Molecular Systematics & Environmental Genomics

• Assessing SSU rDNA Barcodes in Foraminifera: A Case Study using Bolivina quadrata. J. Eukaryot. Microbiol. 2017 Sep;():. 10.1111/jeu.12471. 03-09-2017

  Borrelli C, Hou Y, Pawlowski JW, Holzmann M, Katz ME, Chandler GT, Bowser SS

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  The Small Subunit Ribosomal RNA gene (SSU rDNA) is a widely used tool to reconstruct phylogenetic relationships among foraminiferal species. Recently, the highly variable regions of this gene have been proposed as DNA barcodes to identify foraminiferal species. However, the resolution of these barcodes has not been well established, yet. In this study, we evaluate four SSU rDNA hypervariable regions (37/f, 41/f, 43/e, and 45/e) as DNA barcodes to distinguish among species of the genus Bolivina, with particular emphasis on Bolivina quadrata for which ten new sequences (KY468817-KY468826) were obtained during this study. Our analyses show that a single SSU rDNA hypervariable sequence is insufficient to resolve all Bolivina species and that some regions (37/f and 41/f) are more useful than others (43/e and 45/e) to distinguish among closely related species. In addition, polymorphism analyses reveal a high degree of variability. In the context of barcoding studies, these results emphasize the need to assess the range of intraspecific variability of DNA barcodes prior to their application to identify foraminiferal species in environmental samples; our results also highlight the possibility that a longer SSU rDNA region might be required to distinguish among species belonging to the same taxonomic group (i.e., genus). This article is protected by copyright. All rights reserved.

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• Predicting the ecological quality status of marine environments from eDNA metabarcoding data using supervised machine learning. Environ. Sci. Technol. 2017 Jun;():. 10.1021/acs.est.7b01518. 01-07-2017

  Cordier T, Esling P, Lejzerowicz F, Visco J, Ouadahi A, Martins C, Cedhagen T, Pawlowski J

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  Monitoring biodiversity is essential to assess the impacts of increasing anthropogenic activities in marine environments. Traditionally, marine biomonitoring involves the sorting and morphological identification of benthic macro-invertebrates, which is time-consuming and taxonomic-expertise demanding. High-throughput amplicon sequencing of environmental DNA (eDNA metabarcoding) represents a promising alternative for benthic monitoring. However, an important fraction of eDNA sequences remains unassigned or belong to taxa of unknown ecology, which prevent their use for assessing the ecological quality status. Here, we show that supervised machine learning (SML) can be used to build robust predictive models for benthic monitoring, regardless of the taxonomic assignment of eDNA sequences. We tested three SML approaches to assess the environmental impact of marine aquaculture using benthic foraminifera eDNA, a group of unicellular eukaryotes known to be good bioindicators, as features to infer macro-invertebrates based biotic indices. We found similar ecological status as obtained from macro-invertebrates inventories. We argue that SML approaches could overcome and even bypass the cost and time-demanding morpho-taxonomic approaches in future biomonitoring.

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• Taxonomic revision of freshwater foraminifera with the description of two new agglutinated species and genera. Eur. J. Protistol. 2017 May;60():28-44. S0932-4739(17)30078-0. 10.1016/j.ejop.2017.05.006. 14-06-2017

  Siemensma F, Apothéloz-Perret-Gentil L, Holzmann M, Clauss S, Völcker E, Pawlowski J

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  Most foraminifera inhabit marine habitats, but some species of monothalamids have been described from freshwater environments, mainly from Swiss water bodies over 100 years ago. Recent environmental DNA surveys revealed the presence of four major phylogenetic clades of freshwater foraminifera. However, until now only one of them (clade 2) has been associated to a morphologically described taxon-the family Reticulomyxidae. Here, we present morphological and molecular data for the genera representing the three remaining clades. We describe two new agglutinated freshwater genera from China and the Netherlands, Lacogromia and Limnogromia, which represent clades 3 and 4, respectively. We also report the first ribosomal DNA sequences of the genus Lieberkuehnia, which place this genus within clade 1. Our study provides the first morphotaxonomic documentation of molecular clades of freshwater foraminifera, showing that the environmental DNA sequences correspond to the agglutinated monothalamous species, morphologically similar to those described 100 years ago.

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• Phylogeny and Systematics of Leptomyxid Amoebae (Amoebozoa, Tubulinea, Leptomyxida). Protist 2016 Oct;168(2):220-252. S1434-4610(16)30067-0. 10.1016/j.protis.2016.10.006. 28-03-2017

  Smirnov A, Nassonova E, Geisen S, Bonkowski M, Kudryavtsev A, Berney C, Glotova A, Bondarenko N, Dyková I, Mrva M, Fahrni J, Pawlowski J

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  We describe four new species of Flabellula, Leptomyxa and Rhizamoeba and publish new SSU rRNA gene and actin gene sequences of leptomyxids. Using these data we provide the most comprehensive SSU phylogeny of leptomyxids to date. Based on the analyses of morphological data and results of the SSU rRNA gene phylogeny we suggest changes in the systematics of the order Leptomyxida (Amoebozoa: Lobosa: Tubulinea). We propose to merge the genera Flabellula and Paraflabellula (the genus Flabellula remains valid by priority rule). The genus Rhizamoeba is evidently polyphyletic in all phylogenetic trees; we suggest retaining the generic name Rhizamoeba for the group unifying R. saxonica, R.matisi n. sp. and R. polyura, the latter remains the type species of the genus Rhizamoeba. Based on molecular and morphological evidence we move all remaining Rhizamoeba species to the genus Leptomyxa. New family Rhizamoebidae is established here in order to avoid paraphyly of the family Leptomyxidae. With the suggested changes both molecular and morphological systems of the order Leptomyxida are now fully congruent to each other.

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• Taxonomy-free molecular diatom index for high-throughput eDNA biomonitoring. Mol Ecol Resour 2017 Mar;():. 10.1111/1755-0998.12668. 13-03-2017

  Apothéloz-Perret-Gentil L, Cordonier A, Straub F, Iseli J, Esling P, Pawlowski J

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  Current biodiversity assessment and biomonitoring are largely based on the morphological identification of selected bioindicator taxa. Recently, several attempts have been made to use eDNA metabarcoding as an alternative tool. However, until now, most applied metabarcoding studies have been based on the taxonomic assignment of sequences that provides reference to morphospecies ecology. Usually, only a small portion of metabarcoding data can be used due to a limited reference database and a lack of phylogenetic resolution. Here, we investigate the possibility to overcome these limitations by using a taxonomy-free approach that allows the computing of a molecular index directly from eDNA data without any reference to morphotaxonomy. As a case study, we use the benthic diatoms index, commonly used for monitoring the biological quality of rivers and streams. We analysed 87 epilithic samples from Swiss rivers, the ecological status of which was established based on the microscopic identification of diatom species. We compared the diatom index derived from eDNA data obtained with or without taxonomic assignment. Our taxonomy-free approach yields promising results by providing a correct assessment for 77% of examined sites. The main advantage of this method is that almost 95% of OTUs could be used for index calculation, compared to 35% in the case of the taxonomic assignment approach. Its main limitations are under-sampling and the need to calibrate the index based on the microscopic assessment of diatoms communities. However, once calibrated, the taxonomy-free molecular index can be easily standardized and applied in routine biomonitoring, as a complementary tool allowing fast and cost-effective assessment of the biological quality of watercourses. This article is protected by copyright. All rights reserved.

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• Toward a comprehensive COI DNA barcode library for Swiss Stoneflies (Insecta: Plecoptera) with special emphasis on the genus Leuctra Zoosymposia 11:135-155 18-11-2016

  Gattolliat JL, Vinçon G, Wyler S, Pawlowski J

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  The Swiss Barcode of Life initiative (SwissBOL) aims to inventory the genetic biodiversity in Switzerland using a short DNA sequence. DNA barcoding provides an additional tool for species identification that complements traditional morphological approaches. We report on the establishment of a DNA barcode library for Plecoptera, taxa that are of great importance as bioindicators of water quality and that often present difficulties in species-level identification for larvae and female specimens. Non-destructive DNA extraction, PCR amplification and sequencing of part of the mitochondrial gene Cytochrome Oxidase I (COI) was conducted for 440 individuals (one to eight per species) belonging to 90 species (of the 112 reported from Switzerland). Intra and interspecific distances were calculated and gene trees reconstructed. In most cases, COI was efficient in delimiting stonefly species. Some doubtful specimens were subsequently re-examined and a few misidentifications were found, especially in some problematic groups in the genus Leuctra Stephens, 1836. Larger genetic distances in some species (e.g. Leuctra nigra (Olivier 1811)) indicate the possible presence of sibling species, while in a few cases closely related species are genetically difficult to separate (within the Leuctra fusca species group).

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• Ancient sedimentary DNA reveals past tsunami deposits Marine Geology 381:29-33 01-11-2016

  Szczuciński W, Pawłowska J, Lejzerowicz F, Nishimurad Y, Kokociński M, Majewski W, Nakamura Y, Pawlowski J.

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  Palaeotsunami deposits are the primary sources of information on past large tsunami events and thereby are critical for earthquake and tsunami hazard assessments. They usually form sandy layers preserved in coastal sediments and contain indicators of marine origins, such as microfossils (e.g., diatoms and foraminifera) and geochemical signals of saltwater. However, these indicators are often modified or erased with time. To address this issue, we present the first application of a palaeogenetic approach to a series of up to approximately 2000-year-old sandy palaeotsunami deposits from a coastal wetland on Hokkaido Island (Japan). We found the DNA of marine foraminiferal species preserved in palaeotsunami deposits, in the absence of foraminifera tests. The recovery and analysis of the ancient sedimentary DNA of marine organisms preserved in coastal zone sediments for millennia represents a new and promising tool for the identification of past tsunamis.

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• Phylogeny and Systematics of Leptomyxid Amoebae (Amoebozoa, Tubulinea, Leptomyxida) Protist, DOI:10.1016/j.protis.2016.10.006 15-10-2016

  Alexey Smirnov, Elena Nassonova, Stefan Geisen, Michael Bonkowski, Alexander Kudryavtsev, Cedric Berney, Anna Glotova, Natalya Bondarenko, Iva Dykova, Martin Mrva, Jose Fahrni, Jan Pawlowski

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  We describe four new species of Flabellula, Leptomyxa and Rhizamoeba and publish new SSU rRNA gene and actin gene sequences of leptomyxids. Using these data we provide the most comprehensive SSU phylogeny of leptomyxids to date. Based on the analyses of morphological data and results of the SSU rRNA gene phylogeny we suggest changes in the systematics of the order Leptomyxida (Amoebozoa: Lobosa: Tubulinea). We propose to merge the genera Flabellula and Paraflabellula (the genus Flabellula remains valid by priority rule). The genus Rhizamoeba is evidently polyphyletic in all phylogenetic trees; we suggest retaining the generic name Rhizamoeba for the group unifying R. saxonica, R.matisi n. sp. and R. polyura, the latter remains the type species of the genus Rhizamoeba. Based on molecular and morphological evidence we move all remaining Rhizamoeba species to the genus Leptomyxa. New family Rhizamoebidae is established here in order to avoid paraphyly of the family Leptomyxidae. With the suggested changes both molecular and morphological systems of the order Leptomyxida are now fully congruent to each other.

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• Genetic structure of a morphological species within the amoeba genus Korotnevella (Amoebozoa: Discosea), revealed by the analysis of two genes. Eur. J. Protistol. 2016 Aug;56():102-111. S0932-4739(16)30059-1. 10.1016/j.ejop.2016.08.001. 07-09-2016

  Zlatogursky VV, Kudryavtsev A, Udalov IA, Bondarenko N, Pawlowski J, Smirnov A

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  Amoebae of the genus Korotnevella are covered with scales, the structure of which is believed to be species-specific and allows distinguishing species reliably at the morphological level. We studied members of this genus in order to assess the genetic structure of the local populations of amoebae. For the present study we isolated nine freshwater strains of Korotnevella, belonging to three species, from two locations in North-Western Russia. In order to obtain data on the population structure of these amoebae, we identified all isolates based on the light-microscopic morphology and scale structure and investigated both inter-strain and intra-strain polymorphism of Cox I and 18S rRNA genes. Results show that both genes provide congruent patterns of population structure. The Cox I gene appears to be more reliable DNA barcode while the 18S rRNA gene shows an interesting pattern of polymorphism, which may represent phylotypes of amoebae. Local population of amoebae in every studied species consists of a number of genetic lineages (phylotypes), some shared between the populations while others are unique to a local habitat.

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• 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. 07-09-2016

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

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  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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• Psammophaga fuegia sp. nov., a new monothalamid foraminifera from the Beagle Channel, South America Acta Protozoologica 55:101-110 25-08-2016

  Gschwend F, Majda A, Majewski W, Pawlowski J

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  Psammophaga fuegiais a new monothalamid foraminifera discovered in surface sediment samples in the Beagle Channel, South America. The species is a member of the important, globally distributed genus Psammophaga, which has the ability to ingest and store mineral particles inside the cytoplasm. Its shape is ovoid to pyriform, the size varies from 250 to 600 µm in length and from 200 to 400 µm in width. Like other Psammophaga species P. fuegia has a single aperture. It was found in multiple samples across the Beagle Channel area at water depths of 4 to 220 meters and in environments as variable as fjords, the main channel, and the harbour of Puerto Williams (Chile). The occurrences of the new species in environmental DNA and RNA samples correspond well to its distribution inferred from the microscopic study.

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• DNA barcoding of formalin-fixed aquatic oligochaetes for biomonitoring. BMC Res Notes 2016 ;9():342. 10.1186/s13104-016-2140-1. 10.1186/s13104-016-2140-1. PMC4944268. 15-07-2016

  Vivien R, Ferrari BJ, Pawlowski J

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  Oligochaetes are valuable bioindicators of the quality of watercourse and lake sediments. The morphological identification of aquatic oligochaetes is difficult, prompting the development of new molecular oligochaete indices based on DNA barcoding and Next-generation sequencing of sorted specimens. In general, the samples for DNA barcoding are fixed in absolute ethanol. However, in the case of aquatic oligochaetes, this medium is not appropriate as it can induce a modification of specimen abundances and of the composition of communities. Therefore, we investigated the possibility to amplify and sequence aquatic oligochaetes fixed in formalin for a short time. We performed guanidine extraction and polymerase chain reaction (PCR) amplification/sequencing of the cytochrome c oxydase I (COI) gene on tissue fragments fixed in formalin for different periods of time (from 1 h to 1 week) and in ethanol.

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• Benthic protists: the under-charted majority. FEMS Microbiol. Ecol. 2016 Aug;92(8):. fiw120. 10.1093/femsec/fiw120. 09-06-2016

  Forster D, Dunthorn M, Mahé F, Dolan JR, Audic S, Bass D, Bittner L, Boutte C, Christen R, Claverie JM, Decelle J, Edvardsen B, Egge E, Eikrem W, Gobet A, Kooistra WH, Logares R, Massana R, Montresor M, Not F, Ogata H, Pawlowski J, Pernice MC, Romac S, Shalchian-Tabrizi K, Simon N, Richards TA, Santini S, Sarno D, Siano R, Vaulot D, Wincker P, Zingone A, de Vargas C, Stoeck T

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  Marine protist diversity inventories have largely focused on planktonic environments, while benthic protists have received relatively little attention. We therefore hypothesize that current diversity surveys have only skimmed the surface of protist diversity in marine sediments, which may harbor greater diversity than planktonic environments. We tested this by analyzing sequences of the hypervariable V4 18S rRNA from benthic and planktonic protist communities sampled in European coastal regions. Despite a similar number of OTUs in both realms, richness estimations indicated that we recovered at least 70% of the diversity in planktonic protist communities, but only 33% in benthic communities. There was also little overlap of OTUs between planktonic and benthic communities, as well as between separate benthic communities. We argue that these patterns reflect the heterogeneity and diversity of benthic habitats. A comparison of all OTUs against the Protist Ribosomal Reference database showed that a higher proportion of benthic than planktonic protist diversity is missing from public databases; similar results were obtained by comparing all OTUs against environmental references from NCBI's Short Read Archive. We suggest that the benthic realm may therefore be the world's largest reservoir of marine protist diversity, with most taxa at present undescribed.

