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Franck Lejzerowicz

Postdoctoral fellow in Protists evolution

  • T: +41 22 379 30 77
  • office 4078b (Sciences III)
  • First evaluation of foraminiferal metabarcoding for monitoring environmental impact from an offshore oil drilling site. Mar. Environ. Res. 2016 Aug;120():225-235. S0141-1136(16)30142-8. 10.1016/j.marenvres.2016.08.009.

    Laroche O, Wood SA, Tremblay LA, Ellis JI, Lejzerowicz F, Pawlowski J, Lear G, Atalah J, Pochon X

    abstract

    At present, environmental impacts from offshore oil and gas activities are partly determined by measuring changes in macrofauna diversity. Morphological identification of macrofauna is time-consuming, expensive and dependent on taxonomic expertise. In this study, we evaluated the applicability of using foraminiferal-specific metabarcoding for routine monitoring. Sediment samples were collected along distance gradients from two oil platforms off Taranaki (New Zealand) and their physico-chemical properties, foraminiferal environmental DNA/RNA, and macrofaunal composition analyzed. Macrofaunal and foraminiferal assemblages showed similar shifts along impact gradients, but responded differently to environmental perturbations. Macrofauna were affected by hypoxia, whereas sediment grain size appeared to drive shifts in foraminifera. We identified eight foraminiferal molecular operational taxonomic units that have potential to be used as bioindicator taxa. Our results show that metabarcoding represents an effective tool for assessing foraminiferal communities near offshore oil and gas platforms, and that it can be used to complement current monitoring techniques.

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  • Benthic monitoring of salmon farms in Norway using foraminiferal metabarcoding AEI 8:371-386 (2016) - doi:10.3354/aei00182

    Jan Pawlowski, Philippe Esling, Franck Lejzerowicz, Tristan Cordier, Joana A. Visco, Catarina I. M. Martins, Arne Kvalvik, Knut Staven, Tomas Cedhagen7

    abstract

    The rapid growth of the salmon industry necessitates the development of fast and accurate tools to assess its environmental impact. Macrobenthic monitoring is commonly used to measure the impact of organic enrichment associated with salmon farm activities. However, classical benthic monitoring can hardly answer the rapidly growing demand because the morphological identification of macro-invertebrates is time-consuming, expensive and requires taxonomic expertise. Environmental DNA (eDNA) metabarcoding of meiofauna-sized organisms, such as Foraminifera, was proposed to overcome the drawbacks of macrofauna-based benthic monitoring. Here, we tested the application of foraminiferal metabarcoding to benthic monitoring of salmon farms in Norway. We analysed 140 samples of eDNA and environmental RNA (eRNA) extracted from surface sediment samples collected at 4 salmon farming sites in Norway. We sequenced the variable region 37f of the 18S rRNA gene specific to Foraminifera. We compared our data to the results of macrofaunal surveys of the same sites and tested the congruence between various diversity indices inferred from metabarcoding and morphological data. The results of our study confirm the usefulness of Foraminifera as bioindicators of organic enrichment associated with salmon farming. The foraminiferal diversity increased with the distance to fish cages, and metabarcoding provides an assessment of the ecological quality comparable to the morphological analyses. The foraminiferal metabarcoding approach appears to be a promising alternative to classical benthic monitoring, providing a solution to the morpho-taxonomic bottleneck of macrofaunal surveys.

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  • Protist metabarcoding and environmental biomonitoring: Time for change. Eur. J. Protistol. 2016 Aug;55(Pt A):12-25. S0932-4739(16)30003-7. 10.1016/j.ejop.2016.02.003.

    Pawlowski J, Lejzerowicz F, Apotheloz-Perret-Gentil L, Visco J, Esling P

    abstract

    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.

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  • Next-Generation Sequencing of Aquatic Oligochaetes: Comparison of Experimental Communities. PLoS ONE 2016 ;11(2):e0148644. 10.1371/journal.pone.0148644. PONE-D-15-28411. PMC4750909.

