sign in sign up
<<
Home , Andalucia (Excavata), Apusomonadida, Armophorea, Chilodonella uncinata, Choreotrich, Colponema

NEXT GENERATION SEQUENCING OF IN THE OILSANDS OF ALBERTA (CANADA) Maria Aguilar1, Camilla Nesbo2,3, Julia Foght2, David Bass4 and Joel B. Dacks1,4 1. Department of Cell Biology, University of Alberta, Edmonton, Canada 2. Department of Biological Sciences, University of Alberta, Edmonton, Canada 3. CEES, Department of Biology, University of Oslo, Oslo, Norway 4. Department of Life Sciences, Natural History Museum, London, UK

The Athabasca oil sands are one of the biggest oil deposits in the world. They consist on a mixture of sand, water and very dense petroleum technically known as bitumen. Bitumen extraction is a complex process that involves the addition of hot water and chemicals. As result, huge volumes of sludge containing byproducts of the extraction are produced and stored in artificial reservoirs called the tailings ponds. An adequate understanding of the biological components of this environment is essential for successful management and future land reclamation plans. Although there is evidence of the ability of to process hydrocarbon derivates in the tailings ponds, very little is known about the community of eukaryotic living there. We have carried out a first assessment of eukaryotic organisms in the tailings ponds with next generation sequencing technologies. A comparative analysis of previously existing metagenomes has confirmed the presence of eukaryotic DNA in this environment. However, Bacteria and are clearly dominating the ecosystem and masking the diversity of protists. A more detailed study based on amplicon libraries of the V4 region of the small subunit of the ribosomal DNA has made it possible to detect the presence of a varied community of organisms in this extreme environment, including representatives of most eukaryotic supergroups.

A RECENTLY FORMED SPECIES FLOCK CONTAINS BOTH MARINE AND FRESHWATER

Nataliia V. Annenkova1, Gert Hansen2, Øjvind Moestrup2, Dag Ahrén1, Karin Rengefors1

1. Aquatic Ecology, Department of Biology, Lund University, Ecology Building, 22362 Lund, Sweden 2. Marine Biological Section, Department of Biology, University of Copenhagen, Universitetsparken 4, DK-2100 Copenhagen Ø, Denmark

The process of rapid radiation has been much less studied in protists than among multicellular organisms. We present the first clear example of recent rapid diversification followed by dispersion to environments with different ecological conditions within free-living microeukaryotes. This is a lineage of cold-water dinoflagellates consisting of the marine-brackish Scrippsiella hangoei, S. aff. hangoei and several freshwater species. The limnic species include euryceps and Peridinium baicalense, which are restricted to a few lakes, in particular to the ancient and deepest Lake Baikal, and the cosmopolitan Peridinium aciculiferum. All of them have relatively large morphological differences. However, while SSU rDNA fragments are identical they have distinct but very small differences in the DNA markers (LSU rDNA, ITS-2, COB gene). Our example stands in stark contrast to known examples of closely related protists, in which genetic difference is typically larger than morphological differences. Since some of the species co-occur, and all have small but species-specific sequence differences, we suggest that the differences are not due to phenotypic plasticity. To better understand how these species have diversified we analyzed the transcriptomes from freshwater P. aciculiferum, S. hangoei from Baltic Sea and S. aff. hangoei from Antarctic saline lake. Phylogenetic analysis of 792 gene orthologs allowed us to resolve the relations among the three dinoflagellates. Our data support the idea of the important role of saline barrier for diversification. Further genomic studies of this species flock will help us understand what genetic changes and which processes have led to the different phenotypes.

COMPARATIVE GENOMICS AND PHYLOGENETIC ANALYSIS OF SYNTAXIN 17

Lael D. Barlow1 and Joel B. Dacks1

1. Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Compared to endosymbiotic , the origin of autogenous organelles is obscure. However, it has been mechanistically linked to evolution of protein families acting in the membrane trafficking system, which mediates exchange of material among autogenous organelles. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) constitute a superfamily of proteins involved in fusion of membranes in the membrane trafficking system. SNAREs are essential for fusion in vesicle transport, and for fusion of larger compartments such as endosomes and lysosomes. Formation of -specific complexes composed of SNAREs from different SNARE families on opposing membranes catalyzes fusion. SNAREs have been classified into four families: Qa-, Qb-, Qc-, and R-SNAREs. Previous studies revealed that the last eukaryotic common ancestor (LECA) possessed an unexpectedly complex set of SNARE subfamilies, with, at least five within the Qa-SNARE family. The present study investigates the Qa-SNARE Syntaxin 17 (Syn17). Functional studies in metazoan cells have implicated Syn17 in membrane fusion at the endoplasmic reticulum (ER), and fusion of autophagosomes with lysosomes. Because of the role of Syn17 in autophagy, which is common to all , we hypothesized that Syn17 exists in eukaryotes other than the Metazoa. Homology searching revealed several potential Syn17 homologs in distantly related organisms, including Bigelowiella natans, and gruberi. Orthology of putative Syn17 sequences is supported by phylogenetic analysis, suggesting that Syn17 represents a sixth Qa-SNARE subfamily that was present in the last eukaryotic common ancestor (LECA). This may be important for reconstructing the early evolution of autogenous organelles, including the ER and autophagosome.

CHLOROPLAST GENOMES OF THE EUGLENACEAE Matthew S. Bennett1 and Richard E. Triemer1 1. Department of Biology, Michigan State University Euglenophytes obtained their from (a) secondary endosymbiotic event(s) involving a heterotrophic and a prasinophyte green alga. Over the last few years, multiple studies have been published outlining chloroplast genomes which represent many of the photosynthetic euglenid genera. However, these genomes were scattered throughout the euglenophyte phylogenetic tree, and these studies were focused on the overall chloroplast evolution within the Euglenophyta. Recent phylogenetic analyses, based on both ribosomal and nuclear genes, have determined that the order Euglenales should be further broken down into two families, the Euglenaceae and the Phacaeae. In addition to the genetic evidence, these families share synapomorphic chloroplast characteristics which may help determine chloroplast evolution within the Euglenophyta. Here, we present a study exclusively on taxa within the Euglenaceae. Six new chloroplast genomes were characterized, and were added to the six that have been previously published in order to determine how the evolved within this family. Overall at least one genome has now been characterized for each genus, and we have characterized the genomes of different strains from two taxa to explore intra-species variability. Results indicate that while these genomes do demonstrate a large amount of variability between them, there are common characteristics that are not shared with the chloroplast genomes of the Eutreptiales, the basal most group of photosynthetic . PHYLOGENOMIC PLACEMENT OF THE ORPHANED AMORPHEAN PROTISTS: ANCYROMONADS, MANTAMONADS, COLLODICTYONIDS, AND RIGIFILIDS

Matthew W. Brown1,2, Aaron A. Heiss3, Ryoma Kamikawa4, Akinori Yabuki5, Takashi Shiratori6, Ken-ichiro Ishida6, Yuji Inagaki7, Alastair G.B. Simpson8, Andrew J. Roger9

1. Department of Biological Sciences, Mississippi State University 2. Institute for Genomics, Biocomputing & Biotechnology, Mississippi State University 3. Department of Invertebrate Zoology, American Museum of Natural History 4. Graduate School of Global Environmental Studies and Graduate School of Human and Environmental Studies, Kyoto University 5. Japan Agency for Marine-Earth Science and Technology (JAMSTEC) 6. Graduate School of Life and Environmental Sciences, University of Tsukuba 7. Center for Computational Sciences and Graduate School of Life and Environmental Sciences, University of Tsukuba 8. Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biology, Dalhousie University 9. Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biochemistry and Molecular Biology, Dalhousie University