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• Benthic monitoring of salmon farms in Norway using foraminiferal metabarcoding AEI 8:371-386 (2016) - doi:10.3354/aei00182 01-06-2016

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

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  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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• Cutting the Umbilical: New Technological Perspectives in Benthic Deep-Sea Research J. Mar. Sci. Eng. 2016, 4(2), 36; doi:10.3390/jmse4020036 20-05-2016

  Angelika Brandt, Julian Gutt, Marc Hildebrandt 3, Jan Pawlowski, Jakob Schwendner, Thomas Soltwedel and Laurenz Thomsen

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  Many countries are very active in marine research and operate their own research fleets. In this decade, a number of research vessels have been renewed and equipped with the most modern navigation systems and tools. However, much of the research gear used for biological sampling, especially in the deep-sea, is outdated and dependent on wired operations. The deployment of gear can be very time consuming and, thus, expensive. The present paper reviews wire-dependent, as well as autonomous research gear for biological sampling at the deep seafloor. We describe the requirements that new gear could fulfil, including the improvement of spatial and temporal sampling resolution, increased autonomy, more efficient sample conservation methodologies for morphological and molecular studies and the potential for extensive in situ real-time studies. We present applicable technologies from robotics research, which could be used to develop novel autonomous marine research gear, which may be deployed independently and/or simultaneously with traditional wired equipment. A variety of technological advancements make such ventures feasible and timely. In proportion to the running costs of modern research vessels, the development of such autonomous devices might be already paid off after a discrete number of pioneer expeditions.

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• Reducing long-branch effects in multi-protein data uncovers a close relationship between Alveolata and Rhizaria. Mol. Phylogenet. Evol. 2016 Aug;101():1-7. S1055-7903(16)30067-7. 10.1016/j.ympev.2016.04.033. 02-05-2016

  He D, Sierra R, Pawlowski J, Baldauf SL

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  Rhizaria is a major eukaryotic group of tremendous diversity, including amoebae with spectacular skeletons or tests (Radiolaria and Foraminifera), plasmodial parasites (Plasmodiophorida) and secondary endosymbionts (Chlorarachniophyta). Current phylogeny places Rhizaria in an unresolved trichotomy with Stramenopila and Alveolata (supergroup "SAR"). We assembled a 147-protein data set with extensive rhizarian coverage (M147), including the first transcriptomic data for a euglyphid amoeba. Phylogenetic pre-screening of individual proteins indicated potential problems with radically misplaced sequences due either to contamination of rhizarian sequences amplified from wild collected material and/or extremely long branches (xLBs). Therefore, two data subsets were extracted containing either all proteins consistently recovering rhizarian monophyly (M34) or excluding all proteins with ⩾3 xLBs (defined as ⩾2× the average terminal branch length for the tree). Phylogenetic analyses of M147 give conflicting results depending on the outgroup and method of analysis but strongly support an exclusive Rhizaria+Alveolata (R+A) clade with both data subsets (M34 and M37) regardless of phylogenetic method used. Support for an R+A clade is most consistent when a close outgroup is used and decreases with more distant outgroups, suggesting that support for alternative SAR topologies may reflect a long-branch attraction artifact. A survey of xLB distribution among taxa and protein functional category indicates that small "informational" proteins in particular have highly variable evolutionary rates with no consistent pattern among taxa.

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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. 24-03-2016

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

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  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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• Selective zircon accumulation in a new benthic foraminifer, Psammophaga zirconia, sp. nov. Geobiology, 14: 404–416. doi:10.1111/gbi.12179 01-03-2016

  A. Sabbatini, A. Negri, A. Bartolini, C. Morigi, O. Boudouma, E. Dinelli, F. Florindo, R. Galeazzi, M. Holzmann, P. C. Lurcock, L. Massaccesi, J. Pawlowski, S. Rocchi

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  Benthic foraminifera are single-celled eukaryotes that make a protective organic, agglutinated or calcareous test. Some agglutinated, single-chambered taxa, including Psammophaga Arnold, 1982, retain mineral particles in their cytoplasm, but the selective mechanism of accumulation is not clear. Here, we report the ability of a foraminiferal species to select and accumulate zircons and other heavy minerals in their cytoplasm. In particular, the use of Scanning Electron Microscope coupled with an Energy Dispersive X-ray microanalysis system (SEM–EDS) enabled a representative overview of the mineral diversity and showed that the analysed Psammophaga zirconia sp. nov. individuals contained dominantly crystals of zircon (51%), titanium oxides (27%), and ilmenite (11%) along with minor magnetite and other minerals. The studied specimens occur in the shallow central Adriatic Sea where the sediment has a content of zircon below 1% and of other heavy minerals below 4%. For that reason we hypothesize that: (i) P. zirconia may be able to chemically select minerals, specifically zircon and rutile; (ii) the chemical mechanism allowing the selection is based on electrostatic interaction, and it could work also for agglutinated foraminifera (whether for ingestion, like Xenophyophores, or incorporation in the test as in many other described taxa). In particular, this aptitude for high preferential uptake and differential ingestion or retention of zircon is reported here for the first time, together with the selection of other heavy minerals already described in members of the genus Psammophaga. They are generally counted among early foraminifera, constructing a morphologically simple test with a single chamber. Our molecular phylogenetic study confirms that P. zirconia is a new species, genetically distinctive from other Psammophaga, and occurs in the Adriatic as well as in the Black Sea.

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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. 13-02-2016

  Vivien R, Lejzerowicz F, Pawlowski J

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  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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• Worldwide analysis of sedimentary DNA reveals major gaps in taxonomic knowledge of deep-sea benthos Frontiers in Marine Science 3, 92; 10.3389/fmars.2016.00092 01-01-2016

  Frédéric Sinniger, Jan Pawlowski, Saki Harii, Andrew J. Gooday, Hiroyuki Yamamoto, Pierre Chevaldonné, Tomas Cedhagen, Gary Carvalho and Simon Creer

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  Deep-sea sediments represent the largest but least known ecosystem on earth. With increasing anthropogenic pressure, it is now a matter of urgency to improve our understanding of deep-sea biodiversity. Traditional morpho-taxonomic studies suggest that the ocean floor hosts extraordinarily diverse benthic communities. However, due to both its remoteness and a lack of expert taxonomists, assessing deep-sea diversity is a very challenging task. Environmental DNA (eDNA) metabarcoding offers a powerful tool to complement morpho-taxonomic studies. Here we use eDNA to assess benthic metazoan diversity in 39 deep-sea sediment samples from bathyal and abyssal depths worldwide. The eDNA dataset was dominated by meiobenthic taxa and we identified all animal phyla commonly found in the deep-sea benthos; yet, the diversity within these phyla remains largely unknown. The large numbers of taxonomically unassigned molecular operational taxonomic units (OTUs) were not equally distributed among phyla, with nematodes and platyhelminthes being the most poorly characterized from a taxonomic perspective. While the data obtained here reveal pronounced heterogeneity and vast amounts of unknown biodiversity in the deep sea, they also expose the difficulties in exploiting metabarcoding datasets resulting from the lack of taxonomic knowledge and appropriate reference databases. Overall, our study demonstrates the promising potential of eDNA metabarcoding to accelerate the assessment of deep-sea biodiversity for pure and applied deep-sea environmental research but also emphasizes the necessity to integrate such new approaches with traditional morphology-based examination of deep-sea organisms.

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• Evolutionary Origins of Rhizarian Parasites. Mol. Biol. Evol. 2016 Apr;33(4):980-3. msv340. 10.1093/molbev/msv340. 19-12-2015

  Sierra R, Cañas-Duarte SJ, Burki F, Schwelm A, Fogelqvist J, Dixelius C, González-García LN, Gile GH, Slamovits CH, Klopp C, Restrepo S, Arzul I, Pawlowski J

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  The SAR group (Stramenopila, Alveolata, Rhizaria) is one of the largest clades in the tree of eukaryotes and includes a great number of parasitic lineages. Rhizarian parasites are obligate and have devastating effects on commercially important plants and animals but despite this fact, our knowledge of their biology and evolution is limited. Here, we present rhizarian transcriptomes from all major parasitic lineages in order to elucidate their evolutionary relationships using a phylogenomic approach. Our results suggest that Ascetosporea, parasites of marine invertebrates, are sister to the novel clade Apofilosa. The phytomyxean plant parasites branch sister to the vampyrellid algal ectoparasites in the novel clade Phytorhiza. They also show that Ascetosporea + Apofilosa + Retaria + Filosa + Phytorhiza form a monophyletic clade, although the branching pattern within this clade is difficult to resolve and appears to be model-dependent. Our study does not support the monophyly of the rhizarian parasitic lineages (Endomyxa), suggesting independent origins for rhizarian animal and plant parasites.

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• Widespread intra-specific genetic homogeneity of coastal Antarctic benthic foraminifera Polar Biology 38 (12), 2047-2058; 10.1007/s00300-015-1765-1 01-12-2015

  Wojciech Majewski, Samuel S. Bowser, Jan Pawlowski

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  Benthic foraminifera are a major component of the Antarctic biota. Coastal foraminiferal morphospecies are widely distributed in Antarctic waters. The question is whether these morphotypes are genetically identical or, rather, they represent a cohort of cryptic species. Here, we compared genetically nine benthic foraminiferal morphospecies from Admiralty Bay (South Shetlands) and the western Ross Sea (McMurdo Sound, Terra Nova Bay), separated by a distance of ~4500 km. Additionally, for three of these morphospecies, we included specimens from Rothera (Marguerite Bay), which is located between the two main areas of interest. Our study, based on SSU and ITS rDNA sequence data, shows that all examined morphospecies share the same genotypes despite the presence of considerable intra-individual genetic variability.

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• Molecular Phylogeny and Ecology of Textularia agglutinans d'Orbigny from the Mediterranean Coast of Israel: A Case of a Successful New Incumbent. PLoS ONE 2015 ;10(11):e0142263. 10.1371/journal.pone.0142263. PONE-D-15-21505. PMC4634767. 06-11-2015

  Merkado G, Titelboim D, Hyams-Kaphzan O, Holzmann M, Pawlowski J, Almogi-Labin A, Abdu U, Herut B, Abramovich S

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  Textularia agglutinans d'Orbigny is a non-symbiont bearing and comparatively large benthic foraminiferal species with a widespread distribution across all oceans. In recent years, its populations have considerably expanded along the Israeli Mediterranean coast of the eastern Levantine basin. Despite its exceptionally widespread occurrence, no molecular data have yet been obtained. This study provides the first ribosomal DNA sequences of T. agglutinans complemented with morphological and ecological characterization, which are based on material collected during environmental monitoring of the hard bottom habitats along the Israeli Mediterranean coast, and from the Gulf of Elat (northern Red Sea). Our phylogenetic analyses reveal that all specimens from both provinces belong to the same genetic population, regardless their morphological variability. These results indicate that modern population of T. agglutinans found on the Mediterranean coast of Israel is probably Lessepsian. Our study also reveals that T. agglutinans has an epiphytic life mode, which probably enabled its successful colonization of the hard bottom habitats, at the Mediterranean coast of Israel, which consist of a diverse community of macroalgae. Our study further indicates that the species does not tolerate high SST (> 35°C), which will probably prevent its future expansion in the easternmost Mediterranean in light of the expected rise in temperatures.

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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. 12-09-2015

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

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  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. 05-09-2015

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

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  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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• Description of Paramoeba atlantica n. sp.(Amoebozoa, Dactylopodida)–a marine amoeba from the Eastern Atlantic, with emendation of the dactylopodid families Acta Protozool. (2011) 50: 239–253 27-08-2015

  Alexander Kudryavtsev, Jan Pawlowski, Klaus Hausmann

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  A strain of marine amoeba has been isolated and studied from the bottom sediments of the Great Meteor Seamount (Atlantic Ocean, 29°36.29′N; 28°59.12′W; 267.4 m deep). This amoeba has a typical dactylopodiid morphotype, a coat of delicate, boat-shaped scales, and a Perkinsela-like organism (PLO), an obligatory, deeply-specialized kinetoplastid symbiont near the nucleus. These characters allow us to include this species into the genus Paramoeba. However, it differs from its only described species, P. eilhardi, in the structure of scales. P. atlantica n. sp. is established therefore to accommodate the studied strain. SSU rRNA gene sequence analysis suggests that P. atlantica belongs to the Dactylopodida, and is sister to a monophyletic clade of P. eilhardi and all Neoparamoeba spp., branching separately from P. eilhardi. Therefore, the genera Paramoeba and Neoparamoeba, currently defined based on the cell surface ultrastructure, might be paraphyletic and probably should be synonymized, as further evidence is accumulated. Based on the data available we emend the families Vexilliferidae and Paramoebidae to make them more consistent with the current phylogenetic schemes.

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• Palaeoceanographic changes in Hornsund Fjord (Spitsbergen, Svalbard) over the last millennium: new insights from ancient DNA 13-08-2015

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

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  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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• Flexammina islandica gen. nov. sp. nov. and some new phylotypes of monothalamous foraminifera from the coast of Iceland. Zootaxa 2015 ;3964(2):245-59. zootaxa.3964.2.5. 08-08-2015

  Voltski I, Pawlowski J

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  Monothalamous (single-chambered) foraminifera comprise a poorly known group, the diversity of which is strongly underestimated according to environmental DNA surveys. The gross morphology of monothalamids offers few distinguishing features; their organic-walled or agglutinated tests are often very delicate and make isolation difficult. Here, we use an integrated taxonomic approach, including morphological and molecular analysis, to examine the diversity of monothalamids in a shallow subtidal area on the coast of Iceland. We report nine new phylotypes of single-chambered foraminifera distinguished by SSU rDNA sequences. Among them, we establish a new genus Flexammina and a new species Flexammina islandica, possessing a very pliable, finely agglutinated test capable of extreme shape transformations. According to molecular data, F. islandica belongs to the monothalamid clade M, which also includes the well-known genus Allogromia. In addition, we provide brief descriptions and illustrations of nine other monothalamous foraminifera isolated from the same area. Three of them are closely related to some unidentified environmental sequences and serve as the first microscopic documentation of these anonymous lineages.

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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 01-08-2015

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

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  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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• Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing. Environ. Microbiol. 2015 Oct;17(10):4035-49. 10.1111/1462-2920.12955. 30-06-2015

  Massana R, Gobet A, Audic S, Bass D, Bittner L, Boutte C, Chambouvet A, Christen R, Claverie JM, Decelle J, Dolan JR, Dunthorn M, Edvardsen B, Forn I, Forster D, Guillou L, Jaillon O, Kooistra WH, Logares R, Mahé F, Not F, Ogata H, Pawlowski J, Pernice MC, Probert I, Romac S, Richards T, Santini S, Shalchian-Tabrizi K, Siano R, Simon N, Stoeck T, Vaulot D, Zingone A, de Vargas C

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  Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

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• Environmental Monitoring: Inferring the Diatom Index from Next-Generation Sequencing Data. Environ. Sci. Technol. 2015 Jul;49(13):7597-605. 10.1021/es506158m. 09-06-2015

  Visco JA, Apothéloz-Perret-Gentil L, Cordonier A, Esling P, Pillet L, Pawlowski J

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  Diatoms are widely used as bioindicators for the assessment of water quality in rivers and streams. Classically, the diatom biotic indices are based on the relative abundance of morphologically identified species weighted by their autoecological value. Obtaining such indices is time-consuming, costly, and requires excellent taxonomic expertise, which is not always available. Here we tested the possibility to overcome these limitations using a next-generation sequencing (NGS) approach to identify and quantify diatoms found in environmental DNA and RNA samples. We analyzed 27 river sites in the Geneva area (Switzerland), in order to compare the values of the Swiss Diatom Index (DI-CH) computed either by microscopic quantification of diatom species or directly from NGS data. Despite gaps in the reference database and variations in relative abundance of analyzed species, the diatom index shows a significant correlation between morphological and molecular data indicating similar biological quality status for the majority of sites. This proof-of-concept study demonstrates the potential of the NGS approach for identification and quantification of diatoms in environmental samples, opening new avenues toward the routine application of genetic tools for bioassessment and biomonitoring of aquatic ecosystems.

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• Cunea n. g. (Amoebozoa, Dactylopodida) with two cryptic species isolated from different areas of the ocean. Eur. J. Protistol. 2015 Jun;51(3):197-209. S0932-4739(15)00021-8. 10.1016/j.ejop.2015.04.002. 08-05-2015

  Kudryavtsev A, Pawlowski J

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  This paper describes a new genus, Cunea n. g., of marine naked amoebae with two cryptic species, Cunea profundata and Cunea thuwala, isolated from distant localities in the ocean and different depths (Brazilian abyssal plain, Western Atlantic Ocean, depth >5km and the Red Sea off the Saudi Arabian coast, depth ca. 58.7m). Both species are very similar to each other in the set of light microscopic and ultrastructural characters and might be described as a single species, yet their genetic divergence based on 3 molecular markers (small-subunit ribosomal RNA, actin and cytochrome c oxidase subunit 1) corresponds to the level of variation typically observed between different morphospecies of Amoebozoa. In addition, the studied strains differ strongly in their temperature tolerance ranges, C. profundata isolated from the cold Atlantic deep-sea habitat being able to reproduce under lower temperatures than C. thuwala isolated from the warm Red Sea benthos. Molecular phylogenetic analysis based on SSU rRNA gene shows that the new genus robustly branches within the Dactylopodida, but forms an independent clade within this order that does not group with any of its known genera.

