, we outline that great caution is required when interpreting sequencing data in such cases. In essence, the problems of accidental consumption and secondary ingestion are significant sources of error when investigating omnivorous diets. The integration of multiple high throughput sequencing markers increases the taxonomic breadth of taxa detected but we reveal how some detections may be misleading. Disentangling which taxa have been deliberately or accidentally consumed by the focal omnivore is challenging and can falsely emphasise those that were not intentionally consumed, obscuring biologically meaningful interactions. Although we suggest ways to disentangle these issues, we urge that the results of such analyses should be interpreted with caution and all possible scenarios for the presence of biota within omnivores given due consideration