In most cases phylogenetics clearly places most eukaryotic lineages neatly into one of the several eukaryotic supergroups. However, there are still several orphan lineages that elude clear supergroup affiliations. Some of the greatest holdouts include such as the ancyromonads, mantamonads, and collodictyonids. Previous works have variously proposed that they are somehow related to the or (i.e., Opisthokonta, Breviatea, and ). They have further been placed into the recently proposed ‘megagroup’ . Although there is some transcriptomic sampling of the collodictyonids, their placement is not robust in phylogenomic reconstructions and the rest of these taxa are severely undersampled. Here we provide both 1) an extensive taxon sampling of RNAseq transcriptomic data from orphaned amorpheans, including the flagellates , Fabomonas, Mantamonas, and Diphylleia as well as the small thecate Rigifila, and 2) a new phylogenomic matrix that is constructed of 351 orthologs, consisting of ~100,000 amino acids sites. Analyses of this super-matrix strongly conclude that these orphaned taxa do not fall within either Amoebozoa or Obazoa, but rather that they branch variously amongst Amorphea. Surprisingly, the ancyromonads (Ancyromonas + Fabomonas) branch with the “excavate” , together at the base of a strongly supported Amorphea clade. The flagellate Mantamonas branches as sister to a strongly supported clade composed of Rigifila + Diphylleia + , nested within Amorphea. Collectively, these data strongly support the eukaryotic ‘mega-group’, Amorphea, and that morphological and genomic evolution of the amorpheans will likely provide significant changes to our understanding of deep eukaryotic evolution. TRACKING THE CRYPTIC RECORD OF EARLY EVOLUTION

Nicholas J. Butterfield1

1. Department of Earth Sciences, University of Cambridge

Animals are a monophyletic group of multicellular heterotrophic which dominate and define the modern biosphere. The oldest direct evidence for in the fossil record is from the Ediacaran (~565 million years ago [Ma]), but this is unlikely to approximate their first evolutionary appearance. In the absence of cell walls or other potentially preservable structures, there is vanishingly little likelihood that small non-skeletal animals will produce recognizable fossils. There is, however, a modest record of unambiguously eukaryotic microfossils extending back to ~1600 Ma; on the assumption that even primitive animals will leave co- evolutionary traces, this can be used to reconstruct first-order patterns in early animal evolution. One of the most notable features of the Proterozoic biosphere is its persistently prokaryotic ecological expression despite early eukaryotic diversification. The first substantial shift in this trend appears at around 750 Ma, which sees the introduction of a significant new range of armoured and loricate protists in the fossil record, as well as the first measurable occurrence of steranes (eukaryotic biomarker molecules). Shortly afterwards the Earth experiences its most dramatic interval of climatic and biogeochemical readjustment, out of which emerges the first macrofossil evidence of animals. I will argue here that the impetus for these profound perturbations was the (relatively belated) evolution of animals themselves: in the first instance the assembly of their uniquely complex developmental programmes, giving rise to their uniquely complex grade of organization – with its unique capacity to drive co-evolutionary, macroecological and biogeochemical change. WHAT CAN GENOMICS TELL US ABOUT THE ZOONOTIC WORLD OF TRICHOMONADS?

Jane Carlton

Center for Genomics and Systems Biology, Department of Biology, New York University

Trichomonads are common anaerobic, flagellated protists belonging to the large and diverse groups Trichomonadea and Tritrichomonadea of phylum Parabasalia. Trichomonads infect many vertebrate and invertebrate species, with four species classically recognized as human parasites: , Pentatrichomonas hominis, vaginalis, and Trichomonas tenax. The latter two species are considered human-specific; in contrast, D. fragilis and P. hominis have been isolated from domestic and farm mammals, demonstrating a wide host range and potential zoonotic origin. Several new studies have highlighted this zoonotic dimension of trichomonads (reviewed in Maritz et al., Trends in Parasitology, July 2014). First, species typically known to infect birds and domestic mammals have been identified in human clinical samples. Second, several phylogenetic analyses have identified animal-derived trichomonads as close sister taxa of the two human-specific species. We are undertaking whole genome sequencing of a variety of trichomonad genomes, with subsequent comparative genomic analyses, to facilitate identifying the closest relatives of human trichomonads. In addition we hope to jump-start investigation of the molecular mechanisms behind their apparent zoonotic life-style.

GENOME INVESTIGATION OF METABOLIC LINKS BETWEEN NEOPARAMOEBA PEMAQUIDENSIS AND ITS KINETOPLASTID

Ugo Cenci1, Goro Tanifuji2, Bruce A. Curtis1, Pavel Flegontov3, Julius Lukes3, and John M. Archibald1

1. Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax Nova Scotia, Canada 2. Institute of Parasitology, Biology Centre, ASCR, v.v.i., University of South Bohemia, Czech Republic 3. University of Tsukuba Faculty of life and environmental sciences, Japan

Neoparamoeba pemaquidensis, an amoebozoan parasite of lobster and fish, contains a mysterious intracellular structure historically known as a ‘parasome’ (Hollande 1980). The parasome is in fact a recently-established kinetoplastid endosymbiont related to the ectoparasite Ichtyobodo (Tanifuji et al. 2011 and Dykova et al. 2003) and has been assigned to the genus . In order to elucidate the nature of the endosymbiotic relationship between N. pemaquidensis and Perkinsela, the nuclear genomes of both organisms have been sequenced. The Perkinsela genome is ~8 Mbp in size, significantly reduced compared to those of other kinetoplastids. An analysis of biochemical pathways predicted to exist in the amoeba and its obligate endosymbiont demonstrated the absence of several important pathways of kinetoplastids in Perkinsela, including some lipid and amino acid metabolisms, but also the retention of some kinetoplastid-specific pathways such as enzymes involved in trypanothione metabolism. In addition the Perkinsiela genome encodes a variety of proteins predicted to be secreted into the N. pemaquidensis cytoplasm. These data point to the existence of complex metabolic links between these two organisms, giving molecular clues to the mutual interests of these two endosymbiotic partners. This analysis represents the first attempt to dissect the early stages of a -eukaryote endosymbiosis that does not involve as a possible selective factor in the persistence of the endosymbiont.

Dyková I., Fiala I., Lom J., Lukeš J. (2003). Perkinsiella amoebae-like of Neoparamoeba spp., relatives of the kinetoplastid Ichthyobodo. Eur. J. Protistol. 39:37– 52.

Hollande A. (1980). Identification du parasome (Nebenkern) de Janickina pigmentifera a un symbionte (Perkinsella amoebae nov gen-nov sp.) apparente aux flagelles kinetoplastidies. Protistologica. 16:613–625.

Tanifuji G., Kim E., Onodera N T., Gibeault R., Dlutek M., Cawthorn R. J., Fiala I., Lukes J., Greenwood S. J. and Archibald J. M. (2011). Genomic characterization of Neoparamoeba pemaquidensis (Amoebozoa) and its kinetoplastid endosymbiont. Eukaryot. Cell. 10:1143–1146 ANAERAMOEBA SPP., NOVEL ANAEROBIC AMOEBOFLAGELLATES WITH UNCERTAIN PHYLOGENETIC POSITION

Ivan Čepička1, Petr Táborský1, and Tomáš Pánek1

1. Department of Zoology, Faculty of Science, Charles University in Prague

We have cultured seven strains of anaerobic amoebae (‘Anaeramoeba’) obtained from anoxic/microoxic marine coastal sediments worldwide. The amoebae are highly reminiscent of the genera Flamella (Amoebozoa: Gracilipodida), Flabellula or Paraflabellula (Amoebozoa: ) being extremely flattened and fan-shaped, with trailing uroidal filaments. On the other hand, the cells display a unique combination of ultrastructure features: they possess acristate -related organelles, presumably , closely associated with symbiotic , and a large MTOC, but no flagellar basal bodies in the cytoplasm. The strains of Anaeramoeba morphologically represent three distinct species that differ in the nuclear morphology, extent of the hyaline zone, and cell diameters. Two non-conspecific strains of Anaeramoeba are also able to produce flagellates in the culture, which means that they are true amoeboflagellates. The flagellates are or tetrakont with isokont flagella and their movement is rather fast. The nuclear morphology of the flagellates is identical with that of conspecific amoebae. Interestingly, the flagellates are formed only in cultures where contaminating eukaryotes, mainly various , are present; in monoeukaryotic culture the formation of the flagellates ceases. Phylogenetic analyses of SSU rDNA and five protein-coding genes did not resolve phylogenetic position of Anaramoeba suggesting that it may form a deep eukaryotic lineage. Although Anaeramoeba seems to be common in marine anoxic sediments and is relatively diverse being represented by several distinct species, it is apparently a novel anaerobic lineage of protists. MINORISA MINUTA AND THE TRANSITION TO SECONDARY ENDOSYMBIOSIS IN CHLORARACHNIOPHYTES. A SINGLE CELL GENOMICS APPROACH.