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• Molecular barcoding of aquatic oligochaetes: implications for biomonitoring. PLoS ONE 2015 ;10(4):e0125485. 10.1371/journal.pone.0125485. PONE-D-14-47299. PMC4391796. 10-04-2015

  Vivien R, Wyler S, Lafont M, Pawlowski J

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  Aquatic oligochaetes are well recognized bioindicators of quality of sediments and water in watercourses and lakes. However, the difficult taxonomic determination based on morphological features compromises their more common use in eco-diagnostic analyses. To overcome this limitation, we investigated molecular barcodes as identification tool for broad range of taxa of aquatic oligochaetes. We report 185 COI and 52 ITS2 rDNA sequences for specimens collected in Switzerland and belonging to the families Naididae, Lumbriculidae, Enchytraeidae and Lumbricidae. Phylogenetic analyses allowed distinguishing 41 lineages separated by more than 10 % divergence in COI sequences. The lineage distinction was confirmed by Automatic Barcode Gap Discovery (ABGD) method and by ITS2 data. Our results showed that morphological identification underestimates the oligochaete diversity. Only 26 of the lineages could be assigned to morphospecies, of which seven were sequenced for the first time. Several cryptic species were detected within common morphospecies. Many juvenile specimens that could not be assigned morphologically have found their home after genetic analysis. Our study showed that COI barcodes performed very well as species identifiers in aquatic oligochaetes. Their easy amplification and good taxonomic resolution might help promoting aquatic oligochaetes as bioindicators for next generation environmental DNA biomonitoring of aquatic ecosystems.

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• Transcriptome analyses to investigate symbiotic relationships between marine protists. Front Microbiol 2015 ;6():98. 10.3389/fmicb.2015.00098. PMC4362344. 09-04-2015

  Balzano S, Corre E, Decelle J, Sierra R, Wincker P, Da Silva C, Poulain J, Pawlowski J, Not F

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  Rhizaria are an important component of oceanic plankton communities worldwide. A number of species harbor eukaryotic microalgal symbionts, which are horizontally acquired in the environment at each generation. Although these photosymbioses are determinant for Rhizaria ability to thrive in oceanic ecosystems, the mechanisms for symbiotic interactions are unclear. Using high-throughput sequencing technology (i.e., 454), we generated large Expressed Sequence Tag (EST) datasets from four uncultured Rhizaria, an acantharian (Amphilonche elongata), two polycystines (Collozoum sp. and Spongosphaera streptacantha), and one phaeodarian (Aulacantha scolymantha). We assessed the main genetic features of the host/symbionts consortium (i.e., the holobiont) transcriptomes and found rRNA sequences affiliated to a wide range of bacteria and protists in all samples, suggesting that diverse microbial communities are associated with the holobionts. A particular focus was then carried out to search for genes potentially involved in symbiotic processes such as the presence of c-type lectins-coding genes, which are proteins that play a role in cell recognition among eukaryotes. Unigenes coding putative c-type lectin domains (CTLD) were found in the species bearing photosynthetic symbionts (A. elongata, Collozoum sp., and S. streptacantha) but not in the non-symbiotic one (A. scolymantha). More particularly, phylogenetic analyses group CTLDs from A. elongata and Collozoum sp. on a distinct branch from S. streptacantha CTLDs, which contained carbohydrate-binding motifs typically observed in other marine photosymbiosis. Our data suggest that similarly to other well-known marine photosymbiosis involving metazoans, the interactions of glycans with c-type lectins is likely involved in modulation of the host/symbiont specific recognition in Radiolaria.

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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. 19-02-2015

  Esling P, Lejzerowicz F, Pawlowski J

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  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 31-01-2015

  Franck Lejzerowicz, Ivan Voltski, Jan Pawlowski

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  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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• PROTELPHIDIUM NIVEUM () AND THE TAXONOMY OF “LOWER” ELPHIDIIDS The Journal of Foraminiferal Research 45 (3), 250-263; 10.2113/gsjfr.45.3.250 07-01-2015

  Ivan Voltski, Sergei Korsun, Loïc Pillet, Jan Pawlowski

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  A small planispiral rotaliid, Nonion? niveum, was described by Lafrenz from the Eemian (upper Pleistocene) of the Baltic Sea. Since then, the species has several times been attributed to other genera (Haynesina, Protelphidium), resulting in taxonomic confusion. Recently, a morphologically identical foraminifer has been found in the White Sea, which is the first documented occurrence of the species from this area. In-depth investigation of its test morphology revealed that the species has features common to modern Haynesina representatives and particularly to the extinct Paleogene species Protelphidium hofkeri. This was especially evident in the architecture of the umbilical area. With the help of a comparative morphological study of several other elphidiids, the species in question was attributed to the genus Protelphidium, as its only living member and with the necessary emendation of the diagnosis. We believe that the redescription of Protelphidium niveum helps to clarify the taxonomy of the basal “lower” elphidiids by coupling the revision of morphological features with molecular data.

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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. 21-11-2014

  Pawlowski J, Lejzerowicz F, Esling P

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  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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• Molecular Phylogeny and Morphology of Leannia veloxifera n. gen. et sp. Unveils a New Lineage of Monothalamous Foraminifera. J. Eukaryot. Microbiol. ;62(3):353-61. 10.1111/jeu.12190. 05-11-2014

  Apothéloz-Perret-Gentil L, Pawlowski J

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  Monothalamous (single-chambered) foraminifera have long been considered as the "poor cousins" of multichambered species, which calcareous and agglutinated tests dominate in the fossil record. This view is currently changing with environmental DNA surveys showing that the monothalamids may be as diverse as hard-shelled foraminifera. Yet, the majority of numerous molecular lineages revealed by eDNA studies remain anonymous. Here, we describe a new monothalamous species and genus isolated from the sample of sea grass collected in Gulf of Eilat (Red Sea). This new species, named Leannia veloxifera, is characterized by a tiny ovoid theca (about 50-100 μm) composed of thin organic wall, with two opposite apertures. The examined individuals are multinucleated and show very active reticulopodial movement. Phylogenetic analyses of SSU rDNA, actin, and beta-tubulin (ß-tubulin) show that the species represents a novel lineage branching separately from other monothalamous foraminifera. Interestingly, the SSU rDNA sequence of the new species is very similar to an environmental foraminiferal sequence from Bahamas, suggesting that the novel lineage may represent a group of shallow-water tropical allogromiids, poorly studied until now.

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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 31-10-2014

  Franck Lejzerowicz, Philippe Esling, Jan Pawlowski

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  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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• MOLECULAR PHYLOGENY OF CARTERINA SPICULOTESTA AND RELATED SPECIES FROM NEW CALEDONIA The Journal of Foraminiferal Research 44 (4), 440-450; 10.2113/gsjfr.44.4.440 01-10-2014

  Jan Pawlowski, Maria Holzmann, Jean-Pierre Debenay

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  Carterina spiculotesta is a common tropical benthic foraminifer characterized by fusiform or rod-like calcareous spicules covering the surface of the test. Because of this peculiar wall feature, the genus Carterina was separated from other foraminifera and placed in its own suborder (Caterinina) or order (Carterinida). However, there is no agreement about the origin of Carterina spicules, which are considered either as being secreted by the foraminiferal cell or as agglutinated foreign particles; if the latter case, the genus was placed in the order Trochamminida. Here, we attempted to resolve this controversy by analysis of genetic data from various carterinids collected in New Caledonia. We obtained seven complete and 47 partial small subunit (SSU) rDNA sequences. Our results show that all specimens of spicule-bearing Carterina cluster together in a strongly supported clade. Sister to this clade are undetermined lineages of trochamminid morphospecies. Together with its sister groups, the Carterina clade forms an independent lineage at the base of Globothalamea within the paraphyletic radiation of textulariids and robertinids. Its exact phylogenetic position was difficult to establish because the used SSU rRNA genes lack resolution. As long as experimental data do not contradict the hypothesis of a secreted origin for Carterina spicules, we propose to retain the ordinal status of the Carterina clade, and consider it as one of the orders of the class Globothalamea. In view of our study, the diversity of this order may be much higher than traditionally accepted, including several genera and species, many of them new to science.

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• Wide occurrence of SSU rDNA intragenomic polymorphism in foraminifera and its implications for molecular species identification. Protist 2014 Sep;165(5):645-61. S1434-4610(14)00078-9. 10.1016/j.protis.2014.07.006. 26-08-2014

  Weber AA, Pawlowski J

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  Ribosomal DNA is commonly used as a marker for protist phylogeny and taxonomy because of its ubiquity and its expected species specificity thanks to the mechanism of concerted evolution. However, numerous studies reported the occurrence of intragenomic (intra-individual) polymorphism in various protists and particularly in Foraminifera. To infer to what extent the SSU rDNA intragenomic variability occurs in Foraminifera, we studied 16 foraminiferal species belonging to single-chambered monothalamids and multi-chambered Globothalamea, with one to six individuals per species. We performed single-cell DNA extractions and PCRs of a 600bp fragment of SSU rDNA, and sequenced 9 to 23 clones per individual for a total of 818 sequences. We found intragenomic variability in almost all species, even after excluding singleton mutations. Intra-individual sequence divergence ranged from 0 to 5.15% and was higher than 1% in 11 species. Variability was usually located at the end of stem-loop structures and included compensatory single nucleotide polymorphisms and expansion segments polymorphisms. However, the polymorphisms did not change the secondary structure of the rRNA. Our results suggest a non-concerted evolution of rRNA genes in Foraminifera. The origin of this variability and its implications for species identification in environmental DNA studies are discussed.

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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 07-08-2014

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

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  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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• The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing. PLoS Biol. 2014 Jun;12(6):e1001889. 10.1371/journal.pbio.1001889. PBIOLOGY-D-14-00511. PMC4068987. 25-06-2014

  Keeling PJ, Burki F, Wilcox HM, Allam B, Allen EE, Amaral-Zettler LA, Armbrust EV, Archibald JM, Bharti AK, Bell CJ, Beszteri B, Bidle KD, Cameron CT, Campbell L, Caron DA, Cattolico RA, Collier JL, Coyne K, Davy SK, Deschamps P, Dyhrman ST, Edvardsen B, Gates RD, Gobler CJ, Greenwood SJ, Guida SM, Jacobi JL, Jakobsen KS, James ER, Jenkins B, John U, Johnson MD, Juhl AR, Kamp A, Katz LA, Kiene R, Kudryavtsev A, Leander BS, Lin S, Lovejoy C, Lynn D, Marchetti A, McManus G, Nedelcu AM, Menden-Deuer S, Miceli C, Mock T, Montresor M, Moran MA, Murray S, Nadathur G, Nagai S, Ngam PB, Palenik B, Pawlowski J, Petroni G, Piganeau G, Posewitz MC, Rengefors K, Romano G, Rumpho ME, Rynearson T, Schilling KB, Schroeder DC, Simpson AG, Slamovits CH, Smith DR, Smith GJ, Smith SR, Sosik HM, Stief P, Theriot E, Twary SN, Umale PE, Vaulot D, Wawrik B, Wheeler GL, Wilson WH, Xu Y, Zingone A, Worden AZ

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• Toxisarcon taimyr sp. nov., a new large monothalamous foraminifer from the Kara Sea inner shelf Biodiv (2014) 44: 213. doi:10.1007/s12526-014-0204-9 01-06-2014

  Ivan Voltski, Sergei Korsun, Jan Pawlowski

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  A large monothalamous foraminiferan, Toxisarcon taimyr sp. nov., has been isolated from the benthic samples from the Kara Sea inner shelf near the mouth of Yenisey river estuary, at a depth of 50–100 m. In its overall morphology, the new species closely resembles T. synsuicidica, one of the two species of Toxisarcon described to date. It possesses a large irregularly shaped cell body, covered by a thin layer of a fibrous organic coating. Numerous reticulopodia typically extend from all over the cell surface; the species is very motile and rapidly changes cell shape. Long and thick reticulopodial bundles form in the direction of movement. In the phylogenetic tree based on partial small-subunit ribosomal DNA (SSU rDNA) sequences, T. taimyr branches together with the two other known species of Toxisarcon within the clade C of monothalamous foraminifera.

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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. 17-04-2014

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

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  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 15-04-2014

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

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  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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• A revision of the order Pellitida Smirnov et al., 2011 (Amoebozoa, Discosea) based on ultrastructural and molecular evidence, with description of Endostelium crystalliferum n. sp. Protist 2014 Mar;165(2):208-29. S1434-4610(14)00016-9. 10.1016/j.protis.2014.02.003. 08-04-2014

  Kudryavtsev A, Brown MW, Tice A, Spiegel FW, Pawlowski J, Anderson OR

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  We present the results of an ultrastructural re-investigation of two amoebae strains that can be identified morphologically as previously described species of the genus Pellita, as well as the first molecular phylogenetic analysis of these amoebae based on SSU rRNA and actin gene sequences. The results obtained show close relationships between the genera Pellita, Gocevia, and Endostelium. These relationships are further supported by the description of Endostelium crystalliferum n. sp., which shares morphological characters simultaneously with Pellita spp. and Endostelium zonatum. The three genera form a robust clade that branches deeply within Amoebozoa, among either Flabellinia, or Longamoebia, depending on taxon sampling. The results suggest that Gocevia and Endostelium should not be included in the Himatismenida; therefore, we transfer the family Goceviidae into Pellitida. The type of cell organisation that was considered to be typical of Himatismenida (a lens-shaped cell covered dorsally with a flexible layer of organic material) has most probably evolved in some of these amoebae independently of Cochliopodiidae and Parvamoebidae. The robustness of the Pellitida clade in the molecular trees is consistent with the fine structure cytoplasmic evidence for these taxa, in particular, the presence of a centrosphere (a dictyosome-associated lamellar MTOC) in all these genera.

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• Patterns of rare and abundant marine microbial eukaryotes. Curr. Biol. 2014 Apr;24(8):813-21. S0960-9822(14)00218-8. 10.1016/j.cub.2014.02.050. 08-04-2014

  Logares R, Audic S, Bass D, Bittner L, Boutte C, Christen R, Claverie JM, Decelle J, Dolan JR, Dunthorn M, Edvardsen B, Gobet A, Kooistra WH, Mahé F, Not F, Ogata H, Pawlowski J, Pernice MC, Romac S, Shalchian-Tabrizi K, Simon N, Stoeck T, Santini S, Siano R, Wincker P, Zingone A, Richards TA, de Vargas C, Massana R

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  Biological communities are normally composed of a few abundant and many rare species. This pattern is particularly prominent in microbial communities, in which most constituent taxa are usually extremely rare. Although abundant and rare subcommunities may present intrinsic characteristics that could be crucial for understanding community dynamics and ecosystem functioning, microbiologists normally do not differentiate between them. Here, we investigate abundant and rare subcommunities of marine microbial eukaryotes, a crucial group of organisms that remains among the least-explored biodiversity components of the biosphere. We surveyed surface waters of six separate coastal locations in Europe, independently considering the picoplankton, nanoplankton, and microplankton/mesoplankton organismal size fractions.

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• A plea for DNA barcoding of foraminifera Marine Biodiversity 44 (2), 213-221; DOI: 10.2113/gsjfr.44.1.62 01-01-2014

  Jan Pawlowski, Maria Holzmann

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  DNA barcoding is the molecular identification of species using short, standardized gene sequences. Numerous applications of DNA barcoding in taxonomy, ecology, bioconservation, and biosafety contributed to a spectacular development of this initiative administered by the Consortium for the Barcode of Life (CBOL). Reference databases that assign DNA barcodes to particular morphospecies have been developed for almost all groups of animals, plants, and fungi, as well as some groups of protists (i.e., diatoms, ciliates, amoebae). However, such a database does not exist for foraminifera, despite large numbers of DNA sequences being available. To fill this gap, we initiated the Foram Barcoding (FB) project, whose objective is to create a curated molecular database for modern foraminifera. Each species included in our database is represented by one or several specimens, from which DNA was extracted and sequenced. Species entries include photos of processed specimens, taxonomic references, and DNA barcode sequences. A fragment of the 18S rRNA gene, commonly used in foraminiferal molecular studies, was chosen as the DNA barcode. We believe that the FB project will help resolve at least some misidentification problems that plague foraminiferal taxonomy. We also foresee its further applications in such domains of foraminiferal research as diversity assessment, ecology, biogeography, and biomonitoring. However, we are aware that the impact of the FB database depends mainly on its completeness and accuracy, and thus we appeal to the community of foraminiferologists to support this project by providing material for genetic studies and by contributing their taxonomic expertise in species identification and documentation.

