Environmental diversity surveys are crucial for the bioassessment of anthropogenic impacts on marine ecosystems. Traditional benthic monitoring relying on morphotaxonomic inventories of macrofaunal communities is expensive, time-consuming and expertise-demanding. High-throughput sequencing of environmental DNA barcodes (metabarcoding) offers an alternative to describe biological communities. However, whether the metabarcoding approach meets the quality standards of benthic monitoring remains to be tested. Here, we compared morphological and eDNA/RNA-based inventories of metazoans from samples collected at 10 stations around a fish farm in Scotland, including near-cage and distant zones. For each of 5 replicate samples per station, we sequenced the V4 region of the 18S rRNA gene using the Illumina technology. After filtering, we obtained 841,766 metazoan sequences clustered in 163 Operational Taxonomic Units (OTUs). We assigned the OTUs by combining local BLAST searches with phylogenetic analyses. We calculated two commonly used indices: the Infaunal Trophic Index and the AZTI Marine Biotic Index. We found that the molecular data faithfully reflect the morphology-based indices and provides an equivalent assessment of the impact associated with fish farms activities. We advocate that future benthic monitoring should integrate metabarcoding as a rapid and accurate tool for the evaluation of the quality of marine benthic ecosystems.
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.
This paper presents the reconstruction of climate-driven environmental changes of the last millennium from Hornsund Fjord (Svalbard) based on sedimentological and micropalaeontological records. Our palaeo-investigation was supported by the analysis of 5 foraminiferal ancient DNA (aDNA), focusing on non-fossilised monothalamous species. The main climatic fluctuations over the last millennium were the Medieval Warm Period (MWP, 1000–1600 AD), the Little Ice Age (LIA, 1600–1900 AD), and the Modern Warming (MW, 1900 AD–present). Our study indicated that environmental conditions in Hornsund during the MWP and the early LIA (before ∼ 1800 AD) were relatively 10 stable, resulting from the distant position of glaciers. The beginning of the LIA (∼ 1600 AD) was poorly evidenced by the micropalaeontological record, but well marked in the aDNA data, by an increased proportion of monothalamous foraminifera, especially Bathysiphon sp. The early LIA (∼ 1600–∼ 1800 AD) was marked by the increase in abundance of sequences of Hippocrepinella hirudinea and Cedhagenia saltatus. In the 15 late LIA (after ∼ 1800 AD), conditions in the fjord became glacier-proximal, characterised by increased meltwater outflows, high sedimentation and a high calving rate. This coincided with an increase in the percentages of sequences of Micrometula sp. and Vellaria pellucidus. During the MW, major glaciers fronts retreated rapidly to the inner bays, limiting the iceberg discharge to the fjord centre and causing the shift in the 20 foraminiferal community reflected in both fossil and aDNA records. Palaeoceanographic changes in the Hornsund Fjord over the last millennium were driven mainly by the inflow of shelf-originated water masses and glaciers’ activity. However, the environmental changes were poorly evidenced in the micropalaeontological record, but well documented in our aDNA data. We considerably increased the number 25 of potential proxy species by including monothalamous foraminifera in the palaeoecological studies.
Tagging amplicons with tag sequences appended to PCR primers allow the multiplexing of numerous samples for high-throughput sequencing (HTS). This approach is routinely used in HTS-based diversity analyses, especially in microbial ecology and biomedical diagnostics. However, amplicon library preparation is subject to pervasive sample sequence cross-contaminations as a result of tag switching events referred to as mistagging. Here, we sequenced seven amplicon libraries prepared using various multiplexing designs in order to measure the magnitude of this phenomenon and its impact on diversity analyses. Up to 28.2% of the unique sequences correspond to undetectable (critical) mistags in single- or saturated double-tagging libraries. We show the advantage of multiplexing samples following Latin Square Designs in order to optimize the detection of mistags and maximize the information on their distribution across samples. We use this information in designs incorporating PCR replicates to filter the critical mistags and to recover the exact composition of mock community samples. Being parameter-free and data-driven, our approach can provide more accurate and reproducible HTS data sets, improving the reliability of their interpretations.
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.
Spatial patchiness is a natural feature that strongly influences the level of species richness we perceive in surface sediments sampled in the deep-sea. Recent environmental DNA (eDNA) surveys of benthic micro- and meiofauna confirmed this exceptional richness. However, it is unknown to which extent the results of these studies, based usually on few grams of sediment, are affected by spatial patchiness of deep-sea benthos. Here, we analyse the eDNA diversity of Foraminifera in 42 deep-sea sediment samples collected across different scales in the Southern Ocean. At three stations, we deployed at least twice the multicorer and from each multicorer cast, we subsampled 3 sediment replicates per core for 2 cores. Using high-throughput sequencing (HTS), we generated over 2.35 million high-quality sequences that we clustered into 451 operational taxonomic units (OTUs). The majority of OTUs were assigned to the monothalamous (single-chambered) taxa and environmental clades. On average, a one-gram sediment sample captures 57.9% of the overall OTU diversity found in a single core, while three replicates cover at most 61.9% of the diversity found in a station. The OTUs found in all the replicates of each core gather up to 87.9% of the total sequenced reads, but only represent from 12.2% to 30% of the OTUs found in one core. These OTUs represent the most abundant species, among which dominate environmental lineages. The majority of the OTUs are represented by few sequences comprising several well-known deep-sea morphospecies or remaining unassigned. It is crucial to study wider arrays of sample and PCR replicates as well as RNA together with DNA in order to overcome biases stemming from deep-sea patchiness and molecular methods.