Javier del Campo1, Michael E. Sieracki2, Ramon Massana3, and Patrick Keeling1

1 University of British Columbia, Vancouver, BC, Canada.

2 Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA.

3 Institut de Ciències del Mar, CSIC, Barcelona, Catalonia, Spain

Using Single Cell Sorting and Single Cell Genomics on marine surface water samples we were able to obtain and characterize 17 Chlorarachniophytes Single Amplified Genomes from both the heterotrophic and phototrophic fraction, including among them Minorisa minuta and the well known Bigelowella natans. The tiny uniflagellated Minorisa minuta stands up as one of the smallest bacterial grazers known to date. It has a worldwide planktonic distribution and accounts for 5% of heterotrophic protists communities in coastal waters. Moreover, it apparently represents the only heterotrophic representative within the Chlorarachniophytes, the single photosynthetic lineage within . Chlorarachniophytes are marine amoeboflagellated cercozoans that together with the Euglenophytes represent the two eukaryotic groups that acquired a chloroplast by secondary endosymbiosis with a green alga. The existence of this unpigmented protist could be another evidence indicating that the acquisition of the green chloroplast took place independently in both lineages. Nevertheless, this is not a definitive clue as the absence of chloroplast could also derive from a posterior loss of it. Therefore, the genomic analysis of Minorisa minuta and other basal heterotrophic Chlorarachniophytes retrieved in our study is of primary interest to study the transition to secondary plastid endosymbiosis. The Plant Hormone Ethylene is Functional in Charophyte Green Charles F. Delwiche, Chuanli Ju, Bram Van de Poel, Endymion D. Cooper, James Thierer, Theodore R. Gibbons, and Caren Chang Cell Biology and Molecular Genetics, University of Maryland, College Park, College Park, MD 20742 USA. Land (embryophytes) dominate the terrestrial environment, but are phylogenetically a lineage of charophyte . However, despite their unambiguous evolutionary origin among the charophytes, land plants have a number of distinctive properties that cannot easily be related to the biology of charophytes. Particularly striking is the large, complex, and closely regulated plant body typical of most land plants. Hormones play a key role in patterning and signaling in land plants, but remain quite poorly understood in charophytes. We determined that the genes for many plant hormone pathways were present in charophyte transcriptomes, and undertook the analysis of one of these -- ethylene -- in more detail. Focusing on the plant hormone ethylene and the filamentous charophyte Spirogyra pratensis, we provide bioinformatic and functional evidence that Spirogyra possesses a complete ethylene hormone system, with the pathways needed for both biosynthesis and reception/signal transduction being present and well-conserved inSpirogyra. We determined that Spirogyra does produce ethylene, which in turn induces cell elongation in a dose-dependent manner. We also found that Spirogyra homologs can partially rescue ethylene mutants in the angiosperm Arabidopsis thaliana and/or respond post-translationally to ethylene treatment when expressed in plant cells. These findings indicate that the ethylene- signaling pathways in Spirogyra and Arabidopsis are unambiguously homologous, and imply that the common aquatic ancestor possessed this same pathway. Because cell elongation is also an ethylene response in land plants, it is possible that this represents the ancestral ethylene response. These observations reinforce the predictive value of phylogenetic information, and show how bioinformatic inferences can be validated in vitro and in vivo, and in turn can lead to novel biological insights.

Transcriptome-based profiling of algal nutritional physiology; linking physiology to geochemistry in cultures and field populations

Sonya Dyhrman1

1Department of Earth and Environmental Science and the Lamont-Doherty Earth Observatory, Columbia University

Phytoplankton in the ocean account for roughly half of global primary production, exerting profound control on the Earth system and how it functions. Although nutrient availability is known to play a critical role in driving the distribution and activities of phytoplankton, there are fundamental gaps in understanding how key species and functional groups metabolize nutrients like nitrogen and phosphorus, and how this metabolic potential is expressed and modulated in field populations. Data from the Marine Microbial Eukaryote Transcriptome Project (MMETSP - funded by the Gordon and Betty Moore Foundation) is being used to address this knowledge gap, which will allow for better biogeochemical modeling and prediction of the distribution and activities of phytoplankton in the future ocean. Using transcriptome data from species grown in replete, low N and low P conditions, we are examining how nutrients are metabolized and how these pathways are regulated by resource availability in key species like the bloom forming Pelagophyte, Aureococcus anophagefferens, among others. Although the MMETSP data are not replicated, we employed a Bayesian model called Analysis of Sequence Counts (ASC) with a posterior probability of .95 and a fold change of 2 to conservatively examine differential expression patterns. ASC performs conservatively with few false positives on data without replication. Further leveraging MMETSP data is allowing for the quantitative, species-specific assessment of gene expression patterns in field populations. Metatranscriptome data from diverse systems has been mapped to the MMETSP database to identify which taxa are present, and assess their physiological ecology. ON THE AGE OF EUKARYOTES: EVALUATING EVIDENCE FROM FOSSILS AND MOLECULAR CLOCKS

Laura Eme1, Susan C. Sharpe1, Matthew W. Brown1,2 and Andrew J.Roger1

1. Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Canada

2. Current Address: Department of Biological Sciences, Mississippi State University, Mississippi State, MS, USA

Our understanding of the phylogenetic relationships among extant eukaryotes has improved dramatically over the past few decades owing to the development of sophisticated methods and models of evolution, and the increased availability of sequence data for a variety of lineages. Concurrently, efforts have been made to infer the age of major evolutionary events along the tree of eukaryotes using fossil-calibrated molecular clock-based methods. Here, we present an investigation of molecular clock approaches to estimate the age of the last eukaryotic common ancestor (LECA) and major protistan lineages. After reviewing in detail previous attempts to date deep eukaryote divergences, we present the results of a Bayesian relaxed molecular clock analysis of a large dataset comprised of 159 proteins from 85 taxa representing all major protistan and multicellular eukaryotic lineages, calibrated using 19 eukaryotic fossil dates. We show that for major groups estimated dates of divergence are heavily influenced by the models and methods used, and the nature and treatment of fossil calibrations. Although the estimated age of LECA varied widely, ranging from 1007 to 1898 Ma, all analyses suggested that the eukaryotic super-groups subsequently diverged rapidly (i.e., within 300 Ma of LECA). The extreme variability of these and previously published analyses suggests that age estimates of eukaryotic clades should be treated cautiously. As more reliable fossil data of eukaryotes from the Proterozoic become available and improvements are made in relaxed molecular clock modelling, we may be able to date the age of extant eukaryotes more precisely.