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• The genome of the foraminiferan Reticulomyxa filosa. Curr. Biol. 2014 Jan;24(1):11-8. S0960-9822(13)01446-2. 10.1016/j.cub.2013.11.027. 18-12-2013

  Glöckner G, Hülsmann N, Schleicher M, Noegel AA, Eichinger L, Gallinger C, Pawlowski J, Sierra R, Euteneuer U, Pillet L, Moustafa A, Platzer M, Groth M, Szafranski K, Schliwa M

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  Rhizaria are a major branch of eukaryote evolution with an extensive microfossil record, but only scarce molecular data are available. The rhizarian species Reticulomyxa filosa, belonging to the Foraminifera, is free-living in freshwater environments. In culture, it thrives only as a plasmodium with thousands of haploid nuclei in one cell. The R. filosa genome is the first foraminiferal genome to be deciphered.

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• Molecular evidence for Lessepsian invasion of soritids (larger symbiont bearing benthic foraminifera). PLoS ONE 2013 ;8(10):e77725. 10.1371/journal.pone.0077725. PONE-D-13-03809. PMC3812231. 10-11-2013

  Merkado G, Holzmann M, Apothéloz-Perret-Gentil L, Pawlowski J, Abdu U, Almogi-Labin A, Hyams-Kaphzan O, Bakhrat A, Abramovich S

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  The Mediterranean Sea is considered as one of the hotspots of marine bioinvasions, largely due to the influx of tropical species migrating through the Suez Canal, so-called Lessepsian migrants. Several cases of Lessepsian migration have been documented recently, however, little is known about the ecological characteristics of the migrating species and their aptitude to colonize the new areas. This study focused on Red Sea soritids, larger symbiont-bearing benthic foraminifera (LBF) that are indicative of tropical and subtropical environments and were recently found in the Israeli coast of the Eastern Mediterranean. We combined molecular phylogenetic analyses of soritids and their algal symbionts as well as network analysis of Sorites orbiculus Forskål to compare populations from the Gulf of Elat (northern Red Sea) and from a known hotspot in Shikmona (northern Israel) that consists of a single population of S. orbiculus. Our phylogenetic analyses show that all specimens found in Shikmona are genetically identical to a population of S. orbiculus living on a similar shallow water pebbles habitat in the Gulf of Elat. Our analyses also show that the symbionts found in Shikmona and Elat soritids belong to the Symbiodinium clade F5, which is common in the Red Sea and also present in the Indian Ocean and Caribbean Sea. Our study therefore provides the first genetic and ecological evidences that indicate that modern population of soritids found on the Mediterranean coast of Israel is probably Lessepsian, and is less likely the descendant of a native ancient Mediterranean species.

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• Molecular evidence for β-tubulin neofunctionalization in Retaria (Foraminifera and radiolarians). Mol. Biol. Evol. 2013 Nov;30(11):2487-93. mst150. 10.1093/molbev/mst150. 07-09-2013

  Hou Y, Sierra R, Bassen D, Banavali NK, Habura A, Pawlowski J, Bowser SS

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  Foraminifera and radiolarians are closely related amoeboid protists (i.e., retarians) often characterized by their shells and pseudopodia. Previous studies hypothesized that the unusual "Type 2" β-tubulin (β2) is critically involved in forming helical filaments (HFs), a unique microtubule (MT) assembly/disassembly intermediate found in foraminiferan reticulopodia. Such noncanonical β-tubulin sequences have also been found in two radiolarian species and appear to be closely related to the foraminiferan β2. In this study, we report 119 new β-tubulin transcript sequences from six foraminiferans, four radiolarians, and a related non-retarian species. We found that foraminiferan and radiolarian β2-tubulins share some of the unusual substitutions in the structurally essential and usually conserved domains. In the β-tubulin phylogeny, retarian β2-tubulin forms a monophyletic clade, well separated from the canonical β-tubulin (β1) ubiquitous in eukaryotes. Furthermore, we found that foraminiferan and radiolarian β2-tubulin lineages were under positive selection, and used homology models for foraminiferan α- and β-tubulin hexamers to understand the structural effect of the positively selected substitutions. We suggest that the positively selected substitutions play key roles in the transition of MT to HF by altering the lateral and longitudinal interactions between α- and β-tubulin heterodimers. Our results indicate that the unusual β2-tubulin is a molecular synapomorphy of retarians, and the β-tubulin gene duplication occurred before the divergence of Foraminifera and radiolarians. The duplicates have likely been subjected to neofunctionalization responsible for the unique MT to HF assembly/disassembly dynamics, and/or other unknown physiological processes in retarian protists.

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• Phylogenomics of the intracellular parasite Mikrocytos mackini reveals evidence for a mitosome in rhizaria. Curr. Biol. 2013 Aug;23(16):1541-7. S0960-9822(13)00758-6. 10.1016/j.cub.2013.06.033. 31-07-2013

  Burki F, Corradi N, Sierra R, Pawlowski J, Meyer GR, Abbott CL, Keeling PJ

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  Mikrocytos mackini is an intracellular protistan parasite of oysters whose position in the phylogenetic tree of eukaryotes has been a mystery for many years [1,2]. M. mackini is difficult to isolate, has not been cultured, and has no defining morphological feature. Furthermore, its only phylogenetic marker that has been successfully sequenced to date (the small subunit ribosomal RNA) is highly divergent and has failed to resolve its evolutionary position [2]. M. mackini is also one of the few eukaryotes that lacks mitochondria [1], making both its phylogenetic position and comparative analysis of mitochondrial function particularly important. Here, we have obtained transcriptomic data for M. mackini from enriched isolates and constructed a 119-gene phylogenomic data set. M. mackini proved to be among the fastest-evolving eukaryote lineages known to date, but, nevertheless, our analysis robustly placed it within Rhizaria. Searching the transcriptome for genetic evidence of a mitochondrion-related organelle (MRO) revealed only four mitochondrion-derived genes: IscS, IscU, mtHsp70, and FdxR. Interestingly, all four genes are involved in iron-sulfur cluster formation, a biochemical pathway common to other highly reduced "mitosomes" in unrelated MRO-containing lineages [7]. This is the first evidence of MRO in Rhizaria, and it suggests the parallel evolution of mitochondria to mitosomes in this supergroup.

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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. 01-06-2013

  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

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  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. 10-05-2013

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

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  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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• The new micro-kingdoms of eukaryotes. BMC Biol. 2013 ;11():40. 1741-7007-11-40. 10.1186/1741-7007-11-40. PMC3626909. 17-04-2013

  Pawlowski J

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• Can abundance of protists be inferred from sequence data: a case study of foraminifera. PLoS ONE 2013 ;8(2):e56739. 10.1371/journal.pone.0056739. PONE-D-12-33799. PMC3576339. 23-02-2013

  Weber AA, Pawlowski J

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  Protists are key players in microbial communities, yet our understanding of their role in ecosystem functioning is seriously impeded by difficulties in identification of protistan species and their quantification. Current microscopy-based methods used for determining the abundance of protists are tedious and often show a low taxonomic resolution. Recent development of next-generation sequencing technologies offered a very powerful tool for studying the richness of protistan communities. Still, the relationship between abundance of species and number of sequences remains subjected to various technical and biological biases. Here, we test the impact of some of these biological biases on sequence abundance of SSU rRNA gene in foraminifera. First, we quantified the rDNA copy number and rRNA expression level of three species of foraminifera by qPCR. Then, we prepared five mock communities with these species, two in equal proportions and three with one species ten times more abundant. The libraries of rDNA and cDNA of the mock communities were constructed, Sanger sequenced and the sequence abundance was calculated. The initial species proportions were compared to the raw sequence proportions as well as to the sequence abundance normalized by rDNA copy number and rRNA expression level per species. Our results showed that without normalization, all sequence data differed significantly from the initial proportions. After normalization, the congruence between the number of sequences and number of specimens was much better. We conclude that without normalization, species abundance determination based on sequence data was not possible because of the effect of biological biases. Nevertheless, by taking into account the variation of rDNA copy number and rRNA expression level we were able to infer species abundance, suggesting that our approach can be successful in controlled conditions.

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• Identifying active foraminifera in the Sea of Japan using metatranscriptomic approach Deep-Sea Res. Feb-Mar 2013 01-02-2013

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

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  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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• Deep relationships of Rhizaria revealed by phylogenomics: a farewell to Haeckel's Radiolaria. Mol. Phylogenet. Evol. 2013 Apr;67(1):53-9. S1055-7903(12)00489-7. 10.1016/j.ympev.2012.12.011. 03-01-2013

  Sierra R, Matz MV, Aglyamova G, Pillet L, Decelle J, Not F, de Vargas C, Pawlowski J

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  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.

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• The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy. Nucleic Acids Res. 2013 Jan;41(Database issue):D597-604. gks1160. 10.1093/nar/gks1160. PMC3531120. 30-11-2012

  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 JR, Dunthorn M, Edvardsen B, Holzmann M, Kooistra WH, Lara E, Le Bescot N, Logares R, Mahé F, Massana R, Montresor M, Morard R, Not F, Pawlowski J, Probert I, Sauvadet AL, Siano R, Stoeck T, Vaulot D, Zimmermann P, Christen R

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  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.

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• CBOL protist working group: barcoding eukaryotic richness beyond the animal, plant, and fungal kingdoms. PLoS Biol. 2012 ;10(11):e1001419. 10.1371/journal.pbio.1001419. PBIOLOGY-D-12-01686. PMC3491025. 10-11-2012

  Pawlowski J, Audic S, Adl S, Bass D, Belbahri L, Berney C, Bowser SS, Cepicka I, Decelle J, Dunthorn M, Fiore-Donno AM, Gile GH, Holzmann M, Jahn R, Jirků M, Keeling PJ, Kostka M, Kudryavtsev A, Lara E, Lukeš J, Mann DG, Mitchell EA, Nitsche F, Romeralo M, Saunders GW, Simpson AG, Smirnov AV, Spouge JL, Stern RF, Stoeck T, Zimmermann J, Schindel D, de Vargas C

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• The revised classification of eukaryotes. J. Eukaryot. Microbiol. 2012 Sep;59(5):429-93. 10.1111/j.1550-7408.2012.00644.x. PMC3483872. NIHMS394066. 02-10-2012

  Adl SM, Simpson AG, Lane CE, Lukeš J, Bass D, Bowser SS, Brown MW, Burki F, Dunthorn M, Hampl V, Heiss A, Hoppenrath M, Lara E, Le Gall L, Lynn DH, McManus H, Mitchell EA, Mozley-Stanridge SE, Parfrey LW, Pawlowski J, Rueckert S, Shadwick L, Shadwick L, Schoch CL, Smirnov A, Spiegel FW

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  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.

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• Arnoldiellina fluorescens gen. et sp. nov.--a new green autofluorescent foraminifer from the Gulf of Eilat (Israel). Eur. J. Protistol. 2013 May;49(2):210-6. S0932-4739(12)00061-2. 10.1016/j.ejop.2012.08.005. 25-09-2012

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

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  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.

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• Transcriptome analysis of foraminiferan Elphidium margaritaceum questions the role of gene transfer in kleptoplastidy. Mol. Biol. Evol. 2013 Jan;30(1):66-9. mss226. 10.1093/molbev/mss226. 21-09-2012

  Pillet L, Pawlowski J

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  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 nucleus, it is unknown how the host cell can maintain the photosynthetic activity without this information. It has been proposed that maintenance of kleptoplastids could be explained by horizontal gene transfer (HGT). To test this hypothesis we obtained 17,125 EST sequences of Elphidium margaritaceum, and we screened this data set 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 chloroplast's longevity in Elphidium.

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• Squamamoeba japonica n. g. n. sp. (Amoebozoa): a deep-sea amoeba from the Sea of Japan with a novel cell coat structure. Protist 2013 Jan;164(1):13-23. S1434-4610(12)00078-8. 10.1016/j.protis.2012.07.003. 12-09-2012

  Kudryavtsev A, Pawlowski J

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  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.

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• Intra-genomic ribosomal RNA polymorphism and morphological variation in Elphidium macellum suggests inter-specific hybridization in foraminifera. PLoS ONE 2012 ;7(2):e32373. 10.1371/journal.pone.0032373. PONE-D-11-21607. PMC3290570. 07-03-2012

  Pillet L, Fontaine D, Pawlowski J

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  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.

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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. 27-07-2011

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

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  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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• Bayesian relaxed clock estimation of divergence times in foraminifera. Mol. Phylogenet. Evol. 2011 Oct;61(1):157-66. S1055-7903(11)00275-2. 10.1016/j.ympev.2011.06.008. 05-07-2011

  Groussin M, Pawlowski J, Yang Z

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  Accurate and precise estimation of divergence times during the Neo-Proterozoic is necessary to understand the speciation dynamic of early Eukaryotes. However such deep divergences are difficult to date, as the molecular clock is seriously violated. Recent improvements in Bayesian molecular dating techniques allow the relaxation of the molecular clock hypothesis as well as incorporation of multiple and flexible fossil calibrations. Divergence times can then be estimated even when the evolutionary rate varies among lineages and even when the fossil calibrations involve substantial uncertainties. In this paper, we used a Bayesian method to estimate divergence times in Foraminifera, a group of unicellular eukaryotes, known for their excellent fossil record but also for the high evolutionary rates of their genomes. Based on multigene data we reconstructed the phylogeny of Foraminifera and dated their origin and the major radiation events. Our estimates suggest that Foraminifera emerged during the Cryogenian (650-920 Ma, Neo-Proterozoic), with a mean time around 770 Ma, about 220 Myr before the first appearance of reliable foraminiferal fossils in sediments (545 Ma). Most dates are in agreement with the fossil record, but in general our results suggest earlier origins of foraminiferal orders. We found that the posterior time estimates were robust to specifications of the prior. Our results highlight inter-species variations of evolutionary rates in Foraminifera. Their effect was partially overcome by using the partitioned Bayesian analysis to accommodate rate heterogeneity among data partitions and using the relaxed molecular clock to account for changing evolutionary rates. However, more coding genes appear necessary to obtain more precise estimates of divergence times and to resolve the conflicts between fossil and molecular date estimates.

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• Ovalopodium desertum n. sp. and the phylogenetic relationships of Cochliopodiidae (Amoebozoa). Protist 2011 Oct;162(4):571-89. S1434-4610(11)00028-9. 10.1016/j.protis.2011.04.002. 24-05-2011

  Kudryavtsev A, Wylezich C, Pawlowski J

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  An amoeba isolated from a weakly saline semi-desert pond in Kazakhstan (Central Asia) resembles a small Cochliopodium in the light microscope, but has a dorsal fibrous cell coat without scales. Thus it can be identified morphologically as a new species of Ovalopodium Sawyer, 1980, and it is herein named O. desertum. Phylogenetic analysis of the SSU rRNA gene sequences of the new species and four Cochliopodium spp. sequenced additionally shows that Ovalopodium desertum is a sister clade to a robustly monophyletic Cochliopodium. The close relationship between Ovalopodium and Cochliopodium is also confirmed by the analysis of SSU rRNA secondary structure showing the specific helices in the region V5 in all species of both genera. Analysis of actin gene sequences fails to resolve the position of Ovalopodium but demonstrates that Parvamoeba Rogerson, 1993 is probably related to Cochliopodium. The position of Cochliopodiidae within Amoebozoa remains unresolved, despite our efforts to resolve it using broader taxonomic sampling of Amoebozoa, testing alternative tree topologies and removing the fast-evolving sites. Among sequenced genera, Parvamoeba and Endostelium Olive et al., 1984 are probable relatives to Cochliopodiidae. Molecular trees weakly support an inclusion of the family in Flabellinia (Discosea), but more phylogenomic data are necessary to test this hypothesis.

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• Eukaryotic richness in the abyss: insights from pyrotag sequencing. PLoS ONE 2011 ;6(4):e18169. 10.1371/journal.pone.0018169. PMC3070721. 13-04-2011

  Pawlowski J, Christen R, Lecroq B, Bachar D, Shahbazkia HR, Amaral-Zettler L, Guillou L

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  The deep sea floor is considered one of the most diverse ecosystems on Earth. Recent environmental DNA surveys based on clone libraries of rRNA genes confirm this observation and reveal a high diversity of eukaryotes present in deep-sea sediment samples. However, environmental clone-library surveys yield only a modest number of sequences with which to evaluate the diversity of abyssal eukaryotes.