The cytoplasm of four species of abyssal benthic foraminiferans from the Southern Ocean (around 51°S; 12°W and 50°S; 39°W) was analysed by High Performance Liquid Chromatography (HPLC) and found to contain large concentrations of algal pigments and their degradation products. The composition of the algal pigments in the foraminiferan cytoplasm reflected the plankton community at the surface. Some foraminiferans contained high ratios of chlorophyll a/degraded pigments because they were feeding on fresher phytodetritus. Other foraminiferans contained only degraded pigments which shows that they utilized degraded phytodetritus. The concentration of algal pigment and corresponding degradation products in the foraminiferan cytoplasm is much higher than in the surrounding sediment. It shows that the foraminiferans collect a diluted and sparse food resource and concentrate it as they build up their cytoplasm. This ability contributes to the understanding of the great quantitative success of foraminiferans in the deep sea. Benthic foraminiferans are a food source for many abyssal metazoans. They form a link between the degraded food resources, phytodetritus, back to the active metazoan food chains.
Due to their high abundance and large body size sponges have a central position in Antarctic zoobenthos, where they form the most extensive sponge grounds of the world. Though research on Antarctic benthos communities is quite established, research on sponge-associated infauna communities is scarce. We analyzed associated infauna of fifteen individuals of the sponge species Mycale (Oxymycale) acerata Kirkpatrick, 1907 (Demospongiae: Mycalina), Rossella antarctica Carter, 1872 and R. racovitzae Topsent, 1901 (both Hexactinellida: Lyssacinosida). Samples were collected from the deep Ekström Shelf at 602 m in the South-Eastern Weddell Sea, Antarctica, during the ANT XXIV-2 (SYSTCO I) expedition of RV Polarstern. The number of species, α- and β-diversity and the significantly different species composition of infauna communities related to sponge species were calculated, the latter via cluster analysis. The sponge-associated infauna consisted of five phyla: Foraminifera, Nematoda, Polychaeta, Mollusca and Arthropoda. In total 11,463 infaunal specimens were extracted and we found at least 76 associated species. Highest values of α-diversity were calculated for a sample of R. antarctica with a Shannon-Index of 1.84 and Simpson-Index of 0.72 respectively. Our results of the cluster-analysis show significant differences between infauna communities and a unique species composition for single sponge species. Polychaetes of the genus Syllis Lamarck, 1818 were numerous in M. acerata and genera like Pionosyllis Malmgren, 1867 and Cirratulus Lamarck, 1801 were numerous in R. antarctica. Individuals of the amphipod species Seba cf. dubia Schellenberg, 1926 were often found in R. antarctica and R. racovitzae while Colomastix fissilingua Schellenberg, 1926 was frequent in samples of M. acerata. Molluscs were present in M. acerata and R. antarctica but absent in R. racovitzae.
The measurement of species diversity represents a powerful tool for assessing the impacts of human activities on marine ecosystems. Traditionally, the impact of fish farming on the coastal environment is evaluated by monitoring the dynamics of macrobenthic infaunal populations. However, taxonomic sorting and morphology-based identification of the macrobenthos demand highly trained specialists and are extremely time-consuming and costly, making it unsuitable for large-scale biomonitoring efforts involving numerous samples. Here, we propose to alleviate this laborious task by developing protist metabarcoding tools based on next-generation sequencing (NGS) of environmental DNA and RNA extracted from sediment samples. In this study, we analysed the response of benthic foraminiferal communities to the variation of environmental gradients associated with salmon farms in Scotland. We investigated the foraminiferal diversity based on ribosomal minibarcode sequences generated by the Illumina NGS technology. We compared the molecular data with morphospecies counts and with environmental gradients, including distance to cages and redox used as a proxy for sediment oxygenation. Our study revealed high variations between foraminiferal communities collected in the vicinity of fish farms and at distant locations. We found evidence for species richness decrease in impacted sites, especially visible in the RNA data. We also detected some candidate bioindicator foraminiferal species. Based on this proof-of-concept study, we conclude that NGS metabarcoding using foraminifera and other protists has potential to become a new tool for surveying the impact of aquaculture and other industrial activities in the marine environment.
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.