BIOCHEMICAL CHARACTERIZATION OF BIFUNCTIONAL ADHE ENZYMES IN ENTAMOEBA

Avelina Espinosa1 & Guillermo Paz-y-Miño-C2

1. Department of Biology, Roger Williams University; 2. Department of Biology, University of Massachusetts Dartmouth

Amoeboid protists taxonomic studies have transitioned from single morphological traits (), to single gene (SSU rRNA, phylogenies. Single-gene analyses of metabolic traits (e.g. alcohol dehydrogenase adhe) contribute to conflictive phylogenetic depictions, due to horizontal acquisition (horizontal gene transfer, HGT) of genes from prokaryotes and/or unicellular eukaryotes. The intestinal pathogen Entamoeba histolytica lacks mitochondria and derives energy from the of glucose to ethanol. The last two steps of this pathway are catalyzed by E. histolytica alcohol dehydrogenase 2 (EhADH2), which belongs to the ADHE family. ADHE bifunctional enzymes have separate N-terminal aldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH) domains. All Entamoeba ADHE protein sequences (E. terrapinae, E. invadens, E. moshkovskii, and E. histolytica) branch together next to a cohesive cluster of low G+C Gram positive and γ-proteobacteria. We characterize two E. invadens IP-1 and VK-1:NS ADHE enzymes and compare them to EhADH2. The result shows a similar binding mechanism of acetyl-CoA to the ALDH among all three enzymes suggesting a similar evolutionary origin. However, because all three enzymes show different binding affinities for acetaldehyde to the ADH domain, selective pressures within specific host environments and genetic variability might have influenced the adaptations of ADHE homologs to diverse ecological niches (i.e. genetic adaptation to anoxic conditions in the vertebrate/invertebrate gut). It is conceivable that ancestral amoeba ingested, via phagotrophism, prokaryotes capable of glucose fermentation, and later integrated bacterial metabolic genes into the Entamoeba genome. MITOCHONDRIAL GENOME OF AN ENDOSYMBIOTIC KINETOPLASTID PERKINSELA: U-INSERTION/DELETION EDITING OF RIBOSOMAL RNAS PROCEEDS IN A NUMBER OF ALTERNATIVE PATHWAYS

Pavel Flegontov1, Vojtěch David1,2, Evgeny S. Gerasimov3, Goro Tanifuji4, Naoko T. Onodera4, Ivan Fiala1, Maria Logacheva3, Hassan Hashimi1,2, John Archibald4 & Julius Lukeš1,2 1 Institute of Parasitology, Czech Academy of Sciences, and 2 Faculty of Biology, University of South Bohemia, České Budějovice, Czech Republic; 3 Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia; 4 Department of Biochemistry and Molecular Biology, Dalhousie University, Nova Scotia, Canada

A basally-branching kinetoplastid Perkinsela sp. is an obligate intracellular symbiont of certain amoebae (Paramoeba spp.), parasitizing fish gills. So far little has been known about this peculiar association. Using a combination of second-generation sequencing technologies (Illumina 100 bp and 250 bp paired reads, 454 single reads) we have sequenced total genomes and polyA transcriptomes for two strains of ameoba-Perkinsela, CCAP 1560/4 and GillNor1/I. Using a number of independent approaches, the Perkinsela mitochondrial genome was assembled into few linear fragments with terminal repeats. Both strains have an identical gene content: at least two rRNA fragments, cox1-3, cob, rps12. All nad genes are missing from the nuclear and mitochondrial genomes/transcriptomes, suggesting that complex I of the respiratory chain has been lost. Two extremely divergent rRNA fragments have been revealed as high-coverage transcripts: one of them can be folded into a structure similar to a part of LSU containing polypeptidyl-transferase, the other one possibly represents a fragment of SSU. All transcripts are edited with U-insertions/deletions, a mechanism characteristic for kinetoplastid mitochondria. Similarly to a bodonid Trypanoplasma borreli, editing occurs in separate domains at 5'- and 3'-ends of transcripts, and high coverage Illumina data allowed us to dissect editing events in unprecedented detail. While editing of protein-coding genes proceeds to a mature translated sequence, editing of rRNAs represents a branching pathway with a number of alternative products, conserved in both strains. Only few cases of editing in rRNAs have been described, and Perkinsela rRNA may be a part of the most divergent ribosome known.

DIPLONEMEA () EMERGE AS A MAJOR COMPONENT OF MESOPELAGIC PLANKTON IN THE WORLD OCEAN

Olga Flegontova1,2, Stephane Audic3, Colomban de Vargas3, Patrick Wincker4, Julius Lukeš1,2 & Aleš Horák1,2

1 Institute of Parasitology, Biology Centre ASCR, České Budějovice, Czech Republic 2 Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic 3 Station Biologique de Roscoff, Roscoff, France 4 Genoscope, CEA, Évry, France

Diplonemea remains an obscure group of (Excavata), represented by few benthic bacteriovorous or parasitic species living in marine or freshwater habitats (, Rhynchopus). Metabarcoding studies revealed a large marine clade close to classic diplonemids. We present the first worldwide study on marine diplonemids based on data collected during the Tara Oceans expedition in 2009-2012 (V9 region of 18S rRNA) and Tara Oceans Polar Circle in 2013 (full-length 18S rRNA). Diplonemea ranked the third according to the number of OTUs among all planktonic clades of photic layer, after and Metazoa. However, a great share of diplonemids is concentrated in mesopelagic zone, where they comprise up to 46% of eukaryotic barcodes (usually 10-20%), and are 5- 10x more abundant than in photic zones. Planktonic diplonemids occur mostly within 0.8-5 and 5-20 μm size-fractions, suggesting the small cell size. Full-length rRNA sequences show that marine diplonemids of the Tara dataset fall into the diverse clade of marine diplonemids described earlier. Several small divergent clades may also exist, but require further verification. Transcriptome sequencing of planktonic diplonemids from the richest samples may advance the study of this overlooked group and enlighten their role in the ocean ecosystem. THE RETAINMENT OF THE SECONDARY PLASTID OF LONGA IS CONNECTED TO THE FUNCTIONAL CALVIN CYCLE LOCALIZED TO THIS COMPARTMENT

Zoltán Füssy1, Kristína Záhonová2, Vladimír Klimeš2, Lucia Hadariová3, Erik Birčák3, Eva Kotabová4, Juraj Krajčovič3, Miroslav Oborník1,4,5, and Marek Eliáš2

1 Biology Centre of the Czech Academy of Sciences, Institute of Parasitology, České Budějovice, Czech Republic 2 University of Ostrava, Faculty of Science, Ostrava, Czech Republic 3 Comenius University, Faculty of Science, Bratislava, Slovakia 4 Institute of of the Czech Academy of Sciences, Třeboň, Czech Republic 5 University of South Bohemia, Faculty of Science, České Budějovice, Czech Republic

Euglenida comprise a diversity of species living in various environmental niches. While lives autotrophically or mixotrophically, and tolerates loss of its green plastid upon stress, E. longa is a naturally „bleached“ osmotrophic relative of E. gracilis, keeping its plastid as an essential organelle. E. longa plastidial DNA encodes only for ribulose-bisphosphate carboxylase large subunit (RbcL), proteins of the chloroplast transcriptional and translational systems, and non-coding RNAs. Our bioinformatic analyses of E. longa transcriptome data revealed no predicted plastid- targeted enzymes of the heme, DOXP, or type II fatty acid biosynthesis. However, we were able to predict plastidial localization of a majority of Calvin cycle enzymes, including the small Rbc subunit. We used a Calvin cycle inhibitor, glycolaldehyde (GLA), and observed inhibitory effect of GLA on the growth of E. longa to an extent observed for streptomycin-treated cultures. Mixotrophic E. gracilis controls were tolerant to streptomycin or GLA. Nevertheless, GLA also inhibited the growth of brucei lacking the Calvin cycle, suggesting a general side-effect may play role in our observations. Further, we tested the possibility of E. longa to utilize Rbc for CO2 fixation. Preliminary data show that E. longa does fix CO2 at a considerable pace: about 20 % of mixotrophic E. gracilis fixation rate. Further experiments are in progress. Lastly, we used anti-RbcL antibodies to show the RbcL protein localizes to the of both studied Euglena species, and allows visualization of the E. longa plastid for the first time. Supported by Czech Science Foundation grant GA13-33039S.