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• Molecular identification of sequestered diatom chloroplasts and kleptoplastidy in foraminifera. Protist 2011 Jul;162(3):394-404. S1434-4610(10)00079-9. 10.1016/j.protis.2010.10.001. 07-12-2010

  Pillet L, de Vargas C, Pawlowski J

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  Kleptoplastidy is the ability of heterotrophic organisms to preserve chloroplasts of algal preys they eat and partially digest. As the sequestered chloroplasts stay functional for months, the "host" becomes photosynthetically active. Although remaining a marginal process, kleptoplastidy was observed in different protist lineages, including foraminifera. Previous studies showed at least eight species of the foraminiferal genera Haynesina and Elphidium grazing on diatoms and husbanding their chloroplasts. In order to characterize more precisely the origin of kleptochloroplasts in these genera, we obtained 1027 chloroplastic 16S rDNA sequences from 13 specimens of two Haynesina and five Elphidium species. We identified the foraminiferal kleptochloroplasts using a reference phylogeny made of 87 chloroplastic sequences of known species of diatoms and brown algae. All the analyzed specimens were performing kleptoplastidy and according to our phylogenetic analyses they seem to retain exclusively chloroplasts of diatom origin. There is no apparent specificity for the type of diatom from which chloroplasts originated, however some foraminiferal species seem to accept a wider range of diatoms than others. Possibly the diversity of kleptochloroplasts depends on the type of diatoms the foraminiferans feed on.

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• Evolution of Rhizaria: new insights from phylogenomic analysis of uncultivated protists. BMC Evol. Biol. 2010 ;10():377. 1471-2148-10-377. 10.1186/1471-2148-10-377. PMC3014934. 04-12-2010

  Burki F, Kudryavtsev A, Matz MV, Aglyamova GV, Bulman S, Fiers M, Keeling PJ, Pawlowski J

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  Recent phylogenomic analyses have revolutionized our view of eukaryote evolution by revealing unexpected relationships between and within the eukaryotic supergroups. However, for several groups of uncultivable protists, only the ribosomal RNA genes and a handful of proteins are available, often leading to unresolved evolutionary relationships. A striking example concerns the supergroup Rhizaria, which comprises several groups of uncultivable free-living protists such as radiolarians, foraminiferans and gromiids, as well as the parasitic plasmodiophorids and haplosporids. Thus far, the relationships within this supergroup have been inferred almost exclusively from rRNA, actin, and polyubiquitin genes, and remain poorly resolved. To address this, we have generated large Expressed Sequence Tag (EST) datasets for 5 species of Rhizaria belonging to 3 important groups: Acantharea (Astrolonche sp., Phyllostaurus sp.), Phytomyxea (Spongospora subterranea, Plasmodiophora brassicae) and Gromiida (Gromia sphaerica).

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• Comparing potential COI and SSU rDNA barcodes for assessing the diversity and phylogenetic relationships of cyphoderiid testate amoebae (Rhizaria: Euglyphida). Protist 2011 Jan;162(1):131-41. S1434-4610(10)00050-7. 10.1016/j.protis.2010.05.002. 13-08-2010

  Heger TJ, Pawlowski J, Lara E, Leander BS, Todorov M, Golemansky V, Mitchell EA

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  The mitochondrial Cytochrome Oxidase Subunit 1 gene (COI) has been promoted as an ideal "DNA barcode" for animal species and other groups of eukaryotes. However, the utility of the COI marker for species level discrimination and for phylogenetic analyses has yet to be tested within the Rhizaria. Accordingly, we analysed mitochondrial COI gene sequences and nuclear small subunit rDNA (SSU) sequences from several morphospecies of euglyphid testate amoebae (Cercozoa, Rhizaria) in order to evaluate the utility of these DNA markers for species discrimination and phylogenetic reconstructions. Sequences were obtained from eleven populations belonging to sixCyphoderiamorphospecies that were isolated from field samples in North America and Europe. Mean inter-population COI sequence dissimilarities were on average 2.9 times greater than in the SSU, while the intra-population sequence dissimilarities were higher in the SSU (0-0.95%) than in the COI (0%); this suggests that the COI fragment is valuable for discriminating Cyphoderiidae isolates. Our study also demonstrated that COI sequences are useful for inferring phylogenetic relationships among Cyphoderiidae isolates. COI and SSU tree topologies were very similar even though the COI fragment used in these analyses (500bp) was much shorter than the SSU sequences (1600bp). Altogether, these results demonstrate the utility of the COI as a potential taxonomic DNA barcode for assessing cyphoderiid species diversity and for inferring phylogenetic relationships within the group.

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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. 22-04-2010

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

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  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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• Structure, expression and function of Allomyces arbuscula CDP II (metacaspase) gene. Gene 2010 Jun;457(1-2):25-34. S0378-1119(10)00087-9. 10.1016/j.gene.2010.02.014. 11-03-2010

  Ojha M, Cattaneo A, Hugh S, Pawlowski J, Cox JA

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  Allomyces arbuscula, a primitive chytridiomycete fungus, has two Ca(2+)-dependent cysteine proteases, the CDP I and CDP II. We have cloned and analyzed the nucleotide sequence of CDP II gene and domain structure of the protein. Blast analysis of the sequence has shown that the protein belongs to a newly described member of caspase superfamily protein, the metacaspase, a CD clan of C14 family cysteine protease, we hence-forth name it as AMca 2 (Allomyces metacaspase 2). Southern hybridization studies have shown that the gene exists in a single copy per genome. The transcriptional analysis by Northern hybridization has confirmed our previous results that the protein is developmentally regulated, i.e. present in active growth phase but disappears during nutritional stress which also induces reproductive differentiation, indicating that the protein promotes cell growth, not death. The recombinant gene product expressed in Escherichiacoli has all the catalytic properties of native enzyme, i.e. sensitivity to protease inhibitors and substrate specificity. There is an absolute requirement of Ca(2+) for the activation of catalytic activity and the presence of R residue at the cleavage site (P1 position) in the substrate. The presence of a second basic residue, either R or K, in the P2 position strongly inhibits the catalytic activity which is stimulated by the presence of P and to a lesser extent G at this site. Peptide substrates with D at the cleavage site are not recognised and therefore not cleaved. The enzyme activity is inhibited by EDTA-EGTA, cysteine protease inhibitors and a specific peptide inhibitor Ac GVRCHCL TFA, but not by E64, although a potent inhibitor of cysteine proteases.

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• Short rDNA barcodes for species identification in foraminifera. J. Eukaryot. Microbiol. ;57(2):197-205. JEU468. 10.1111/j.1550-7408.2009.00468.x. 02-02-2010

  Pawlowski J, Lecroq B

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  Ribosomal DNA (rDNA) sequences have been shown to be very useful for identification of microbial eukaryotes. Usually, complete or long partial sequences of the rDNA genes are analysed. However, the development of new massive sequencing technologies producing a large amount of relatively short sequences raises the question about the minimum length of rDNA fragments necessary for species distinction in environmental sampling. To answer this question, we compared six variable regions of the small subunit (SSU) rDNA of foraminifera, known to have rapidly evolving ribosomal genes. For each region, we analysed (1) the sequence divergence between and within foraminiferal morphospecies, (2) the intraspecific polymorphism, and (3) the ability of each region to recognize the phylotypes inferred from analysis of a longer fragment. Our results show that although the variable regions differ considerably between taxonomic groups, most of them perform very well as species identifiers. Taking into account different analyses, the expansion segment of Helix 37 appears to be the best candidate for barcoding foraminifera. We propose that this relatively short region, averaging 50-60 nt in length, could be an ideal barcode for identification of foraminifera in environmental samples using massive sequencing approach.

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• Morphologic and molecular diversity of the foraminiferal genus Globocassidulina in Admiralty Bay, King George Island 01-01-2010

  Majewski W, Pawlowski J

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• The Parazoanthidae (Hexacorallia: Zoantharia) DNA taxonomy: description of two new genera 01-01-2010

  Sinniger F, Reimer JD, Pawlowski J

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• Molecular phylogeny of euglyphid testate amoebae (Cercozoa: Euglyphida) suggests transitions between marine supralittoral and freshwater/terrestrial environments are infrequent. Mol. Phylogenet. Evol. 2010 Apr;55(1):113-22. S1055-7903(09)00491-6. 10.1016/j.ympev.2009.11.023. 17-12-2009

  Heger TJ, Mitchell EA, Todorov M, Golemansky V, Lara E, Leander BS, Pawlowski J

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  Marine and freshwater ecosystems are fundamentally different regarding many biotic and abiotic factors. The physiological adaptations required for an organism to pass the salinity barrier are considerable. Many eukaryotic lineages are restricted to either freshwater or marine environments. Molecular phylogenetic analyses generally demonstrate that freshwater species and marine species segregate into different sub-clades, indicating that transitions between these two environments occur only rarely in the course of evolution. It is, however, unclear if the transitions between freshwater and environments characterized by highly variable salinities, such as the marine supralittoral zone, are also infrequent. Here, we use testate amoebae within the Euglyphida to assess the phylogenetic interrelationships between marine supralittoral and freshwater taxa. Euglyphid testate amoebae are mainly present in freshwater habitats but also occur in marine supralittoral environments. Accordingly, we generated and analyzed partial SSU rRNA gene sequences from 49 new marine/supralittoral and freshwater Cyphoderiidae sequences, 20 sequences of the Paulinellidae, Trinematidae, Assulinidae, and Euglyphidae families as well as 21 GenBank sequences of unidentified taxa derived from environmental PCR surveys. Both the molecular and morphological data suggest that the diversity of Cyphoderiidae is strongly underestimated. The results of our phylogenetic analyses demonstrated that marine supralittoral and freshwater euglyphid testate amoeba species are segregated into distinct sub-clades, suggesting that transitions between these two habitats occurred only infrequently.

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• Semimorula liquescens is a modified echinostelid myxomycete (Mycetozoa). Mycologia ;101(6):773-6. 26-11-2009

  Fiore-Donno AM, Haskins EF, Pawlowski J, Cavalier-Smith T

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  The enigmatic Semimorula liquescens E.F. Haskins, McGuin. & C.S. Berry has been isolated repeatedly from dried infructescences of Lythrum salicaria collected from Seattle and Kirkland, Washington. Detailed developmental, morphological and ultrastructural studies suggested that it represents a taxon within Mycetozoa, closely allied with Myxogastria (Myxomycetes) but with unique characteristics. Phylogeny based on two genes places it with confidence in family Echinosteliidae. This species differs from a typical Echinostelium in the way spores germinate and in the lack of a stalked sporophore, the latter being a secondary loss. Semimorula liquescens therefore might be a useful negative model to search for genes inducing stalk formation during sporulation.

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• Description and phylogenetic relationships of Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. (Amoebozoa, Arcellinida). J. Eukaryot. Microbiol. ;56(6):495-503. JEU430. 10.1111/j.1550-7408.2009.00430.x. 04-11-2009

  Kudryavtsev A, Pawlowski J, Hausmann K

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  Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. were isolated and described from dry epiphytic moss. The morphology and ultrastructure of both species clearly demonstrate that they belong to the genus Spumochlamys (family Microchlamyiidae). They differ from its only described member, Spumochlamys iliensis (as well as from species of Microchlamys), in the relief of the dorsal surface of the test, revealed by scanning electron microscopy, which can represent a good characteristic for species identification. They also differ in the structure of the dorsal part of the test wall (especially S. perforata). Small subunit ribosomal DNA-based molecular phylogenetic analyses show that Spumochlamys is a deeply branching lineage of the Arcellinida, without any close affinities. Actin gene sequence analysis places this genus within the Tubulinea, close to two other arcellinid lineages but without forming a monophyletic group with them. These data together strongly suggest that the lack of resolution in the arcellinid molecular phylogenies is due to serious undersampling of taxa, a limited number of sequence data, and high divergence rates in most of the species.

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• Barcoding amoebae: comparison of SSU, ITS and COI genes as tools for molecular identification of naked lobose amoebae. Protist 2010 Jan;161(1):102-15. S1434-4610(09)00055-8. 10.1016/j.protis.2009.07.003. 13-10-2009

  Nassonova E, Smirnov A, Fahrni J, Pawlowski J

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  Morphological identification of naked lobose amoebae has always been a problem, hence the development of reliable molecular tools for species distinction is a priority for amoebae systematics. Previous studies based on SSU rDNA sequences provided a backbone for the phylogeny of Amoebozoa but were of little help for the species distinctions in this group. On one hand, the SSU rDNA sequences were rather conserved between closely related species; on the other hand, the intra-strain polymorphism of the SSU gene obscured species identification. In the present study, a 3' fragment of the SSU, a complete ITS1-5.8S-ITS2 block and a 5' fragment of COI gene were cloned and sequenced for six Vannella morphospecies, of which V. simplex was represented by six different isolates. SSU rDNA and ITS were found to be inappropriate for species differentiation, while distinctive and homogenous COI sequences were obtained for each well-defined morphospecies. Moreover, a number of distinct COI genotypes have been identified among V. simplex isolates. This suggests that COI may be a good candidate for DNA barcoding of amoebae, but further studies are necessary to confirm the accurateness of the COI gene as a barcode in other gymnamoebae, and to understand the taxonomic meaning of COI variations.

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• Deep phylogeny and evolution of slime moulds (mycetozoa). Protist 2010 Jan;161(1):55-70. S1434-4610(09)00044-3. 10.1016/j.protis.2009.05.002. 07-08-2009

  Fiore-Donno AM, Nikolaev SI, Nelson M, Pawlowski J, Cavalier-Smith T, Baldauf SL

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  Mycetozoa, characterized by spore-bearing fruiting bodies, are the most diverse Amoebozoa. They traditionally comprise three taxa: Myxogastria, Dictyostelia and Protostelia. Myxogastria and Dictyostelia typically have multispored fruiting bodies, but controversy exists whether they are related or arose independently from different unicellular ancestors. Protostelid slime moulds, with single-spored fruiting bodies, are possible evolutionary intermediates between them and typical amoebae, but have received almost no molecular study. Protostelid morphology is so varied that they might not be monophyletic. We therefore provide 38 new 18S rRNA and/or EF-1alpha gene sequences from Mycetozoa and related species, including four protostelids and the enigmatic Ceratiomyxa fruticulosa. Phylogenetic analyses support the monophyly of Dictyostelia, Myxogastria, and Ceratiomyxa (here collectively called "macromycetozoa") and show that protostelids are Amoebozoa, mostly related to non-fruiting amoebae of the class Variosea, but may not be monophyletic; some phylogenetic relationships remain poorly resolved. Ceratiomyxa fruticulosa, originally regarded as a myxogastrid, but in recent decades included in Protostelia, is a deeply diverging sister to Myxogastria. The protostelids studied here plus varipodid amoebae and the flagellates Phalansterium and Multicilia together probably form the outgroup to macromycetozoa plus Archamoebae. Thus protostelids and Variosea are especially significant for understanding the evolutionary transition from solitary amoebae to macromycetozoa.

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• Rhizamoeba neglecta n. sp. (Amoebozoa, Tubulinea) from the bottom sediments of freshwater Lake Leshevoe (Valamo Island, North-Western Russia), with notes on the phylogeny of the order Leptomyxida. Eur. J. Protistol. 2009 Nov;45(4):251-9. S0932-4739(09)00030-3. 10.1016/j.ejop.2009.04.002. 05-08-2009

  Smirnov A, Nassonova E, Fahrni J, Pawlowski J

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  A new species of Leptomyxida, named Rhizamoeba neglecta was found during studies of the amoeba fauna of the inner Lake Leshevoe located at Valamo archipelago (The Lake Ladoga, North-Western Russia). Light-microscopical and ultrastructural studies indicated that it represents a new species of Leptomyxida. The partial 18S rDNA sequence of this amoeba is very similar to that of Leptomyxa reticulata.. These organisms, however, are very different in LM morphology and biology. Organisms assigned to the genus Rhizamoeba do not form a single clade in the 18S rDNA tree. This may indicate that the genus is an artificial grouping or that a number of studied strains were misidentified. The phylogeny and the systematics of leptomyxids require further investigation.

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• Untangling the phylogeny of amoeboid protists. J. Eukaryot. Microbiol. ;56(1):16-25. JEU379. 10.1111/j.1550-7408.2008.00379.x. 02-04-2009

  Pawlowski J, Burki F

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  The amoebae and amoeboid protists form a large and diverse assemblage of eukaryotes characterized by various types of pseudopodia. For convenience, the traditional morphology-based classification grouped them together in a macrotaxon named Sarcodina. Molecular phylogenies contributed to the dismantlement of this assemblage, placing the majority of sarcodinids into two new supergroups: Amoebozoa and Rhizaria. In this review, we describe the taxonomic composition of both supergroups and present their small subunit rDNA-based phylogeny. We comment on the advantages and weaknesses of these phylogenies and emphasize the necessity of taxon-rich multigene datasets to resolve phylogenetic relationships within Amoebozoa and Rhizaria. We show the importance of environmental sequencing as a way of increasing taxon sampling in these supergroups. Finally, we highlight the interest of Amoebozoa and Rhizaria for understanding eukaryotic evolution and suggest that resolving their phylogenies will be among the main challenges for future phylogenomic analyses.

