Lejzerowicz F, Esling P, Majewski W, Szczucinski W, Decelle J, Obadia C, Arbizu P M, Pawlowski J

Department of Genetics and Evolution, University of Geneva, , 1211 Geneve 4, Switzerland.

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.

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

(a) Department of Genetics and Evolution, University of Geneva, Sciences III, 30, Quai Ernest Ansermet, CH 1211 Genève 4, Switzerland (b) Department of Invertebrate Zoology, Faculty of Biology and...

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.

Pawlowski J

Department of Genetics and Evolution, University of Geneva, 30, Quai Ernest Ansermet, Sciences 3, CH-1211 Geneva, Switzerland. Jan.Pawlowski@unige.ch.

Sierra R, Matz M V, Aglyamova G, Pillet L, Decelle J, Not F, Vargas C D, Pawlowski J

Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland. Electronic address: roberto.sierra@unige.ch.

Rhizaria is one of the six supergroups of eukaryotes, which comprise the majority of amoeboid and skeleton-building protists living in freshwater and marine ecosystems. There is an overall lack of molecular data for the group and therefore the deep phylogeny of rhizarians is unresolved. Molecular data are particularly scarce for the clade of Retaria, which include two prominent groups of microfossils: foraminiferans and radiolarians. To fill this gap, we have produced and sequenced EST libraries for 14 rhizarian species including seven foraminiferans, Gromia and six taxa belonging to traditional Haeckel's Radiolaria: Acantharea, Polycystinea, and Phaeodarea. A matrix was constructed for phylogenetic analysis based on 109 genes and a total of 56 species, of which 22 are rhizarians. Our analyses provide the first multigene evidence for branching of Phaeodarea within Cercozoa, confirming the polyphyly of Haeckel's Radiolaria. It confirms the monophyly of Retaria, a clade grouping Foraminifera with other lineages of Radiolaria. However, contrary to what could be expected from morphological observations, Foraminifera do not form a sister group to radiolarians, but branch within them as sister to either Acantharea or Polycystinea depending on the multigene data set. While the monophyly of Foraminifera and Acantharea is well supported, that of Polycystinea, represented in our data by Spumellaria and Collodaria is questionable. In view of our study, Haeckel's Radiolaria appears as both, a polyphyletic and paraphyletic assemblage of independent groups that should be considered as separate lineages in protist classification.

Guillou L, Bachar D, Audic S, Bass D, Berney C, Bittner L, Boutte C, Burgaud G, de Vargas C, Decelle J, Del Campo J, Dolan J R, Dunthorn M, Edvardsen B, Holzmann M, Kooistra W H, Lara E, Le Bescot ...

CNRS, UMR 7144, Adaptation et Diversite en Milieu Marin, 29682 Roscoff, France, UPMC Universite Paris 06, UMR 7144, Station Biologique de Roscoff, 29682 Roscoff, France, CNRS, UMR 7138, Systematiqu...

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR(2), http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

Pawlowski J, Audic S, Adl S, Bass D, Belbahri L, Berney C, Bowser S S, Cepicka I, Decelle J, Dunthorn M, Fiore-Donno A M, Gile G H, Holzmann M, Jahn R, Jirku M, Keeling P J, Kostka M, Kudryavtsev A...

Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland.

A group of protist experts proposes a two-step DNA barcoding approach, comprising a universal eukaryotic pre-barcode followed by group-specific barcodes, to unveil the hidden biodiversity of microbial eukaryotes.

Adl S M, Simpson A G, Lane C E, Lukes J, Bass D, Bowser S S, Brown M W, Burki F, Dunthorn M, Hampl V, Heiss A, Hoppenrath M, Lara E, le Gall L, Lynn D H, McManus H, Mitchell E A, Mozley-Stanridge S...

Department of Soil Science, University of Saskatchewan, Saskatoon, SK, S7N 5A8, Canada; Department of Biology, Dalhousie University, Halifax, NS, B3H 4R2, Canada.

This revision of the classification of eukaryotes, which updates that of Adl et al. [J. Eukaryot. Microbiol. 52 (2005) 399], retains an emphasis on the protists and incorporates changes since 2005 that have resolved nodes and branches in phylogenetic trees. Whereas the previous revision was successful in re-introducing name stability to the classification, this revision provides a classification for lineages that were then still unresolved. The supergroups have withstood phylogenetic hypothesis testing with some modifications, but despite some progress, problematic nodes at the base of the eukaryotic tree still remain to be statistically resolved. Looking forward, subsequent transformations to our understanding of the diversity of life will be from the discovery of novel lineages in previously under-sampled areas and from environmental genomic information.