SEASONALITY OF ARCTIC IN WESTERN SPITSBERGEN FJORDS

Tove M. Gabrielsen1, 2, Helga B. Kristiansen1, Miriam Marquardt1, Archana R. Meshram1, Stuart Thomson1, Anna Vader1

1. The University Centre in Svalbard, Department of Arctic Biology, Norway 2. University of Bergen, Department of Biology, Norway

The western Spitsbergen fjords, Svalbard are alternatingly dominated by warm and saline Atlantic water and colder and less saline Arctic water, and are thus well suited for studying the effect of climate shifts on pelagic protist communities. The Adventfjorden time series station (ISA) in Isfjorden, western Spitsbergen was sampled monthly throughout 2012, and protists sized 0.45-10 µm from 25 m depth were analysed using high throughput sequencing (HTS) of the 18S V4 region amplified from both DNA and cDNA. From the same samples, the abundance and diversity of Marine Group II (MALV II) and marine fungi were investigated using qPCR and HTS of the 18S V4 region and the ITS, respectively. At the ISA station the protist community of size 0.45-10 µm was dominated by Dinophyceae throughout the year. The winter communities were diverse and fairly stable, whereas the composition of the spring and summer protist communities varied considerably between sampling dates. The abundance of MALV II was high in the spring, but declined considerably prior to the spring bloom at the time when a hydrographic change occurred. The fungal communities were predominantly structured by Julian date, and were dominated by Mortierellales. A comparison of the ISA data to HTS data of picoplankton communities from two other western Spitsbergen fjords showed considerable differences in both community composition and potential ecosystem function. Although Arctic pelagic protist communities are strongly seasonal, a thorough knowledge of the hydrography of the area is necessary to understand the changes of these communities.

A UNIQUE H2-PRODUCING MITOCHONDRION IN A NOVEL ANAEROBIC CERCOMONAD.

Ryan M.R. Gawryluk1, Courtney W. Stairs2, Laura Eme2, Michelle M. Leger2, Matthew W. Brown3, Jeffrey D. Silberman4, Andrew J. Roger2.

1. Department of Botany, University of British Columbia 2. Department of Biochemistry & Molecular Biology, Dalhousie University 3. Department of Biological Sciences, Mississippi State University 4. Department of Biological Sciences, University of Arkansas

Mitochondria are eukaryotic organelles that are famous as the site of aerobic adenosine triphosphate (ATP) production via the coupled action of the electron transport chain (ETC; CI-CIV) and FoF1 ATP synthase (CV). In contrast, numerous anaerobes have independently evolved functionally reduced mitochondrion-related organelles (MROs), including hydrogenosomes. Hydrogenosomes, which lack mitochondrial DNA (mtDNA), the tricarboxylic acid (TCA) cycle, and an ETC, generate ATP via substrate-level phosphorylation. The apparent evolutionary chasm between mitochondria and MROs was narrowed considerably by the characterization of H2-producing mitochondria (HPM) in the Nyctotherus ovalis, and the stramenopile Blastocystis hominis. In each case, the HPM retains mtDNA, a partial TCA cycle, and an incomplete ETC that lacks CIII- CV, suggesting that ATP is generated via hydrogenosomal-type metabolism alone. In order to better comprehend the evolution of anaerobic mitochondria, and their evolution within Rhizaria, we undertook a sequencing-based metabolic reconstruction of the mitochondrion of a novel anaerobic cercomonad, DMV, identifying >300 candidate mitochondrial proteins. We demonstrate that DMV possesses mtDNA that codes for a small number of mitochondrial RNAs and proteins, and a nearly complete TCA cycle. Like HPM, DMV mitochondria are predicted to house hydrogenosomal energy generation enzymes, and seemingly lack functional ETC complexes III-IV. Uniquely, however, DMV has retained CV – albeit the most unusual one described to date – indicating the potential for true oxidative phosphorylation. Together, our analyses indicate that DMV possesses complex HPM, with properties that are intermediate between aerobic mitochondria and previously described HPM, giving further insight into the early stages of hydrogenosomal evolution. SOIL PROTISTAN COMMUNITIES IN CONTRASTING ECOSYSTEMS FROM THE COASTAL TEMPERATE RAINFOREST OF CALVERT ISLAND (BRITISH COLUMBIA, CANADA)

Thierry J. Heger1, Ian Giesbrecht2, Kira M. Hoffman3, William W. Mohn4, Colleen T.E Kellogg4, Ken Lertzman 2,5, and Patrick J. Keeling1

1 Biodiversity Research Centre, University of British Columbia 2 Hakai Beach Institute, Heriot Bay, British Columbia 3 School of Environmental Studies, University of Victoria 4 Microbiology and Immunology Departments, University of British Columbia 5 Faculty of Environment, Simon Fraser University

Abstract

Although widely recognized as key players in ecosystems and representing a large part of the Earth's biodiversity, unicellular eukaryotes (protists) are still poorly characterized, in particular in soil ecosystems. Here, we assess protistan diversity and community structure across distinct ecosystems on Calvert Island (British Columbia, Canada). Soil samples have been collected from twelve Ecosystem Comparison Plots (ECPs) established in blanket bogs, bog woodlands, bog forests and zonal forests. High-throughput sequencing analyses of protists and microscope-based identification of testate amoebae (Arcellinida and Euglyphida) are underway to characterize the protistan communities of these samples. Preliminary microscopy-based results indicate testate amoeba community structure differs between ecosystems and are particularly abundant in bog ecosystems. Overall, this study should 1) provide extensive data on protistan diversity across distinct terrestrial ecosystems of the temperate rainforest of Calvert Island and 2) highlight which environmental factors structure protistan communities. INTRODUCING A NEW SPECIES WITH A PREDATORY LIFESTYLE

Elisabeth Hehenberger1, Denis V. Tikhonenkov1, Jan Janouŝkovec2, and Patrick J. Keeling1

1. Canadian Institute for Advanced Research, Botany Department, University of British Columbia, Vancouver, British Columbia, Canada 2. Biology Department, San Diego State University, San Diego, California, USA

The origin of the multicellular metazoans from their unicellular ancestors has received a large amount of attention in recent years, with the continuous emergence of new genomic information from unicellular relatives being of essential importance for the understanding of this process. In this context we are introducing a new unicellular opisthokont species which exhibits a predatory lifestyle, preying on various other eukaryotes. Initial phylogenetic analysis employing extensive sampling of small ribosomal subunit RNA sequences from opisthokonts as well as from apusozoans and amoebae, including a large number of environmental sequences for all subgroups, preliminarily positioned the new species either at the base of fungi or at the base of all opisthokonts. Using transcriptome data generated from this organism we are in the process of generating a multi-gene phylogenomic analysis to reliably position this new opisthokont in the eukaryotic tree of life.

MALAWIMONAD ULTRASTRUCTURE AND MOLECULAR PHYLOGENETICS

Aaron A. Heiss1, Martin Kolisko2, Flemming Ekelund3, Matthew W. Brown4,5, Andrew J. Roger6, Alastair G. B. Simpson7

1. Department of Invertebrate Zoology, American Museum of Natural History 2. Department of Botany, University of British Columbia 3. Department of Terrestrial Ecology, Zoological Institute, University of Copenhagen 4. Department of Biological Sciences, Mississippi State University 5. Institute for Genomics, Biocomputing & Biotechnology, Mississippi State University 6. Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biology, Dalhousie University 7. Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biochemistry and Molecular Biology, Dalhousie University

Malawimonads are enigmatic eukaryotes that are understood to have ‘typical excavate’ morphology. They usually do not branch with other excavates (divisible into Metamonada and Discoba) in molecular phylogenies. To date, they have been known only from one described species, Malawimonas jakobiformis, and one undescribed strain, M. ‘californiana’. We describe a new strain, basal to both of these. Electron microscopy shows that this strain bears all canonical features of ‘typical excavates’, including a probable composite fibre (unreported in M. jakobiformis), and like most excavates specifically, has a second, dorsal flagellar vane (not present in M. jakobiformis). Phylogenomic analyses recover a sister relationship between malawimonads and collodictyonids, remote from other excavates, but the topologies vary based on taxon selection. We thus confirm both the morphological similarity between malawimonads and other ‘typical excavates’, and the separation between the two groups in standard phylogenomic analyses. CONTRASTING THE GENOMES OF THE HARMLESS AMOEBA NAEGLERIA GRUBERI AND THE DEADLY, NEUROPATHOGENIC