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• Potential of DNA sequences to identify zoanthids (Cnidaria: Zoantharia). Zool. Sci. 2008 Dec;25(12):1253-60. 10.2108/zsj.25.1253. 10-03-2009

  Sinniger F, Reimer JD, Pawlowski J

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  The order Zoantharia is known for its chaotic taxonomy and difficult morphological identification. One method that potentially could help for examining such troublesome taxa is DNA barcoding, which identifies species using standard molecular markers. The mitochondrial cytochrome oxidase subunit I (COI) has been utilized to great success in groups such as birds and insects; however, its applicability in many other groups is controversial. Recently, some studies have suggested that barcoding is not applicable to anthozoans. Here, we examine the use of COI and mitochondrial 16S ribosomal DNA for zoanthid identification. Despite the absence of a clear barcoding gap, our results show that for most of 54 zoanthid samples, both markers could separate samples to the species, or species group, level, particularly when easily accessible ecological or distributional data were included. Additionally, we have used the short V5 region of mt 16S rDNA to identify eight old (13 to 50 years old) museum samples. We discuss advantages and disadvantages of COI and mt 16S rDNA as barcodes for Zoantharia, and recommend that either one or both of these markers be considered for zoanthid identification in the future.

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• Precambrian biota: protistan origin of trace fossils? Curr. Biol. 2009 Jan;19(1):R28-30. S0960-9822(08)01434-6. 10.1016/j.cub.2008.11.003. 14-01-2009

  Pawlowski J, Gooday AJ

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  Some Precambrian trace fossils have been presented as evidence for the early origin of bilaterians; the recent finding that large amoeboid protists leave macroscopic traces at the bottom of the deep ocean questions the metazoan nature of early trace fossils, stressing the importance of single-cell organisms in Precambrian biota.

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• Large-scale phylogenomic analyses reveal that two enigmatic protist lineages, telonemia and centroheliozoa, are related to photosynthetic chromalveolates. Genome Biol Evol 2009 ;1():231-8. 10.1093/gbe/evp022. PMC2817417. 01-01-2009

  Burki F, Inagaki Y, Bråte J, Archibald JM, Keeling PJ, Cavalier-Smith T, Sakaguchi M, Hashimoto T, Horak A, Kumar S, Klaveness D, Jakobsen KS, Pawlowski J, Shalchian-Tabrizi K

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  Understanding the early evolution and diversification of eukaryotes relies on a fully resolved phylogenetic tree. In recent years, most eukaryotic diversity has been assigned to six putative supergroups, but the evolutionary origin of a few major "orphan" lineages remains elusive. Two ecologically important orphan groups are the heterotrophic Telonemia and Centroheliozoa. Telonemids have been proposed to be related to the photosynthetic cryptomonads or stramenopiles and centrohelids to haptophytes, but molecular phylogenies have failed to provide strong support for any phylogenetic hypothesis. Here, we investigate the origins of Telonema subtilis (a telonemid) and Raphidiophrys contractilis (a centrohelid) by large-scale 454 pyrosequencing of cDNA libraries and including new genomic data from two cryptomonads (Guillardia theta and Plagioselmis nannoplanctica) and a haptophyte (Imantonia rotunda). We demonstrate that 454 sequencing of cDNA libraries is a powerful and fast method of sampling a high proportion of protist genes, which can yield ample information for phylogenomic studies. Our phylogenetic analyses of 127 genes from 72 species indicate that telonemids and centrohelids are members of an emerging major group of eukaryotes also comprising cryptomonads and haptophytes. Furthermore, this group is possibly closely related to the SAR clade comprising stramenopiles (heterokonts), alveolates, and Rhizaria. Our results link two additional heterotrophic lineages to the predominantly photosynthetic chromalveolate supergroup, providing a new framework for interpreting the evolution of eukaryotic cell structures and the diversification of plastids.

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• Molecular phylogeny of common cibicidids and related Rotaliida (Foraminifera) based on small subunit rDNA sequences 01-01-2009

  Schweizer M, Pawlowski J, Kouwenhoven T, van der Zwaan B

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• Global genetic homogeneity in deep-sea foraminiferan Epistominella exigua (Rotaliida:Pseudoparrellidae) 01-01-2009

  Lecroq B, Gooday AJ, Pawlowski J

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• A new genus of xenophyophores (Foraminifera) from Japan Trench: morphological description, molecular phylogeny and elemental analysis 01-01-2009

  Lecroq B, Gooday A, Tsuchiya M, Pawlowski J

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• The partial mitochondrial genome of Leiopathes glaberrima (Hexacorallia: Antipatharia) and the first report of the presence of an intron in COI in black corals 01-01-2009

  Sinniger F, Pawlowski J

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• Three new species of deep-sea Gromia (Protista, Rhizaria) from the bathyal and abyssal Weddell Sea, Antarctica 01-01-2009

  Rothe N, Gooday A.J., Cedhagen T, Fahrni J, Hughes AJ

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• A new genus and two new species of saccamminid foraminiferans (Protista, Rhizaria) from the deep Southern Ocean 01-01-2009

  Cedhagen T, Gooday AJ, Pawlowski J

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• Molecular analyses reveal high levels of eukaryotic richness associated with enigmatic deep-sea protists (Komokiacea) 01-01-2009

  Lecroq B, Gooday AJ, Cedhagen T, Sabbatini A, Pawlowski J

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• Phylogenomics reveals a new 'megagroup' including most photosynthetic eukaryotes. Biol. Lett. 2008 Aug;4(4):366-9. J355M22278421402. 10.1098/rsbl.2008.0224. PMC2610160. 05-06-2008

  Burki F, Shalchian-Tabrizi K, Pawlowski J

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  Advances in molecular phylogeny of eukaryotes have suggested a tree composed of a small number of supergroups. Phylogenomics recently established the relationships between some of these large assemblages, yet the deepest nodes are still unresolved. Here, we investigate early evolution among the major eukaryotic supergroups using the broadest multigene dataset to date (65 species, 135 genes). Our analyses provide strong support for the clustering of plants, chromalveolates, rhizarians, haptophytes and cryptomonads, thus linking nearly all photosynthetic lineages and raising the question of a possible unique origin of plastids. At its deepest level, the tree of eukaryotes now receives strong support for two monophyletic megagroups comprising most of the eukaryotic diversity.

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• Evolution of dark-spored Myxomycetes (slime-molds): molecules versus morphology. Mol. Phylogenet. Evol. 2008 Mar;46(3):878-89. S1055-7903(07)00451-4. 10.1016/j.ympev.2007.12.011. 29-01-2008

  Fiore-Donno AM, Meyer M, Baldauf SL, Pawlowski J

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  The Myxomycetes are a major component of soil amoebae, displaying a complex life cycle that terminates in the formation of often macroscopic fruiting bodies. The classification of Myxomycetes is controversial and strongly depends on the weight given by different authors to morphological and developmental characters. We used a molecular approach to establish the phylogenetic relationships in the dark-spored orders Stemonitales and Physarales. Twenty-five small subunit ribosomal RNA gene sequences were obtained, with focus on two Stemonitales genera, Lamproderma and Comatricha. Unexpectedly, our results show that Stemonitales are paraphyletic with Physarales arising from within a Lamproderma clade. The genus Lamproderma itself is polyphyletic and can be divided into two distinct clades. Additionally, we found that Comatricha nigricapillitia comprises two cryptic species, both related to Enerthenema. Our study allows the reappraisal of morphological and developmental characters in the light of molecular data and sets foundations for a new classification of Myxomycetes.

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• Phylogenomics reshuffles the eukaryotic supergroups. PLoS ONE 2007 ;2(8):e790. 10.1371/journal.pone.0000790. PMC1949142. 30-08-2007

  Burki F, Shalchian-Tabrizi K, Minge M, Skjaeveland A, Nikolaev SI, Jakobsen KS, Pawlowski J

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  Resolving the phylogenetic relationships between eukaryotes is an ongoing challenge of evolutionary biology. In recent years, the accumulation of molecular data led to a new evolutionary understanding, in which all eukaryotic diversity has been classified into five or six supergroups. Yet, the composition of these large assemblages and their relationships remain controversial.

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• Bipolar gene flow in deep-sea benthic foraminifera. Mol. Ecol. 2007 Oct;16(19):4089-96. MEC3465. 10.1111/j.1365-294X.2007.03465.x. 30-08-2007

  Pawlowski J, Fahrni J, Lecroq B, Longet D, Cornelius N, Excoffier L, Cedhagen T, Gooday AJ

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  Despite its often featureless appearance, the deep-ocean floor includes some of the most diverse habitats on Earth. However, the accurate assessment of global deep-sea diversity is impeded by a paucity of data on the geographical ranges of bottom-dwelling species, particularly at the genetic level. Here, we present molecular evidence for exceptionally wide distribution of benthic foraminifera, which constitute the major part of deep-sea meiofauna. Our analyses of nuclear ribosomal RNA genes revealed high genetic similarity between Arctic and Antarctic populations of three common deep-sea foraminiferal species (Epistominella exigua, Cibicides wuellerstorfi and Oridorsalis umbonatus), separated by distances of up to 17, 000 km. Our results contrast with the substantial level of cryptic diversity usually revealed by molecular studies, of shallow-water benthic and planktonic marine organisms. The very broad ranges of the deep-sea foraminifera that we examined support the hypothesis of global distribution of small eukaryotes and suggest that deep-sea biodiversity may be more modest at global scales than present estimates suggest.

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• Higher-level phylogeny of Foraminifera inferred from the RNA polymerase II (RPB1) gene. Eur. J. Protistol. 2007 Aug;43(3):171-7. S0932-4739(07)00007-7. 10.1016/j.ejop.2007.01.003. 29-05-2007

  Longet D, Pawlowski J

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  Macroevolutionary relations among main lineages of Foraminifera have traditionally been inferred from the small subunit ribosomal genes (SSU rDNA). However, important discrepancies in the rates of SSU rDNA evolution between major lineages led to difficulties in accurate interpretation of SSU-based phylogenetic reconstructions. Recently, actin and beta-tubulin sequences have been used as alternative markers of foraminiferal phylogeny and their analyses globally confirm results obtained with SSU rDNA. In order to test new protein markers, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1), a nuclear encoded single copy gene, for 8 foraminiferal species representing major orders of Foraminifera. Analyses of our data robustly confirm previous SSU rDNA and actin phylogenies and show (i) the paraphyly and ancestral position of monothalamid Foraminifera; (ii) the independent origin of miliolids; (iii) the monophyly of rotaliids, including buliminids and globigerinids; and (iv) the polyphyly of planktonic families Globigerinidae and Candeinidae. Additionally, the RPB1 phylogeny suggests Allogromiidae as the most ancestral foraminiferal lineage. In the light of our study, RPB1 appears as a valuable phylogenetic marker, particularly useful for groups of protists showing extreme variations of evolutionary rates in ribosomal genes.

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• First insights into the biodiversity and biogeography of the Southern Ocean deep sea. Nature 2007 May;447(7142):307-11. nature05827. 10.1038/nature05827. 18-05-2007

  Brandt A, Gooday AJ, Brandão SN, Brix S, Brökeland W, Cedhagen T, Choudhury M, Cornelius N, Danis B, De Mesel I, Diaz RJ, Gillan DC, Ebbe B, Howe JA, Janussen D, Kaiser S, Linse K, Malyutina M, Pawlowski J, Raupach M, Vanreusel A

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  Shallow marine benthic communities around Antarctica show high levels of endemism, gigantism, slow growth, longevity and late maturity, as well as adaptive radiations that have generated considerable biodiversity in some taxa. The deeper parts of the Southern Ocean exhibit some unique environmental features, including a very deep continental shelf and a weakly stratified water column, and are the source for much of the deep water in the world ocean. These features suggest that deep-sea faunas around the Antarctic may be related both to adjacent shelf communities and to those in other oceans. Unlike shallow-water Antarctic benthic communities, however, little is known about life in this vast deep-sea region. Here, we report new data from recent sampling expeditions in the deep Weddell Sea and adjacent areas (748-6,348 m water depth) that reveal high levels of new biodiversity; for example, 674 isopods species, of which 585 were new to science. Bathymetric and biogeographic trends varied between taxa. In groups such as the isopods and polychaetes, slope assemblages included species that have invaded from the shelf. In other taxa, the shelf and slope assemblages were more distinct. Abyssal faunas tended to have stronger links to other oceans, particularly the Atlantic, but mainly in taxa with good dispersal capabilities, such as the Foraminifera. The isopods, ostracods and nematodes, which are poor dispersers, include many species currently known only from the Southern Ocean. Our findings challenge suggestions that deep-sea diversity is depressed in the Southern Ocean and provide a basis for exploring the evolutionary significance of the varied biogeographic patterns observed in this remote environment.

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• Analysis of expressed sequence tags from a naked foraminiferan Reticulomyxa filosa. Genome 2006 Aug;49(8):882-7. g06-048. 10.1139/g06-048. 13-10-2006

  Burki F, Nikolaev SI, Bolivar I, Guiard J, Pawlowski J

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  Foraminifers are a major component of modern marine ecosystems and one of the most important oceanic producers of calcium carbonate. They are a key phylogenetic group among amoeboid protists, but our knowledge of their genome is still mostly limited to a few conserved genes. Here, we report the first study of expressed genes by means of expressed sequence tag (EST) from the freshwater naked foraminiferan Reticulomyxa filosa. Cluster analysis of 1630 valid ESTs enabled the identification of 178 groups of related sequences and 871 singlets. Approximately 50% of the putative unique 1059 ESTs could be annotated using Blast searches against the protein database SwissProt + TrEMBL. The EST database described here is the first step towards gene discovery in Foraminifera and should provide the basis for new insights into the genomic and transcriptomic characteristics of these interesting but poorly understood protists.

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• Monophyly of Rhizaria and multigene phylogeny of unicellular bikonts. Mol. Biol. Evol. 2006 Oct;23(10):1922-30. msl055. 10.1093/molbev/msl055. 11-07-2006

  Burki F, Pawlowski J

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  Reconstructing a global phylogeny of eukaryotes is an ongoing challenge of molecular phylogenetics. The availability of genomic data from a broad range of eukaryotic phyla helped in resolving the eukaryotic tree into a topology with a rather small number of large assemblages, but the relationships between these "supergroups" are yet to be confirmed. Rhizaria is the most recently recognized "supergroup," but, in spite of this important position within the tree of life, their representatives are still missing in global phylogenies of eukaryotes. Here, we report the first large-scale analysis of eukaryote phylogeny including data for 2 rhizarian species, the foraminiferan Reticulomyxa filosa and the chlorarachniophyte Bigelowiella natans. Our results confirm the monophyly of Rhizaria (Foraminifera + Cercozoa), with very high bootstrap supports in all analyses. The overall topology of our trees is in agreement with the current view of eukaryote phylogeny with basal division into "unikonts" (Opisthokonts and Ameobozoa) and "bikonts" (Plantae, alveolates, stramenopiles, and excavates). As expected, Rhizaria branch among bikonts; however, their phylogenetic position is uncertain. Depending on the data set and the type of analysis, Rhizaria branch as sister group to either stramenopiles or excavates. Overall, the relationships between the major groups of unicellular bikonts are poorly resolved, despite the use of 85 proteins and the largest taxonomic sampling for this part of the tree available to date. This may be due to an acceleration of evolutionary rates in some bikont phyla or be related to their rapid diversification in the early evolution of eukaryotes.

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• A molecular time-scale for eukaryote evolution recalibrated with the continuous microfossil record. Proc. Biol. Sci. 2006 Aug;273(1596):1867-72. U1HN07N0R3RQ4101. 10.1098/rspb.2006.3537. PMC1634798. 11-07-2006

  Berney C, Pawlowski J

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  Recent attempts to establish a molecular time-scale of eukaryote evolution failed to provide a congruent view on the timing of the origin and early diversification of eukaryotes. The major discrepancies in molecular time estimates are related to questions concerning the calibration of the tree. To limit these uncertainties, we used here as a source of calibration points the rich and continuous microfossil record of dinoflagellates, diatoms and coccolithophorids. We calibrated a small-subunit ribosomal RNA tree of eukaryotes with four maximum and 22 minimum time constraints. Using these multiple calibration points in a Bayesian relaxed molecular clock framework, we inferred that the early radiation of eukaryotes occurred near the Mesoproterozoic-Neoproterozoic boundary, about 1100 million years ago. Our results indicate that most Proterozoic fossils of possible eukaryotic origin cannot be confidently assigned to extant lineages and should therefore not be used as calibration points in molecular dating.