    Vivien R, Lejzerowicz F, Pawlowski J

    abstract

    Aquatic oligochaetes are a common group of freshwater benthic invertebrates known to be very sensitive to environmental changes and currently used as bioindicators in some countries. However, more extensive application of oligochaetes for assessing the ecological quality of sediments in watercourses and lakes would require overcoming the difficulties related to morphology-based identification of oligochaetes species. This study tested the Next-Generation Sequencing (NGS) of a standard cytochrome c oxydase I (COI) barcode as a tool for the rapid assessment of oligochaete diversity in environmental samples, based on mixed specimen samples. To know the composition of each sample we Sanger sequenced every specimen present in these samples. Our study showed that a large majority of OTUs (Operational Taxonomic Unit) could be detected by NGS analyses. We also observed congruence between the NGS and specimen abundance data for several but not all OTUs. Because the differences in sequence abundance data were consistent across samples, we exploited these variations to empirically design correction factors. We showed that such factors increased the congruence between the values of oligochaetes-based indices inferred from the NGS and the Sanger-sequenced specimen data. The validation of these correction factors by further experimental studies will be needed for the adaptation and use of NGS technology in biomonitoring studies based on oligochaete communities.

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  • High-throughput sequencing and morphology perform equally well for benthic monitoring of marine ecosystems. Sci Rep 2015 ;5():13932. srep13932. 10.1038/srep13932. PMC4564730.

    Lejzerowicz F, Esling P, Pillet L, Wilding TA, Black KD, Pawlowski J

    abstract

    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.

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  • Accurate assessment of the impact of salmon farming on benthic sediment enrichment using foraminiferal metabarcoding. Mar. Pollut. Bull. 2015 Nov;100(1):370-82. S0025-326X(15)00526-3. 10.1016/j.marpolbul.2015.08.022.

    Pochon X, Wood SA, Keeley NB, Lejzerowicz F, Esling P, Drew J, Pawlowski J

    abstract

    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.

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  • Palaeoceanographic changes in Hornsund Fjord (Spitsbergen, Svalbard) over the last millennium: new insights from ancient DNA

    J. Pawłowska (1), M. Zajączkowski (1), M. Łącka (1), F. Lejzerowicz (2), P. Esling (2,3), and J. Pawlowski (2)

    abstract

    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.

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  • Palaeoceanographic changes in Hornsund Fjord (Spitsbergen, Svalbard) over the last millennium: new insights from ancient DNA Climate of the Past Discussions 11, 3665-3698; 10.5194/cpd-11-3665-2015

    J Pawłowska, M Zajączkowski, M Łącka, F Lejzerowicz, P Esling, J Pawlowski

    abstract

    This paper presents a reconstruction of climatedriven environmental changes over the last millennium in Hornsund Fjord (Svalbard), based on sedimentological and micropalaeontological records. Our palaeo-investigation was supported by an analysis of foraminiferal ancient DNA (aDNA), focusing on the non-fossilized monothalamous species. The main climatic fluctuations during the last millennium were the Medieval Warm Period (MWP, AD 1000– 1600), the Little Ice Age (LIA, AD 1600–1900) and the modern warming (MW, AD 1900 to present). Our study indicates that the environmental conditions in Hornsund during the MWP and the early LIA (before ∼ AD 1800) were relatively stable. The beginning of the LIA (∼ AD 1600) was poorly evidenced by the micropalaeontological record but was well marked in the aDNA data by an increased proportion of monothalamous foraminifera, especially Bathysiphon sp. The early LIA (∼ 1600 to ∼ AD 1800) was marked by an increase in the abundance of sequences of Hippocrepinella hirudinea and Cedhagenia saltatus. In the late LIA (after ∼ AD 1800), the conditions in the fjord became glacierproximal and were characterized by increased meltwater out- flows, 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, the major glacier fronts retreated rapidly to the inner bays, which limited the iceberg discharge to the fjord’s centre and caused a shift in the foraminiferal community that was reflected in both the fossil and aDNA records.