Emily K. Herman1, Alex L. Greninger2, Govinda S. Visvesvara3, Francine Marciano- Cabral4, Joel B. Dacks1, and Charles Y. Chiu2,5,6

1. Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada 2. UCSF-Abbott Viral Diagnostics and Discovery Center, University of California San Francisco, San Francisco, California, USA 3. Division of Foodborne, Waterborne and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA 4. Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA 5. Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, California, USA 6. Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA

Naegleria fowleri is an opportunistic pathogen of humans and animals, and is a member of the supergroup Excavata. It is found in stagnant tropical, subtropical, and thermal waters around the world. It causes primary amoebic meningoencephalitis, killing 99% of those infected, usually within two weeks. Infection occurs when contaminated water enters the nose (e.g. when swimming), and N. fowleri passes through the cribriform plate to the olfactory bulb in the brain. N. fowleri is the only species of Naegleria that regularly infects humans, and no clear pathogenicity factor has been identified. We have sequenced the N. fowleri genome, and are using comparative genomics to identify differences between it and that of its harmless relative, Naegleria gruberi, in order to understand N. fowleri’s unique pathogenic ability. We have observed several striking differences between the two genomes. First, the N. fowleri genome is smaller in both total size and predicted gene complement. Secondly, confirming the findings of our previous work on a small region of the N. fowleri genome, there is evidence of significant genomic rearrangement in the two species since their divergence from a common Naegleria ancestor. Finally, we used comparative genomics to show 6.7% of predicted N. fowleri genes and 9.5% of N. gruberi genes do not have a clear homologue in N. gruberi or N. fowleri, respectively. Our ongoing genomic analysis will further explore the ways in which variation between the two Naegleria species may be related to pathogenicity in the deadly N. fowleri. THE VIRIDIRAPTORIDAE - A NOVEL FAMILY OF ALGIVOROUS FLAGELLATES (GLISSOMONADIDA, RHIZARIA)

Sebastian Hess1 and Michael Melkonian1

1. Department of Botany, Cologne Biocenter, University of Cologne, Zülpicher Str. 47b, 50674 Cologne, Germany

Microalgae represent a major source of biologically available carbon in aquatic ecosystems. Consequently, there exist diverse heterotrophic microeukaryotes that specifically feed on microalgae. Due to difficulties in isolation and long-term maintenance our present knowledge about these obligate algivores is limited. Recently, a new family of algivorous amoeboflagellates inhabiting freshwater ecosystems, the Viridiraptoridae, has been described (Hess and Melkonian 2013). As revealed by molecular phylogenetic analyses the family represents a previously uncharacterised lineage within the Glissomonadida (, Rhizaria). Both genera currently known, Viridiraptor and Orciraptor, perforate algal cell walls and thus exploit a new food source compared to their bacterivorous relatives. The phylogeny, fascinating feeding strategies of Viridiraptor and Orciraptor, autecological aspects (e.g. food range specificity) as well as ultrastructural results will be presented. These data are compared with those from other cercozoan flagellates and discussed in the light of viridiraptorid evolution. CHARACTERIZATION OF TSET, AN ANCIENT AND WIDESPREAD MEMBRANE TRAFFICKING COMPLEX

Jennifer Hirst1, Alexander Schlacht2, John P. Norcott3, David Traynor,4 Gareth Bloomfield4, Robin Antrobus1, Robert R Kay4, Joel B Dacks2, and Margaret S Robinson1

1 University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK 2 Department of Cell Biology, University of Alberta, Edmonton, Canada 3 University of Cambridge, Department of Engineering, Cambridge, UK 4 MRC Laboratory of Molecular Biology, Cambridge, UK

We report the identification of a novel heterotetrameric coat complex, TSET, which is made up of four core subunits: TPLATE, TSAUCER, TCUP, and TSPOON. Functional analysis in the Dictyostelium discoideum indicates that his complex functions as a heterotetramer, with two components (TTRAY1 & TTRAY2) containing the protocoatomer membrane deformation architecture. Comparative genomic analysis identified complete complexes in representatives of three euakryotic supergroups and partial complexes across eukaryotic diversity, suggesting that this complex was present in the Last Eukaryotic Common Ancestor. Interestingly, only a remnant of the medium subunit has been retained in animals and fungi, giving rise to the muniscins (i.e. FCHo, Syp1), mu-homology domain-containing proteins important for endocytosis. Phylogenetic analysis indicates that TSET is related to both the F-COPI subcomplex and the Adaptins. In vivo work indicates that TSET acts at the plasma membrane and is involved in endocytosis, likely trafficking material to the vacuole. Oddly, TCUP-knockouts do not seem to adversely affect cell survival or growth, suggesting functional redundancy. The addition of this novel complex to the repertoire of ancient heterotetrameric complexes allows us to more accurately deduce the evolutionary path from the original heterotetramer- protocoatomer coat to its multiple manifestations in modern organisms. WHENCE THE ? PHYLOGENETIC ANALYSES OF PHOTOSYNTHETIC GENES GIVE US A HINT

Aleš Horák1, Heather J. Esson1, Petra Dufková1 and Miroslav Oborník1,2

1Institute of Parasitology, Academy of Sciences of the Czech Republic. Branišovská 31, České Budějovice, Czech Republic 2Faculty of Science,University of South Bohemia. Branišovská 31, České Budějovice, Czech Republic

The evolutionary history of plastids is a complicated one, beginning with the primary endosymbiosis of a cyanobacterial prey cell and continuing with multiple secondary and tertiary endosymbioses involving eukaryotic prey. Although phylogenetic analyses of nuclear and plastid genes have identified several major trends in plastid evolution, many relationships remain ambiguous – especially in those lineages with secondary red plastids. We retrieved protein and EST sequences for 28 photosynthetic taxa from the ncbi+ and Kegg databases, using them to construct alignments for the component genes of photosystem I, photosystem II, the cytochrome b6/f complex, and the atp synthase complex. These alignments were combined to produce a concatenated alignment of ~60 genes that was subsequently used to infer a maximum-likelihood phylogeny in RAxML. The analysis recovered several previously resolved relationships with strong statistical support, including those between euglenoid plastids and prasinophytes, and the Prochlorococcus/Synechococcus clade and the plastids of Paulinella chromatophora. Taxa with secondary red plastids formed a monophyletic sister group to the . Interestingly, our analysis recovered a sister relationship between the plastids of chromerids (comprised of and ) and those of the eustigmatophyte Nannochloropsis gaditana. Although the relationship has weak statistical support, the similar carotenoid profiles of chromerids and eustigmatophytes also suggest a close relationship between their plastids.

TRANSCRIPTION AND POST-TRANSCRIPTIONAL PROCESSING IN THE CHLOROPLAST.

Christopher Howe, Erin Butterfield, Davy Kurniawan, Harrison Bowers, Ellen Nisbet

Department of Biochemistry, University of Cambridge, UK

Malaria is caused by infection with the Plasmodium parasite. All Plasmodium species contain a remnant chloroplast known as the apicoplast. This represents an attractive drug target, and a great deal of research has gone into understanding its metabolic capabilities. However, little is known about transcription and post- transcriptional processing within the apicoplast. By analogy with gene expression in algae and plants, we would expect an important role for transcript processing. We have analysed transcripts from much of the 35 kbp genome of the Plasmodium apicoplast. We found that transcription is typically polycistronic, followed by cleavage into shorter molecules, as with other chloroplasts. We found no evidence for polyuridylylation, in contrast to the situation in the closely related chromerids and dinoflagellates. We identified significant levels of antisense transcription, extending over much of the apicoplast genome. It remains unclear whether antisense transcripts have a biological function, or are simply the result of ‘accidental’ transcription.