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• Tempo and mode of spliceosomal intron evolution in actin of foraminifera. J. Mol. Evol. 2006 Jul;63(1):30-41. 10.1007/s00239-005-0061-z. 07-06-2006

  Flakowski J, Bolivar I, Fahrni J, Pawlowski J

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  Spliceosomal introns are present in almost all eukaryotic genes, yet little is known about their origin and turnover in the majority of eukaryotic phyla. There is no agreement whether most introns are ancestral and have been lost in some lineage or have been gained recently. We addressed this question by analyzing the spatial and temporal distribution of introns in actins of foraminifera, a group of testate protists whose exceptionally rich fossil record permits the calibration of molecular phylogenies to date intron origins. We identified 24 introns dispersed along the sequence of two foraminiferan actin paralogues and actin deviating proteins, an unconventional type of fast-evolving actin found in some foraminifera. Comparison of intron positions indicates that 20 of 24 introns are specific to foraminifera. Four introns shared between foraminifera and other eukaryotes were interpreted as parallel gains because they have been found only in single species belonging to phylogenetically distinctive lineages. Moreover, additional recent intron gain due to the transfer between the actin paralogues was observed in two cultured species. Based on a relaxed molecular clock timescale, we conclude that intron gains in actin took place throughout the evolution of foraminifera, with the oldest introns inserted between 550 and 500 million years ago and the youngest ones acquired less than 100 million years ago.

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• Phylogenetic position of Multicilia marina and the evolution of Amoebozoa. Int. J. Syst. Evol. Microbiol. 2006 Jun;56(Pt 6):1449-58. 56/6/1449. 10.1099/ijs.0.63763-0. 02-06-2006

  Nikolaev SI, Berney C, Petrov NB, Mylnikov AP, Fahrni JF, Pawlowski J

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  Recent molecular phylogenetic studies have led to the erection of the phylum Amoebozoa, uniting naked and testate lobose amoebae, the mycetozoan slime moulds and amitochondriate amoeboid protists (Archamoebae). Molecular data together with ultrastructural evidence have suggested a close relationship between Mycetozoa and Archamoebae, classified together in the Conosea, which was named after the cone of microtubules that, when present, is characteristic of their kinetids. However, the relationships of conoseans to other amoebozoans remain unclear. Here, we obtained the complete small-subunit (SSU) rRNA gene sequence (2746 bp) of the enigmatic, multiflagellated protist Multicilia marina, which has formerly been classified either in a distinct phylum, Multiflagellata, or among lobose amoebae. Our study clearly shows that Multicilia marina belongs to the Amoebozoa. Phylogenetic analyses including 60 amoebozoan SSU rRNA gene sequences revealed that Multicilia marina branches at the base of the Conosea, together with another flagellated amoebozoan, Phalansterium solitarium, as well as with Gephyramoeba sp., Filamoeba nolandi and two unidentified amoebae. This is the first report showing strong support for a clade containing all flagellated amoebozoans and we discuss the position of the root of the phylum Amoebozoa in the light of this result.

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• Molecular evidence for host-symbiont specificity in soritid foraminifera. Protist 2005 Dec;156(4):399-412. S1434-4610(05)00059-3. 10.1016/j.protis.2005.08.003. 29-11-2005

  Garcia-Cuetos L, Pochon X, Pawlowski J

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  Symbiosis between the dinoflagellate genus Symbiodinium and various invertebrates and protists is an ubiquitous phenomenon in shallow tropical and subtropical waters. Molecular studies undertaken on cnidarian symbionts revealed the presence of several distinctive lineages or subgeneric clades of Symbiodinium whose taxonomic level provides limited information about the specificity between invertebrate hosts and their symbionts. This contrasts with the finding of several Symbiodinium clades being present almost exclusively in foraminifera and belonging to the subfamily Soritinae. To test whether such specificity also exists at a lower taxonomic level within Soritinae, we obtained the SSU rDNA sequences from 159 soritid individuals collected in nine localities worldwide and representing all known morphospecies of this subfamily. For each individual, the symbionts were determined either by sequencing or by RFLP analysis. We distinguished 22 phylotypes of Soritinae in relation with a number of symbiont "groups" corresponding to 3 clades and 5 subclades of Symbiodinium. Among the 22 soritid phylotypes, 14 show strict symbiont specificity and only one was found to be a host for more than two "groups" of Symbiodinium. It is suggested that the strong host-symbiont specificity observed in Soritinae is a combined effect of a selective recognition mechanism, vertical transmission of symbionts, and biogeographical isolation.

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• Phylogenetic analysis and real time PCR detection of a presumbably undescribed Peronospora species on sweet basil and sage. Mycol. Res. 2005 Nov;109(Pt 11):1276-87. 11-11-2005

  Belbahri L, Calmin G, Pawlowski J, Lefort F

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  Downy mildew of sweet basil (Ocimum basilicum) has become a serious disease issue for the producers of sweet basil in Switzerland since it was first recorded in 2001. Reported in Africa in Uganda as early as 1933, major outbreaks of this disease in Europe were first noted in Italy in 1999 and in the USA from 1993. Previous reports have named the pathogen as Peronospora lamii. Its preferential hosts belong to the Lamiaceae family including basils (Ocimum spp.), mints (Menta spp.), sages (Salvia spp.) and other aromatics. This study investigated the taxonomic status of the downy mildew pathogen, using both morphological characters and molecular analysis of the ITS region of the rDNA. The inherent variability of conidial dimensions made species differentiation difficult. Sequence homology and phylogenetic analysis of nine collections of the Peronospora on sweet basil showed unique ITS sequences distinct from those of P. lamii and any other sequenced Peronospora species. This paper describes and illustrates the morphology of this presumably undescribed species of Peronospora. Its taxonomic position and relationships with other related species in the same genus are presented and discussed. In addition to this work, PCR primers for real time PCR analysis have been developed for the specific detection of this downy mildew pathogen from infected tissues or seeds. It is shown that these primers can also be used in classic PCR.

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• The testate lobose amoebae (order Arcellinida Kent, 1880) finally find their home within Amoebozoa. Protist 2005 Aug;156(2):191-202. 21-09-2005

  Nikolaev SI, Mitchell EA, Petrov NB, Berney C, Fahrni J, Pawlowski J

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  Testate lobose amoebae (order Arcellinida Kent, 1880) are common in all aquatic and terrestrial habitats, yet they are one of the last higher taxa of unicellular eukaryotes that has not found its place in the tree of life. The morphological approach did not allow to ascertain the evolutionary origin of the group or to prove its monophyly. To solve these challenging problems, we analyzed partial small-subunit ribosomal RNA (SSU rRNA) genes of seven testate lobose amoebae from two out of the three suborders and seven out of the 13 families belonging to the Arcellinida. Our data support the monophyly of the order and clearly establish its position among Amoebozoa, as a sister-group to the clade comprising families Amoebidae and Hartmannellidae. Complete SSU rRNA gene sequences from two species and a partial actin sequence from one species confirm this position. Our phylogenetic analyses including representatives of all sequenced lineages of lobose amoebae suggest that a rigid test appeared only once during the evolution of the Amoebozoa, and allow reinterpretation of some morphological characters used in the systematics of Arcellinida.

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• Polyubiquitin insertions and the phylogeny of Cercozoa and Rhizaria. Protist 2005 Aug;156(2):149-61. 21-09-2005

  Bass D, Moreira D, López-García P, Polet S, Chao EE, von der Heyden S, Pawlowski J, Cavalier-Smith T

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  A single or double amino acid insertion at the monomer-monomer junction of the universal eukaryotic protein polyubiquitin is unique to Cercozoa and Foraminifera, closely related 'core' phyla in the protozoan infrakingdom Rhizaria. We screened 11 other candidate rhizarians for this insertion: Radiozoa (polycystine and acantharean radiolaria), a 'microheliozoan', and Apusozoa; all lack it, supporting suggestions that Foraminifera are more closely related to Cercozoa than either is to other eukaryotes. The insertion's size was ascertained for 12 additional Cercozoa to help resolve their basal branching order. The earliest branching Cercozoa generally have a single amino acid insertion, like all Foraminifera, but a large derived clade consisting of all Monadofilosa except Metopion, Helk-esimastix, and Cercobodo agilis has two amino acids, suggesting one doubling event and no reversions to a single amino acid. Metromonas and Sainouron, cercozoans of uncertain position, have a double insertion, suggesting that they belong in Monadofilosa. An alternative interpretation, suggested by the higher positions for Metopion and Cercobodo on Bayesian trees compared with most distance trees, cannot be ruled out, i.e. that the second insertion took place earlier, in the ancestral filosan, and was followed by three independent reversions to a single amino acid in Chlorarachnea, Metopion and Cercobodo.

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• Molecular phylogeny and classification of the lobose amoebae. Protist 2005 Aug;156(2):129-42. S1434-4610(05)00041-6. 10.1016/j.protis.2005.06.002. 21-09-2005

  Smirnov A, Nassonova E, Berney C, Fahrni J, Bolivar I, Pawlowski J

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• Molecular phylogeny, evolutionary rates, and divergence timing of the symbiotic dinoflagellate genus Symbiodinium. Mol. Phylogenet. Evol. 2006 Jan;38(1):20-30. S1055-7903(05)00171-5. 10.1016/j.ympev.2005.04.028. 28-06-2005

  Pochon X, Montoya-Burgos JI, Stadelmann B, Pawlowski J

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  Symbiotic dinoflagellates belonging to the genus Symbiodinium are found in association with a wide variety of shallow-water invertebrates and protists dwelling in tropical and subtropical coral-reef ecosystems. Molecular phylogeny of Symbiodinium, initially inferred using nuclear ribosomal genes, was recently confirmed by studies of chloroplastic and mitochondrial genes, but with limited taxon sampling and low resolution. Here, we present the first complete view of Symbiodinium phylogeny based on concatenated partial sequences of chloroplast 23S-rDNA (cp23S) and nuclear 28S-rDNA (nr28S) genes, including all known Symbiodinium lineages. Our data produced a well resolved phylogenetic tree and provide a strong statistical support for the eight distinctive clades (A-H) that form the major taxa of Symbiodinium. The relative-rate tests did not show particularly high differences between lineages and both analysed markers. However, maximum likelihood ratio tests rejected a global molecular clock. Therefore, we applied a relaxed molecular clock method to infer the divergence times of all extant lineages of Symbiodinium, calibrating its phylogenetic tree with the fossil record of soritid foraminifera. Our analysis suggests that Symbiodinium originated in early Eocene, and that the majority of extant lineages diversified since mid-Miocene, about 15 million years ago.

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• higher-order phylogeny of plasmodial slime molds (Myxogastria) based on elongation factor 1-A and small subunit rRNA gene sequences. J. Eukaryot. Microbiol. ;52(3):201-10. JEU05-3363. 10.1111/j.1550-7408.2005.00032.x. 02-06-2005

  Fiore-Donno AM, Berney C, Pawlowski J, Baldauf SL

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  The Myxogastria are common soil microorganisms with a life cycle comprised of a plasmodial trophic stage and large fruiting bodies generally visible with the unaided eye. Until now, their classification has been based exclusively on a combination of morphological, ultrastructural, and developmental characters. Our study is the first attempt to examine phylogenetic relationships among these taxa using molecular data. Partial small-subunit ribosomal RNA and/or elongation factor 1-alpha gene sequences were obtained from eleven, mostly field-collected species representing the five orders of Myxogastria. Nineteen sequences were obtained and subjected to phylogenetic analysis together with 10 sequences available from GenBank. Separate and combined analyses of the two data sets support the division of Myxogastria into three distinct groups. The most basal clade consists of the Echinosteliales, an order considered to have affinities with Protostelia. The three species examined possess unpigmented or slightly pigmented spores. The second group consists of Liceales and Trichiales, taxa characterized by the presence of clear, but pigmented, spores. The third group consists of the two remaining orders, Physarales and Stemonitales, both possessing dark spores. This suggests that spore pigmentation is an evolutionarily conservative character in myxogastrians, and that the simple morphology of echinostelids is not a derived feature.

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• Molecular phylogenetic analysis places Percolomonas cosmopolitus within Heterolobosea: evolutionary implications. J. Eukaryot. Microbiol. ;51(5):575-81. 13-11-2004

  Nikolaev SI, Mylnikov AP, Berney C, Fahrni J, Pawlowski J, Aleshin VV, Petrov NB

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  Percolomonas cosmopolitus is a common free-living flagellate of uncertain phylogenetic position that was placed within the Heterolobosea on the basis of ultrastructure studies. To test the relationship between Percolomonas and Heterolobosea, we analysed the primary structure of the actin and small-subunit ribosomal RNA (SSU rRNA) genes of P. cosmopolitus as well as the predicted secondary structure of the SSU rRNA. Percolomonas shares common secondary structure patterns of the SSU rRNA with heterolobosean taxa, which, together with the results of actin gene analysis, confirms that it is closely related to Heterolobosea. Phylogenetic reconstructions based on the sequences of the SSU rRNA gene suggest Percolomonas belongs to the family Vahlkampfiidae. The first Bayesian analysis of a large taxon sampling of heterolobosean SSU rRNA genes clarifies the phylogenetic relationships within this group.

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• How many novel eukaryotic 'kingdoms'? Pitfalls and limitations of environmental DNA surveys. BMC Biol. 2004 ;2():13. 10.1186/1741-7007-2-13. 1741-7007-2-13. PMC428588. 05-06-2004

  Berney C, Fahrni J, Pawlowski J

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  Over the past few years, the use of molecular techniques to detect cultivation-independent, eukaryotic diversity has proven to be a powerful approach. Based on small-subunit ribosomal RNA (SSU rRNA) gene analyses, these studies have revealed the existence of an unexpected variety of new phylotypes. Some of them represent novel diversity in known eukaryotic groups, mainly stramenopiles and alveolates. Others do not seem to be related to any molecularly described lineage, and have been proposed to represent novel eukaryotic kingdoms. In order to review the evolutionary importance of this novel high-level eukaryotic diversity critically, and to test the potential technical and analytical pitfalls and limitations of eukaryotic environmental DNA surveys (EES), we analysed 484 environmental SSU rRNA gene sequences, including 81 new sequences from sediments of the small river, the Seymaz (Geneva, Switzerland).

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• The twilight of Heliozoa and rise of Rhizaria, an emerging supergroup of amoeboid eukaryotes. Proc. Natl. Acad. Sci. U.S.A. 2004 May;101(21):8066-71. 10.1073/pnas.0308602101. 0308602101. PMC419558. 19-05-2004

  Nikolaev SI, Berney C, Fahrni JF, Bolivar I, Polet S, Mylnikov AP, Aleshin VV, Petrov NB, Pawlowski J

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  Recent molecular phylogenetic studies revealed the extraordinary diversity of single-celled eukaryotes. However, the proper assessment of this diversity and accurate reconstruction of the eukaryote phylogeny are still impeded by the lack of molecular data for some major groups of easily identifiable and cultivable protists. Among them, amoeboid eukaryotes have been notably absent from molecular phylogenies, despite their diversity, complexity, and abundance. To partly fill this phylogenetic gap, we present here combined small-subunit ribosomal RNA and actin sequence data for the three main groups of "Heliozoa" (Actinophryida, Centrohelida, and Desmothoracida), the heliozoan-like Sticholonche, and the radiolarian group Polycystinea. Phylogenetic analyses of our sequences demonstrate the polyphyly of heliozoans, which branch either as an independent eukaryotic lineage (Centrohelida), within stramenopiles (Actinophryida), or among cercozoans (Desmothoracida), in broad agreement with previous ultrastructure-based studies. Our data also provide solid evidence for the existence of the Rhizaria, an emerging supergroup of mainly amoeboid eukaryotes that includes desmothoracid heliozoans, all radiolarians, Sticholonche, and foraminiferans, as well as various filose and reticulose amoebae and some flagellates.

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• Small-subunit ribosomal RNA gene sequences of Phaeodarea challenge the monophyly of Haeckel's Radiolaria. Protist 2004 Mar;155(1):53-63. 18-05-2004

  Polet S, Berney C, Fahrni J, Pawlowski J

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  In his grand monograph of Radiolaria, Ernst Haeckel originally included Phaeodarea together with Acantharea and Polycystinea, all three taxa characterized by the presence of a central capsule and the possession of axopodia. Cytological and ultrastructural studies, however, questioned the monophyly of Radiolaria, suggesting an independent evolutionary origin of the three taxa, and the first molecular data on Acantharea and Polycystinea brought controversial results. To test further the monophyly of Radiolaria, we sequenced the complete small subunit ribosomal RNA gene of three phaeodarians and three polycystines. Our analyses reveal that phaeodarians clearly branch among the recently described phylum Cercozoa, separately from Acantharea and Polycystinea. This result enhances the morphological variability within the phylum Cercozoa, which already contains very heterogeneous groups of protists. Our study also confirms the common origin of Acantharea and Polycystinea, which form a sister-group to the Cercozoa, and allows a phylogenetic reinterpretation of the morphological features of the three radiolarian groups.