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  • Accurate multiplexing and filtering for high-throughput amplicon-sequencing. Nucleic Acids Res. 2015 Mar;43(5):2513-24. gkv107. 10.1093/nar/gkv107. PMC4357712.

    Esling P, Lejzerowicz F, Pawlowski J

    abstract

    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.

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  • Foraminifera of the Kuril–Kamchatka Trench area: The prospects of molecular study Deep Sea Research Part II: Topical Studies in Oceanography 108, 17-26; 10.1016/j.dsr2.2014.10.003

    Franck Lejzerowicz, Ivan Voltski, Jan Pawlowski

    abstract

    Foraminifera remain poorly studied from deep-sea sediment settings, although they often dominate meiofaunal communities and represent an important part of the functional deep-sea diversity. Moreover, there is a striking gap in our knowledge of deep-sea Foraminifera since most of the foraminiferal diversity corresponds to single-chambered monothalamids displaying inconspicuous morphologies. The previous expeditions suggest that the foraminiferal biomass is large in the Kuril–Kamchatka Trench area, including many macrofaunal-size xenophyophoreans and komokiaceans. However, the on-site foraminiferal diversity remains poorly described and was never genetically examined. During the KuramBio expedition, we collected over 1400 specimens representing all major foraminiferal groups, focusing on monothalamids and particularly komokiaceans. From the deep-sea sediments, using four different sampling gears, we sorted, identified and photographed single specimens for DNA (or RNA) extraction. The material we report here will be used in our ongoing research on estimation and visualisation of the diversity of deep-sea monothalamous foraminifera and enigmatic taxa such as the komokiaceans, the origin of which is yet to be determined.

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  • Next-generation environmental diversity surveys of foraminifera: preparing the future. Biol. Bull. 2014 Oct;227(2):93-106. 227/2/93.

    Pawlowski J, Lejzerowicz F, Esling P

    abstract

    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.

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  • Patchiness of deep-sea benthic Foraminifera across the Southern Ocean: insights from high-throughput DNA sequencing 10.1016/j.dsr2.2014.07.018

    Franck Lejzerowicz, Philippe Esling, Jan Pawlowski

    abstract

    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.

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  • Patchiness of deep-sea benthic Foraminifera across the southern ocean: Insights from High-throughput DNA sequencing http://dx.doi.org/10.1016/j.dsr2.2014.07.018

    Franck Lejzerowicz (a), Philippe Esling (a,b), Jan Pawlowski (a)

    abstract

    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.

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  • Algal pigments in Southern Ocean abyssal foraminiferans indicate pelagobenthic coupling (2)

    Tomas Cedhagen (a), Wee Cheah (b), Astrid Bracher (b), Franck Lejzerowicz (c)

    abstract

    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.

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  • The infauna of three widely distributed sponge species (Hexactinellida and Demospongiae) from the deep Ekström Shelf in the Weddell Sea, Antarctica

    Daniel Kersken, Christian Göcke, Angelika Brandt, Franck Lejzerowicz, Enrico Schwabe, Meike Anna Seefeldt, Gritta Veit-Köhler, Dorte Janussen

    abstract

    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.

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  • Environmental monitoring through protist next-generation sequencing metabarcoding: assessing the impact of fish farming on benthic foraminifera communities. Mol Ecol Resour 2014 Nov;14(6):1129-40. 10.1111/1755-0998.12261.

    Pawlowski J, Esling P, Lejzerowicz F, Cedhagen T, Wilding TA

    abstract

    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.

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  • Ancient DNA sheds new light on the Svalbard foraminiferal fossil record of the last millennium Geobiology, 12: 277–288. doi:10.1111/gbi.12087

    J Pawłowska, F Lejzerowicz, P Esling, W Szczuciński, M Zajączkowski, J Pawlowski

    abstract

    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.

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  • Foraminiferal survival after long term experimentally induced anoxia Biogeosciences Discuss., 10, 9243-9284, doi:10.5194/bgd-10-9243-2013, 2013.