OBSERVATION ON THE DEVELOPMENT OF THE BOTHROSOME AFTER SETTLEMENT, WHICH CHARACTERIZED THE LABYRINTHULOMYCETES (STRAMENOPILES)

Izumi IWATA 1 and Daiske HONDA2

1. Graduate School of Natural Science, Konan University, 2. Faculty of Science and Engineering, Konan University, Japan.

The Labyrinthulomycetes is characterized by ectoplasmic net system. This structure is superficially similar to pseudopod, but there are not only any organelles but also ribosomes in the net. The nets originate from a unique organelle, bothrosome, which is a complex of the electron dense material with the endoplasmic reticulum near the cell surface. We investigated the process from zoospore to vegetative cell with ectoplasmic net of Schizochytrium aggregatum, especially focused on the development of the bothrosome in order to reveal the evolutionary origin of this unique organelle. First, the absolute configuration of the organelles (e.g., four flagellar roots, Golgi body, mitochondria and nucleus) in the zoospore was determined. After zoospore settlement, the flagellar roots became shorter and the cell gradually rounded. After the flagella became shorter and were subsequently taken in the cell, the bothrosome appeared de novo at the anterior-ventral region of the cells. In the same stage, the two Golgi bodies were observed and the bothrosome is close to both Golgi bodies. Moreover, the bothrosome located at the position of flagellar root No. 4, which is related to the membranous organelle, , in some other stramenopiles. It suggests that there is the possibility of the evolutionary relationship of the bothrosome with the Golgi body and/or the contractile vacuole. INTERDOMAIN LATERAL GENE TRANSFER AND THE ANIMAL GERM LINE

Lindy Jensen1, Jessica R. Grant1, H. Dail Laughinghouse IV1, and Laura A. Katz1,2 1. Department of Biological Sciences, Smith College, Northampton, Massachusetts 01063, USA 2. Program in Organismic and Evolutionary Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA

Lateral Gene Transfer (LGT) has long been known to be a major contributor to genome innovation and evolution in bacteria, but remains understudied in animals. Typically, it’s suggested that the evolution of a sequestered germ line in animals creates a nigh insurmountable obstacle to LGT, but this assumption is yet to be tested. Though studies discovering evidence of LGT in certain animal genomes are becoming more common, none have attempted to describe the patterns of LGT across the animal clade. Here we test the hypothesis that the evolution of sequestered and differentiated germ line in led to a reduction in lateral gene transfer events. We generate and analyze single-gene phylogenies built from publically available sequence data from over 900 taxa across all three domains to identify well-supported interdomain LGTs in animals. Further, the scope of sampled animals enables us to estimate the placement of lateral gene transfer events in animal evolutionary history and in relation to the evolution of a sequestered germ line. EXTENDED PHYLOGENY OF AT THE BASIS OF METAZOAN EVOLUTION

Alexandra Jeuck1, Hartmut Arndt1 and Frank Nitsche1

1.University of Cologne, Biocenter Cologne, Department of General Ecology, Zuelpicher Straße 47b, D-50674 Cologne, Germany

Choanoflagellates are small heterotrophic protists ubiquitously distributed in marine and freshwater. They possess a single apical surrounded by a collar of microvilli. As being the closest non-animal relatives to Metazoa (within the group of Opisthokonta), the interest in the evolutionary biology of choanoflagellates has recently increased. Phylogenetic and morphologic studies of choanoflagellates might help reconstructing the origin of multicellularity and the cell biology and genome composition of the first animals. Choanoflagellates are currently classified into two orders according to the presence or absence of a – Acanthoecida (loricates) and (non-loricates). Molecular data, mainly based on SSU rDNA, show that on the one hand the phylogeny of loricate species is well defined and monophyletic families exist. On the other hand the two craspedid families of and Codosigidae, based on morphologic characters only, were abandoned as they were clearly not monophyletic. In this study, the sequencing of the SSU and LSU rDNA of seven isolates (marine, brackish, and freshwater) has revealed new insights into the and systematics (phylogeny) of the Craspedida. Interestingly, one of the isolates (River Rhine, Germany) was classified to the craspedid choanoflagellates due to its Monosiga-like morphology. In contrast to that, the phylogenetic analysis (SSU + LSU rDNA) of this species shows a close relationship to Acanthoecida and helps to classify a biologically and evolutionary particular group of up to now undescribed choanoflagellates. This extended phylogeny will hopefully help to get new insights into the evolution of choanoflagellates and of metazoan origins, too.

CELLULAR AND METABOLIC INTEGRATION OF SYMBIOTIC ORGANELLES IN RUBRUM

Matthew D. Johnson1 and Erica Lasek-Nesselquist2 1. Woods Hole Oceanographic Institution; 2. University of Scranton

Mesodinium rubrum is a mixotrophic marine ciliate, commonly encountered in coastal zones and known for forming red-tides. M. rubrum acquires chloroplasts, mitochondria, and other organelles from cryptophyte prey, and is capable of regulating and dividing them in a symbiotic state. Key to this relationship is the acquisition of the cryptophyte nucleus, which does not divide, but while transcriptionally active enables the ciliate to function like a phototroph. Here we present preliminary efforts to decipher the integrated transcriptome, proteome, and metabolome of an M. rubrum strain that steals organelles from Geminigera cryophila. The global transcriptome and proteome of M. rubrum are both dominated by cryptophyte proteins. Relative to free-living cryptophyte prey, the cryptophyte nucleus in M. rubrum over-expresses transcripts for metabolic proteins, while under-expressing proteins involved in genetic information and cellular processes. Both transcriptome and proteome data reveal high expression of proteins involved in photosynthesis, particularly those coding for components of the plastid antennae complex. Relative to its closest heterotrophic relative, M. pulex, M. rubrum shows reduced expression of genes involved in glycolysis, which further emphasizes its metabolic dependency on its symbiotic organelles. Metabolomic analysis indicates major differences in fatty acid and lipid metabolism between M. rubrum and G. cryophila, indicating that the ciliate is capable of harnessing the metabolic products of photosynthesis for its anabolic pathways. Using this multifaceted “omics” approach, along with targeted cell biology, we hope to use M. rubrum as a model organism for understanding adaptation to phototrophy and stable plastid acquisitions.

A SNAP SHOT OF THE GENE REPLACEMENT AFTER ENDOSYMBIOTIC GENE TRANSFER: PLASTID GAPDH GENES IN DINOFLAGELLETE BREVIS AS A CASE STUDY

Ryoma Kamikawa1, Eriko Matsuo2, Euki Yazaki2, Michiru Tahara3, Takaya Sakura3, Kisaburo Nagamune3, Yuji Inagaki2,4.