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• Unexpected foraminiferal diversity revealed by small-subunit rDNA analysis of Antarctic sediment. J. Eukaryot. Microbiol. ;51(2):173-9. 12-05-2004

  Habura A, Pawlowski J, Hanes SD, Bowser SS

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  Studies of benthic Foraminifera typically rely on the morphological identification of dried specimens. This approach can introduce sampling bias against small, delicate, or morphologically ambiguous forms. To overcome this limitation, we extracted total DNA from sediment followed by PCR using group- and species-specific primers. Phylogenetic analyses revealed that approximately ninety percent of the PCR products represented previously undescribed sequence types that group with undersampled members of the allogromiid Foraminifera. We also used a modification of this technique to track individual species in sediment fractions too fine for normal morphological identification, and to confirm species placement of morphologically ambiguous foraminiferans. We were able to identify the DNA of several large foraminiferal species in fine fractions in a seasonally-dependent manner, indicating that in some seasons the majority of the standing stock of these species exists as gametes/juveniles. The approach outlined here represents a powerful strategy for exploring the total diversity of benthic foraminiferal communities.

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• Revised small subunit rRNA analysis provides further evidence that Foraminifera are related to Cercozoa. J. Mol. Evol. 2003 ;57 Suppl 1():S120-7. 10.1007/s00239-003-0015-2. 11-03-2004

  Berney C, Pawlowski J

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  There is accumulating evidence that the general shape of the ribosomal DNA-based phylogeny of Eukaryotes is strongly biased by the long-branch attraction phenomenon, leading to an artifactual basal clustering of groups that are probably highly derived. Among these groups, Foraminifera are of particular interest, because their deep phylogenetic position in ribosomal trees contrasts with their Cambrian appearance in the fossil record. A recent actin-based phylogeny of Eukaryotes has proposed that Foraminifera might be closely related to Cercozoa and, thus, branch among the so-called crown of Eukaryotes. Here, we reanalyze the small-subunit ribosomal RNA gene (SSU rDNA) phylogeny by removing all long-branching lineages that could artifactually attract foraminiferan sequences to the base of the tree. Our analyses reveal that Foraminifera branch together with the marine testate filosean Gromia oviformis as a sister group to Cercozoa, in agreement with actin phylogeny. Our study confirms the utility of SSU rDNA as a phylogenetic marker of megaevolutionary history, provided that the artifacts due to the heterogeneity of substitution rates in ribosomal genes are circumvented.

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• Small subunit ribosomal DNA suggests that the xenophyophorean Syringammina corbicula is a foraminiferan. J. Eukaryot. Microbiol. ;50(6):483-7. 22-01-2004

  Pawlowski J, Holzmann M, Fahrni J, Richardson SL

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  Xenophyophorea are giant deep-sea rhizopodial protists of enigmatic origins. Although species were described as Foraminifera or sponges in the early literature, the xenophyophoreans are currently classified either as a class of Rhizopoda or an independent phylum. To establish the phylogenetic position of Xenophyophorea, we analysed the small subunit (SSU) rRNA gene sequence of Syringammina corbicula Richardson, a newly described xenophyophorean species from the Cape Verde Plateau. The SSUrDNA analyses showed that S. corbicula is closely related to Rhizammina algaeformis, a tubular deep-sea foraminiferan. Both species branch within a group of monothalamous (single-chambered) Foraminifera, which include also such agglutinated genera as Toxisarcon, Rhabdammina, and Saccammina, and the organic-walled genera Gloiogullmia and Cylindrogullmia. Our results are congruent with observations of similar cytoplasmic organisation in Rhizammina and Syringammina. Thus, the Xenophyophorea appear to be a highly specialised group of deep-sea Foraminifera.

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• A marine dinoflagellate, Amphidinium eilatiensis n. sp., from the benthos of a mariculture sedimentation pond in Eilat, Israel. J. Eukaryot. Microbiol. ;50(6):439-48. 22-01-2004

  Lee JJ, Olea R, Cevasco M, Pochon X, Correia M, Shpigel M, Pawlowski J

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  A species of Amphidinium bloomed in a mariculture sedimentation pond that was used to grow bivalves near the Gulf of Eilat, Israel. Its overall length averaged 13 microm, the hypocone was 11 microm, and its width was 8 microm. It has a ventral ridge. The sulcus begins at the longitudinal flagellar pore and does not project forward in the apex toward the transverse flagellar pore and left margin of the cingulum. The sulcus is a very shallow groove that projects variably about a third of the body length toward the antapex. The cingulum is a deep groove as it circles the cell from the left ventral side to the dorsal side and then becomes very shallow on the right ventral side as it arches posterior toward the longitudinal flagellar pore. Using a modified method for studying dinoflagellate chromosomes in the SEM, we observed 31 chromosomes. The plastid is dorsal and peripheral with 6 ventrally projecting peripheral digital lobes that wrap around the sides of the ventral and posterior nucleus. Amphidinium eilatiensis n. sp. is morphologically closest to Amphidinium carterae and Amphidinium rhynchocephalum, but it does not have the obvious thecal plates or polygonal units described for the former species. Instead, it has a series of spicules, bumps, and ridges on its surface. It differs from A. rhynchocephalum by two morphological characters: surface morphology and gross plastid architecture. The amplified fragments of the rDNA from A. eilatiensis n. sp. isolated from 2 separate sedimentation ponds in Eilat include the 3'- end of the SSU rDNA (about 100 nt), the whole ITS region (ITS1 + 5.8S + ITS2) and the 5'-end of the LSU rDNA (about 900 nts). The total length of the sequences ranged from 1,460 nt. (A. eilatiensis isolate #1) to 1,461 nts. (A. eilatiensis isolate #2). The latter sequences are identical, the difference in length being due to three insertions. Amphidinium eilatiensis is genetically more closely related to A. carterae than to A. klebsii, with respectively 2.36% and 6.93% of sequence divergence.

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• Foraminifera and Cercozoa share a common origin according to RNA polymerase II phylogenies. Int. J. Syst. Evol. Microbiol. 2003 Nov;53(Pt 6):1735-9. 10.1099/ijs.0.02597-0. 06-12-2003

  Longet D, Archibald JM, Keeling PJ, Pawlowski J

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  Phylogenetic analysis of small and large subunits of rDNA genes suggested that Foraminifera originated early in the evolution of eukaryotes, preceding the origin of other rhizopodial protists. This view was recently challenged by the analysis of actin and ubiquitin protein sequences, which revealed a close relationship between Foraminifera and Cercozoa, an assemblage of various filose amoebae and amoeboflagellates that branch in the so-called crown of the SSU rDNA tree of eukaryotes. To further test this hypothesis, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1) from five foraminiferans, two cercozoans and the testate filosean Gromia oviformis. Analysis of our data confirms a close relationship between Foraminifera and Cercozoa and points to Gromia as the closest relative of Foraminifera.

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• The evolution of early Foraminifera. Proc. Natl. Acad. Sci. U.S.A. 2003 Sep;100(20):11494-8. 10.1073/pnas.2035132100. 2035132100. PMC208786. 25-09-2003

  Pawlowski J, Holzmann M, Berney C, Fahrni J, Gooday AJ, Cedhagen T, Habura A, Bowser SS

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  Fossil Foraminifera appear in the Early Cambrian, at about the same time as the first skeletonized metazoans. However, due to the inadequate preservation of early unilocular (single-chambered) foraminiferal tests and difficulties in their identification, the evolution of early foraminifers is poorly understood. By using molecular data from a wide range of extant naked and testate unilocular species, we demonstrate that a large radiation of nonfossilized unilocular Foraminifera preceded the diversification of multilocular lineages during the Carboniferous. Within this radiation, similar test morphologies and wall types developed several times independently. Our findings indicate that the early Foraminifera were an important component of Neoproterozoic protistan community, whose ecological complexity was probably much higher than has been generally accepted.

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• Phylogeny of lobose amoebae based on actin and small-subunit ribosomal RNA genes. Mol. Biol. Evol. 2003 Nov;20(11):1881-6. 10.1093/molbev/msg201. msg201. 02-09-2003

  Fahrni JF, Bolivar I, Berney C, Nassonova E, Smirnov A, Pawlowski J

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  Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyze actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the "Gymnamoebia sensu stricto" and the Archamoebae (pelobionts + entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.

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• Freshwater foraminiferans revealed by analysis of environmental DNA samples. J. Eukaryot. Microbiol. ;50(2):135-9. 15-05-2003

  Holzmann M, Habura A, Giles H, Bowser SS, Pawlowski J

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  Sediment-dwelling protists are among the most abundant meiobenthic organisms, ubiquitous in all types of aquatic ecosystems. Yet, because their isolation and identification are difficult, their diversity remains largely unknown. In the present work, we applied molecular methods to examine the diversity of freshwater Foraminifera, a group of granuloreticulosan protists largely neglected until now. By using specific PCR primers, we detected the presence of Foraminifera in all sediment samples examined. Phylogenetic analysis of amplified SSU rDNA sequences revealed two distinct groups of freshwater foraminiferans. All obtained sequences branched within monothalamous (single-chambered), marine Foraminifera, suggesting a repeated colonization of freshwater environments. The results of our study challenge the traditional view of Foraminifera as essentially marine organisms, and provide a conceptual framework for charting the molecular diversity of freshwater granuloreticulosan protists.

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• A novel polyubiquitin structure in Cercozoa and Foraminifera: evidence for a new eukaryotic supergroup. Mol. Biol. Evol. 2003 Jan;20(1):62-6. 10-01-2003

  Archibald JM, Longet D, Pawlowski J, Keeling PJ

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  Ubiquitin is a 76 amino acid protein with a remarkable degree of evolutionary conservation. Ubiquitin plays an essential role in a large number of eukaryotic cellular processes by targeting proteins for proteasome-mediated degradation. Most ubiquitin genes are found as head-to-tail polymers whose products are posttranslationally processed to ubiquitin monomers. We have characterized polyubuiquitin genes from the photosynthetic amoeboflagellate Chlorarachnion sp. CCMP 621 (also known as Bigelowiella natans) and found that they deviate from the canonical polyubiquitin structure in having an amino acid insertion at the junction between each monomer, suggesting that polyubiquitin processing in this organism is unique among eukaryotes. The gene structure indicates that processing likely cleaves monomers at the amino terminus of the insertion. We examined the phylogenetic distribution of the insertion by sequencing polyubiquitin genes from several other eukaryotic groups and found it to be confined to Cercozoa (including Chlorarachnion, Lotharella, Cercomonas, and Euglypha) and Foraminifera (including Reticulomyxa and Haynesina). This character strongly suggests that Cercozoa and Foraminifera are close relatives and form a new "supergroup" of eukaryotes.

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• Phylogenetic position of Gromia oviformis Dujardin inferred from nuclear-encoded small subunit ribosomal DNA. Protist 2002 Sep;153(3):251-60. 23-10-2002

  Burki F, Berney C, Pawlowski J

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  Gromia oviformis Dujardin is a common marine protist characterised by a large, globular test and filose pseudopodia. First considered a foraminifer, Gromia was later placed within the Filosea and recently included among amoebae of uncertain affinities. In order to clarify the phylogenetic position of this genus, we sequenced the complete small-subunit ribosomal DNA gene of G. oviformis collected at five different geographic localities. The high divergence of obtained sequences suggests that G. oviformis is a species complex composed of several genetically distinct sibling species. Sequence analyses show Gromia to be a member of the Cercozoa, a heterogeneous assemblage which includes filose amoebae, the amoeboflagellate cercomonads, the chlorarachniophytes and the plasmodiophorid plant pathogens. Contrary to traditional classification, Gromia is not closely related to other testate filose amoebae (the Euglyphida), but seems to branch early among the Cercozoa. Our analyses also show a close relationship between the Cercozoa and the Acantharea. Because the Cercozoa are related to the Foraminifera based on other molecular data, we propose that most protists possessing filopodia, reticulopodia and axopodia have a common origin.

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• Molecular identification of algal endosymbionts in large miliolid Foraminifera: 2. Dinofiagellates. J. Eukaryot. Microbiol. ;48(3):368-73. 20-06-2001

  Pawlowski J, Holzmann M, Fahrni JF, Pochon X, Lee JJ

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  Large miliolid foraminifers of the subfamily Soritinae bear symbiotic dinoflagellates morphologically similar to the species of the "Symbiodinium" complex, commonly found in corals and other marine invertebrates. Soritid foraminifers are abundant in coral reefs and it has been proposed that they share their symbionts with other dinoflagellate-bearing reef dwellers. In order to test this hypothesis, we have analysed partial large subunit ribosomal DNA sequences from dinoflagellates symbionts obtained from 28 foraminiferal specimens, and compared them to the corresponding sequences of Symbiodinium-like endosymbionts from various groups of invertebrates. Phylogenetic analysis of our data shows that all soritid symbionts belong to the "Symbiodinium" species complex, within which they form seven different molecular types (Frl-Fr7). Only one of these types (Fr1) branches within a group of invertebrate symbionts, previously described as type C. The remaining six types form sister groups to coral symbionts previously designed as types B, C, and D. Our data indicate a high genetic diversity and specificity of Symbiodinium-like symbionts in soritids. Except for type C, we have found no evidence for the transmission of symbionts between foraminifers and other symbiont-bearing invertebrates from the same localities. However, exchanges must have occurred frequently between the different species of Soritinae, as suggested by the lack of host specificity and some biogeographical patterns observed in symbiont distribution. Our data suggest that members of the subfamily Soritinae acquired their symbionts at least three times during their history, each acquisition being followed by a rapid diversification and independent radiation of symbionts within the foraminiferal hosts.

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• Molecular identification of algal endosymbionts in large miliolid foraminifera: 1. Chlorophytes. J. Eukaryot. Microbiol. ;48(3):362-7. 20-06-2001

  Pawlowski J, Holzmann M, Fahrni JF, Hallock P

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  résumé

  Large miliolid foraminifers bear various types of algal endosymbionts including chlorophytes, dinoflagellates, rhodophytes, and diatoms. Symbiosis plays a key role in the adaptation of large foraminifera to survival and growth in oligotrophic seas. The identity and diversity of foraminiferal symbionts, however, remain largely unknown. In the present work we use ribosomal DNA (rDNA) sequences to identify chlorophyte endosymbionts in large miliolid foraminifera of the superfamily Soritacea. Partial 18S and complete Internal Transcribed Spacer (ITS) rDNA sequences were obtained from symbionts of eight species representing all genera of extant chlorophyte-bearing Soritacea. Phylogenetic analysis of the sequences confirms the previous fine structure-based identification of these endosymbionts as belonging to the genus Chlamydomonas. All foraminiferal symbionts form a monophyletic group closely related to Chlamydomonas noctigama. The group is composed of seven types identified in this study, including one previously morphologically described species, Chlamydomonas hedleyi. Each of these types can be considered as a separate species, based on the comparison of genetic differences observed between other established Chlamydomonas species. Several foraminiferal species share the same symbiont type, but only one species, Archaias angulatus, was found to bear more than one type.

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• Molecular evidence that Reticulomyxa filosa is a freshwater naked foraminifer. J. Eukaryot. Microbiol. ;46(6):612-7. 24-11-1999

  Pawlowski J, Bolivar I, Fahrni JF, De Vargas C, Bowser SS

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  résumé

  Reticulomyxa filosa is a freshwater protist possessing fine granular, branching and anastomosing pseudopodia and therefore traditionally placed in the class Granuloreticulosea, order Athalamida, as a sister group to the order Foraminiferida. Recent studies have revealed remarkable similarities in pseudopodial motility and ultrastructure between R. filosa and foraminifera (e.g. Allogromia laticollaris), prompting us to conduct a molecular phylogenetic analysis of these seemingly disparate organisms. We sequenced the complete small-subunit of the ribosomal DNA of the cultured strain of R. filosa and compared it to the corresponding sequences of other protists including 12 species of foraminifera. We also sequenced and analyzed the actin coding genes from R. filosa and two species of foraminifera, Allogromia sp. and Ammonia sp. The analysis of both data sets clearly shows that R. filosa branches within the clade of foraminifera, suggesting that R. filosa is in fact a freshwater naked foraminiferan.

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