Environmental DNA metabarcoding for benthic monitoring: A review of sediment sampling and DNA extraction methods.

Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.

Protist metabarcoding and environmental biomonitoring: Time for change.

High-throughput amplicon sequencing of environmental DNA and/or RNA proved to be a powerful tool to describe protist diversity. This new approach called also the metabarcoding has totally transformed our view of protist diversity, revealing a large number of novel lineages and expanding the range of protist phylogenetic diversity at almost every taxonomic level. However, until now the objectives of the vast majority of metabarcoding studies were purely academic. Practical applications of protist metabarcoding are surprisingly scarce, despite the fact that several groups of protists are commonly used as bioindicators of environmental impacts in freshwater or marine ecosystems. Here, we are reviewing studies that examine the ecological applications of metabarcoding for two groups of well-known protist bioindicators: diatoms and foraminifera. The results of these studies show that despite some biological and technical biases, molecular data quite faithfully reflect the morphology-based biotic indices and provide a similar assessment of ecosystem status. In view of these results, protist metabarcoding appears as a rapid and accurate tool for the evaluation of the quality of aquatic ecosystems. Hence, we plead for integration of protist metabarcoding in future biomonitoring projects as a complement of traditional methods and a source of new biosensors for environmental impact assessment.

Accurate assessment of the impact of salmon farming on benthic sediment enrichment using foraminiferal metabarcoding.

Assessing the environmental impact of salmon farms on benthic systems is traditionally undertaken using biotic indices derived from microscopic analyses of macrobenthic infaunal (MI) communities. In this study, we tested the applicability of using foraminiferal-specific high-throughput sequencing (HTS) metabarcoding for monitoring these habitats. Sediment samples and physico-chemical data were collected along an enrichment gradient radiating out from three Chinook salmon (Oncorhynchus tshawytscha) farms in New Zealand. HTS of environmental DNA and RNA (eDNA/eRNA) resulted in 1,875,300 sequences that clustered into 349 Operational Taxonomic Units. Strong correlations were observed among various biotic indices calculated from MI data and normalized fourth-root transformed HTS data. Correlations were stronger using eRNA compared to eDNA data. Quantile regression spline analyses identified 12 key foraminiferal taxa that have potential to be used as bioindicator species. This study demonstrates the huge potential for using this method for biomonitoring of fish-farming and other marine industrial activities.

Next-generation environmental diversity surveys of foraminifera: preparing the future.

Foraminifera are commonly defined as marine testate protists, and their diversity is mainly assessed on the basis of the morphology of their agglutinated or mineralized tests. Diversity surveys based on environmental DNA (eDNA) have dramatically changed this view by revealing an unexpected diversity of naked and organic-walled lineages as well as detecting foraminiferal lineages in soil and freshwater environments. Moreover, single-cell analyses have allowed discrimination among genetically distinctive types within almost every described morphospecies. In view of these studies, the foraminiferal diversity appeared to be largely underestimated, but its accurate estimation was impeded by the low speed and coverage of a cloning-based eDNA approach. With the advent of high-throughput sequencing (HTS) technologies, these limitations disappeared in favor of exhaustive descriptions of foraminiferal diversity in numerous samples. Yet, the biases and errors identified in early HTS studies raised some questions about the accuracy of HTS data and their biological interpretation. Among the most controversial issues affecting the reliability of HTS diversity estimates are (1) the impact of technical and biological biases, (2) the sensitivity and specificity of taxonomic sequence assignment, (3) the ability to distinguish rare species, and (4) the quantitative interpretation of HTS data. Here, we document the lessons learned from previous HTS surveys and present the current advances and applications focusing on foraminiferal eDNA. We discuss the problems associated with HTS approaches and predict the future trends and avenues that hold promises for surveying foraminiferal diversity accurately and efficiently.

Ancient DNA sheds new light on the Svalbard foraminiferal fossil record of the last millennium.

Recent palaeogenetic studies have demonstrated the occurrence of preserved ancient DNA (aDNA) in various types of fossilised material. Environmental aDNA sequences assigned to modern species have been recovered from marine sediments dating to the Pleistocene. However, the match between the aDNA and the fossil record still needs to be evaluated for the environmental DNA approaches to be fully exploited. Here, we focus on foraminifera in sediments up to one thousand years old retrieved from the Hornsund fjord (Svalbard). We compared the diversity of foraminiferal microfossil assemblages with the diversity of aDNA sequenced from subsurface sediment samples using both cloning and high-throughput sequencing (HTS). Our study shows that 57% of the species archived in the fossil record were also detected in the aDNA data. However, the relative abundance of aDNA sequence reads and fossil specimens differed considerably. We also found a limited match between the stratigraphic occurrence of some fossil species and their aDNA sequences, especially in the case of rare taxa. The aDNA data comprised a high proportion of non-fossilised monothalamous species, which are known to dominate in modern foraminiferal communities of the Svalbard region. Our results confirm the relevance of HTS for studying past micro-eukaryotic diversity and provide insight into its ability to reflect fossil assemblages. Palaeogenetic studies including aDNA analyses of non-fossilised groups expand the range of palaeoceanographical proxies and therefore may increase the accuracy of palaeoenvironmental reconstructions.