    D. Langlet 1, E. Geslin 1, C. Baal 2, E. Metzger 1, F. Lejzerowicz 3, B. Riedel 4, M. Zuschin 2, J. Pawlowski 3, M. Stachowitsch 4, and F. J. Jorissen 1

    abstract

    Anoxia has been successfully induced in four benthic chambers installed on the Northern Adriatic seafloor from 1 week to 10 months. To accurately determine whether benthic foraminifera can survive experimentally induced prolonged anoxia, the CellTrackerGreen method has been applied. Numerous individuals have been found living at all sampling times and at all sampling depths, showing that benthic foraminifera can survive up to 10 months of anoxia with co-occurring hydrogen sulphides. However, foraminiferal standing stocks decrease with sampling time in an irregular way. A large difference in standing stock between two cores samples in initial conditions indicates the presence of a large spatial heterogeneity of the foraminiferal faunas. An unexpected increase in standing stocks after 1 month is tentatively interpreted as a reaction to increased food availability due to the massive mortality of infaunal macrofaunal organisms. After this, standing stocks decrease again in a core sampled after 2 months of anoxia, to attain a minimum in the cores sampled after 10 months. We speculate that the trend of overall decrease of standing stocks is not due to the adverse effects of anoxia and hydrogen sulphides, but rather due to a continuous diminution of labile organic matter.

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  • Ancient DNA complements microfossil record in deep-sea subsurface sediments. Biol. Lett. 2013 Aug;9(4):20130283. rsbl.2013.0283. 10.1098/rsbl.2013.0283. PMC3730641.

    Lejzerowicz F, Esling P, Majewski W, Szczuciński W, Decelle J, Obadia C, Arbizu PM, Pawlowski J

    abstract

    Deep-sea subsurface sediments are the most important archives of marine biodiversity. Until now, these archives were studied mainly using the microfossil record, disregarding large amounts of DNA accumulated on the deep-sea floor. Accessing ancient DNA (aDNA) molecules preserved down-core would offer unique insights into the history of marine biodiversity, including both fossilized and non-fossilized taxa. Here, we recover aDNA of eukaryotic origin across four cores collected at abyssal depths in the South Atlantic, in up to 32.5 thousand-year-old sediment layers. Our study focuses on Foraminifera and Radiolaria, two major groups of marine microfossils also comprising diverse non-fossilized taxa. We describe their assemblages in down-core sediment layers applying both micropalaeontological and environmental DNA sequencing approaches. Short fragments of the foraminiferal and radiolarian small subunit rRNA gene recovered from sedimentary DNA extracts provide evidence that eukaryotic aDNA is preserved in deep-sea sediments encompassing the last glacial maximum. Most aDNA were assigned to non-fossilized taxa that also dominate in molecular studies of modern environments. Our study reveals the potential of aDNA to better document the evolution of past marine ecosystems and opens new horizons for the development of deep-sea palaeogenomics.

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  • Identifying active foraminifera in the Sea of Japan using metatranscriptomic approach Deep-Sea Res. Feb-Mar 2013

    Franck Lejzerowicz (a), Ivan Voltsky (b, 1), Jan Pawlowski (a)

    abstract

    Metagenetics represents an efficient and rapid tool to describe environmental diversity patterns of microbial eukaryotes based on ribosomal DNA sequences. However, the results of metagenetic studies are often biased by the presence of extracellular DNA molecules that are persistent in the environment, especially in deep-sea sediment. As an alternative, short-lived RNA molecules constitute a good proxy for the detection of active species. Here, we used a metatranscriptomic approach based on RNA-derived (cDNA) sequences to study the diversity of the deep-sea benthic foraminifera and compared it to the metagenetic approach. We analyzed 257 ribosomal DNA and cDNA sequences obtained from seven sediments samples collected in the Sea of Japan at depths ranging from 486 to 3665 m. The DNA and RNA-based approaches gave a similar view of the taxonomic composition of foraminiferal assemblage, but differed in some important points. First, the cDNA dataset was dominated by sequences of rotaliids and robertiniids, suggesting that these calcareous species, some of which have been observed in Rose Bengal stained samples, are the most active component of foraminiferal community. Second, the richness of monothalamous (single-chambered) foraminifera was particularly high in DNA extracts from the deepest samples, confirming that this group of foraminifera is abundant but not necessarily very active in the deep-sea sediments. Finally, the high divergence of undetermined sequences in cDNA dataset indicate the limits of our database and lack of knowledge about some active but possibly rare species. Our study demonstrates the capability of the metatranscriptomic approach to detect active foraminiferal species and prompt its use in future high-throughput sequencing-based environmental surveys.