Apotheloz-Perret-Gentil L, Holzmann M, Pawlowski J

Department of Genetics and Evolution, University of Geneva, 1211 Geneva 4, Switzerland.

A new monothalamous (single-chambered) soft-walled foraminiferal species, Arnoldiellina fluorescens gen. et sp. nov., was isolated from samples collected in the Gulf of Eilat, Israel. The species is characterized by a small elongate organic theca with a single aperture of allogromiids. It is characterized by the emission of green autofluorescence (GAF) that has so far not been reported from foraminifera. Phylogenetic analysis of a fragment of the 18S rDNA indicates that the species is related to a group of monothalamous foraminiferans classified as clade I. Although the morphology of the new species is very different compared to the other members of this clade, a specific helix in 18S rRNA secondary structure strongly supports this position.

Pillet L, Pawlowski J

Department of Genetics and Evolution, University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland.

Foraminifera from the genus Elphidium are heterotrophic protists that graze on diatoms and sequester chloroplasts from their algal preys, while digesting the rest of the diatom cell. During that process, known as kleptoplastidy, the acquired plastids remain active inside the foraminiferan cell for several months. As most of the genes required to sustain the activity of the chloroplasts are encoded in the diatom nuclei, it is unknown how can the host cell maintain the photosynthetic activity without this information. It has been proposed that maintenance of kleptoplastidy could be explained by horizontal gene transfer. To test this hypothesis we obtained 17,125 EST sequences of Elphidium margaritaceum and we screened this dataset for diatom nuclear-encoded proteins having a function in photosynthetic activity or plastid maintenance. Our analyses show no evidence for the presence of such transcriptionally active genes and suggest that HGT hypothesis alone cannot explain the chloroplasts longevity in Elphidium.

Kudryavtsev A, Pawlowski J

Department of Genetics and Evolution, University of Geneva, Sciences III, 1211 Geneva, Switzerland; Department of Invertebrate Zoology, Faculty of Biology and Soil Science, St. Petersburg State Uni...

Squamamoeba japonica n. g. n. sp. was isolated and described from marine bottom sediments collected at a depth of ca. 2700 m in the Sea of Japan. Trophic amoebae of this species are elongated and flattened, with a wide anterior hyaloplasm producing numerous ventral subpseudopodia for adhesion to the substratum. The cell coat consists of flat oval scales tightly packed together to form a continuous layer separated from the plasma membrane. Amoebae can form cytoplasmic projections protruding through the scale layer and having tips covered only with the plasma membrane. Small subunit ribosomal RNA gene phylogeny shows that S. japonica forms a long branch in the amoebozoan tree, robustly grouping with the marine strain 'Pessonella' sp. PRA-29. Morphological data available for the latter, although scarce, give additional support for the relatedness of both species. The resulting clade comprising the two taxa shows no close relationships to other Amoebozoa and seems to be a novel lineage that developed an ability to temporarily liberate local areas of the plasma membrane from the cell coat independently from Himatismenida, Trichosida, Pellitida and Dermamoeba.

Pillet L, Fontaine D, Pawlowski J

Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland.

Elphidium macellum is a benthic foraminifer commonly found in the Patagonian fjords. To test whether its highly variable morphotypes are ecophenotypes or different genotypes, we analysed 70 sequences of the SSU rRNA gene from 25 specimens. Unexpectedly, we identified 11 distinct ribotypes, with up to 5 ribotypes co-occurring within the same specimen. The ribotypes differ by varying blocks of sequence located at the end of stem-loop motifs in the three expansion segments specific to foraminifera. These changes, distinct from typical SNPs and indels, directly affect the structure of the expansion segments. Their mosaic distribution suggests that ribotypes originated by recombination of two or more clusters of ribosomal genes. We propose that this expansion segment polymorphism (ESP) could originate from hybridization of morphologically different populations of Patagonian Elphidium. We speculate that the complex geological history of Patagonia enhanced divergence of coastal foraminiferal species and contributed to increasing genetic and morphological variation.

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

Department of Genetics and Evolution, University of Geneva, CH-1211 Geneva 4, Switzerland.

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.