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  • Ultra-deep sequencing of foraminiferal microbarcodes unveils hidden richness of early monothalamous lineages in deep-sea sediments. Proc. Natl. Acad. Sci. U.S.A. 2011 Aug;108(32):13177-82. 1018426108. 10.1073/pnas.1018426108. PMC3156150.

    Lecroq B, Lejzerowicz F, Bachar D, Christen R, Esling P, Baerlocher L, Østerås M, Farinelli L, Pawlowski J

    abstract

    Deep-sea floors represent one of the largest and most complex ecosystems on Earth but remain essentially unexplored. The vastness and remoteness of this ecosystem make deep-sea sampling difficult, hampering traditional taxonomic observations and diversity assessment. This problem is particularly true in the case of the deep-sea meiofauna, which largely comprises small-sized, fragile, and difficult-to-identify metazoans and protists. Here, we introduce an ultra-deep sequencing-based metagenetic approach to examine the richness of benthic foraminifera, a principal component of deep-sea meiofauna. We used Illumina sequencing technology to assess foraminiferal richness in 31 unsieved deep-sea sediment samples from five distinct oceanic regions. We sequenced an extremely short fragment (36 bases) of the small subunit ribosomal DNA hypervariable region 37f, which has been shown to accurately distinguish foraminiferal species. In total, we obtained 495,978 unique sequences that were grouped into 1,643 operational taxonomic units, of which about half (841) could be reliably assigned to foraminifera. The vast majority of the operational taxonomic units (nearly 90%) were either assigned to early (ancient) lineages of soft-walled, single-chambered (monothalamous) foraminifera or remained undetermined and yet possibly belong to unknown early lineages. Contrasting with the classical view of multichambered taxa dominating foraminiferal assemblages, our work reflects an unexpected diversity of monothalamous lineages that are as yet unknown using conventional micropaleontological observations. Although we can only speculate about their morphology, the immense richness of deep-sea phylotypes revealed by this study suggests that ultra-deep sequencing can improve understanding of deep-sea benthic diversity considered until now as unknowable based on a traditional taxonomic approach.

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  • Molecular evidence for widespread occurrence of Foraminifera in soils. Environ. Microbiol. 2010 Sep;12(9):2518-26. EMI2225. 10.1111/j.1462-2920.2010.02225.x.

    Lejzerowicz F, Pawlowski J, Fraissinet-Tachet L, Marmeisse R

    abstract

    Environmental SSU rDNA-based surveys are contributing to the dramatic revision of eukaryotic high-level diversity and phylogeny as the number of sequence data increases. This ongoing revolution gives the opportunity to test for the presence of some eukaryotic taxa in environments where they have not been found using classical microscopic observations. Here, we test whether the foraminifera, a group of single-celled eukaryotes, considered generally as typical for the marine ecosystems are present in soil. We performed foraminiferal-specific nested PCR on 20 soil DNA samples collected in contrasted environments. Unexpectedly, we found that the majority of the samples contain foraminiferal SSU rDNA sequences. In total, we obtained 49 sequences from 17 localities. Phylogenetic analysis clusters them in four groups branching among the radiation of early foraminiferal lineages. Three of these groups also include sequences originated from previous freshwater surveys, suggesting that there were up to four independent colonization events of terrestrial and/or freshwater ecosystems by ancestral foraminifera. As shown by our data, foraminifera are a widespread and diverse component of soil microbial communities. Yet, identification of terrestrial foraminiferal species and understanding of their ecological role represent an exciting challenge for future research.

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