1. Graduate School of Global Environmental Studies and Graduate School of Human and Environmental Studies, Kyoto University 2. Graduate School of Life and Environmental Sciences, University of Tsukuba 3. Department of , National Institute of Infectious Diseases 4. Center for Computational Sciences, University of Tsukuba

Unlike the vast majority of photosynthetic dinoflagellates containing ‘peridinin-type’ plastids, members of the genus Karenia possess atypical ‘-derived’ plastids. The ancestral (peridinin-type) plastid was most likely replaced by the endosymbiotic haptophyte, of which cellular structures were degraded except plastid. The plastid replacement likely associated with transfer of ‘haptophyte’ genes to the host () genome, and a part of those replaced their endogenous orthologues in the host genome. Karenia brevis are reported to possess haptophyte-type and original (perinin-type) ‘plastid’ GAPDH genes—the former was transferred from the endosymbiont, while the latter has resided in the host genome prior to the plastid replacement. We here investigated the structures and quantities of the two gene transcripts, and expressed the two GAPDHs in the model apicomplexan parasite to predict their cellular localizations. Both ‘plastid’ GAPDH gene transcripts carried the GAPDH-coding region with the N-terminal extension, which potentially acts as plastid-targeting signal. Indeed, the green fluorescent proteins fused to the two N-terminal extensions were found to be localized in apicoplast in T. gondii, suggesting that both GAPDHs can be targeted to the K. brevis plastid. Nevertheless, the peridinin-type gene appeared to be transcriptionally suppressed, comparing to the haptophyte-type gene. These results strongly suggest that the two ‘plastid’ GAPDH genes in K. brevis represent an intermediate step of the gene replacement after endosymbiotic gene transfer, in which the exogenous (haptophyte-type) gene is about to replace the endogenous (peridinin-type) gene. GENOMIC STUDY OF MONOCERCOMONOIDES REVEALS A BONA FIDE AMITOCHONDRIATE

Anna Karnkowska 1, Zuzana Zubáčová 1, Vojtech Vacek 1, Lukás Novak1, Sebastian Treitli1, Lael Barlow2, Pavel Doležal1, Andrew Roger3, Miluše Hroudová 4, Joel B. Dacks 2, Čestmir Vlček 4 and Vladimír Hampl 1

1. Department of Parasitology, Faculty of Science, Charles University in Prague 2. Department of Cell Biology, University of Alberta 3. Department of Biochemistry and Molecular Biology, Dalhousie University 4. Institute of Molecular Genetics, Academy of Sciences of the Czech Republic

Mitochondria evolved from an α-proteobacterial symbiont prior to the divergence of the last eukaryotic common ancestor. During eukaryotic evolution, these organelles diversified in structures and functions and in many anaerobic/microaerophilic lineages we can observe variety of reduced forms - mitochondrion related organelles (MROs). Not only the mitochondrial genome but virtually every conceivable function has been shown to be lost in one or another eukaryotic lineage and the question arises as to whether or not the eukaryotic cell can lose this organelle completely. Several eukaryotes have been proposed as amitochondriates but for all examined cases to date the presence of MRO have been eventually demonstrated. Monocercomonoides and the whole group of remained as one of a few cases where no mitochondrion has been revealed. We generated genomic and transcriptomic data of Monocercomonoides to resolve the presence of mitochondria in this organism. The genome assembly contained 2174 scaffolds with average coverage 40x. We searched 16751 predicted proteins for mitochondrial candidates based on homology search to known mitochondrial proteins. So far among 2000 candidates we identified proteins involved in various metabolic pathways, but all of them have also cytosolic homologues, and there is no evidence of their mitochondrial localization. As well we failed to identify any hallmark proteins of mitochondria like those involved in protein import and processing. The most striking is lost of canonical mitochondrial Fe-S cluster biosynthesis pathway (ISC) and substitution by the putatively cytosolic pathway (SUF). Thus, we conclude that Monocercomonoides is the first report of genuine amitochondriate eukaryote. TAXON-RICH ANALYSES USING A PHYLOGENOMIC PIPELINE RESOLVE THE EUKARYOTIC TREE OF LIFE AND REVEAL THE POWER OF SUBSAMPLING BY SITES

Laura A. Katz1,2 and Jessica R. Grant1

1. Department of Biological Sciences, Smith College, Northampton, MA 01063 2. Program in Organismic and Evolutionary Biology, UMass-Amherst, Amherst MA 01003

ABSTRACT: Most eukaryotic lineages are microbial, and many have only recently been sampled for phylogenetic studies or remain in the ‘dark area’ of the tree of life where there are no molecular data. To assess relationships among eukaryotic lineages, we perform a taxon- rich analysis using our custom phylogenomic pipeline and including 232 eukaryotes selected to maximize taxonomic diversity and up to 1554 genes chosen as putatively vertically inherited based on their broad distribution. We also include sequences from 486 bacteria and 84 archaea to assess the impact of endosymbiotic gene transfer (EGT) from plastids and to detect contamination. Overall, our analyses are consistent with less taxon-rich estimates of eukaryotic tree of life and we recover strong support for five major clades: Amoebozoa, Excavata (without the genus Malawimonas), Opisthokonta, and SAR (Stramenopila, Alveolata and Rhizaria). Our analyses also highlight the existence of ‘orphan’ lineages, lineages that lack robust placement in the eukaryotic tree of life and indicate the possibility as of yet undiscovered diversity. In analyses including bacteria and archaea, we find that ~10% of the 1554 genes appear to have been acquired from cyanobacteria through EGT. Removing these EGT genes places the green algae as sister to the instead of the red algae, suggesting that inclusion of genes of endosymbiotic origin may mislead phylogenetic estimates. Finally, the large size of our dataset allows comparative analyses of subsets of data; alignments built from randomly sampled sites provide greater support at deep nodes than do equivalent sized datasets built from randomly sampled genes.

CONTRASTING OUTCOMES OF THE EVOLUTIONARY TRANSITION TO : MIKROCYTOS AND HELICOSPORIDIUM

Patr ick K eeling 1, J ean-François Pombert 1, Fabien Burki 1, C hris L ane 2, C athyrn Abbott 3, Dr ion B oucias 4

1 Botany Department, University of British Columbia,Vancouver, BC, V6T 1Z4, Canada 2 Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881, USA 3 Department of Fisheries and Oceans, Pacific Biological, Station, Nanaimo, BC V9R 5K6, Canada 4 Entomology and Nematology Department, University of Florida, PO Box 110620, Gainesville, FL 32611-0620, USA

The transformation of a free-living organism to an obligate intracellular parasite can be one of the more dramatic evolutionary transitions. Using genomics and transcriptomics, we have examined this transitions at the genomic and metabolic levels in two particularly mysterious protists, Mikrocytos and Helicosporidium, which provide contrasting examples of the process. Mikrocytos is a microcell parasite of oysters with a highly reduced cellular complexity and no recognizable mitochondrion, but whose position in the tree of eukaryotes has remained unresolved for lack of structural characters and highly accelerated substitution rates. Using transcripomics of enriched infections, we have shown Mikrocytos to be a member of the Rhizaria and that it is highly-reduced functionally. For example, the Mikrocytos mitochondrion appears to have been reduced to a relict “”-like organelle functionally restricted to iron- sulfur cluster assembly, and so evolved in parallel with in other lineages (e.g. ). Helicosporidium is an obligate and lethal parasite of insects, and has been determined to have evolved from green algae. We sequenced the complete nuclear and organelle genomes from Helicosporidium and examined how its plastid metabolism changed during its evolutionary transition. Here, we found almost no evidence of reduction except for the very specific loss of genes related to light harvesting and photosystems. The Helicosporidium genome is small, but overall possess nearly all the same functional classes of genes as its free-living relatives, having reduced only housekeeping functions and gene family complexity.

A FIRST LOOK AT THE MEMBRANE TRAFFICKING COMPLEMENT OF THE APICOMPLEXAN-RELATED ALGAE CHROMERA VELIA AND VITRELLA BRASSICAFORMIS

Christen M. Klinger1, Arnab Pain2, and Joel B. Dacks1

1. Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta 2. Computational Bioscience Research Center, King Abdullah University of Science and Technology, Thuwal, of Saudi Arabia

The presence of a relict plastid in several species of apicomplexan parasites hinted at the existence of a photosynthetic ancestor. Recent environmental surveys have revealed a large diversity of algae forming sister groups to the , including the two described species Chromera velia and Vitrella brassicaformis. Despite vastly different morphologies and trophic strategies, these organisms possess structures reminiscent of the apical complex (a unique cytoskeletal apparatus) in Apicomplexa, and organelles that bear similarities to (a key piece of the apicomplexan arsenal). Work from our own lab, as well as many others, has demonstrated modification of membrane trafficking machinery associated with the presence of apical organelles ( and micronemes) in Apicomplexa, mainly in the endocytic system. We hypothesize this to be an adaptation concurrent with a switch emphasizing the exo- rather than endocytic function of these modified endolysosomes. The completion of the C. velia and V. brassicaformis genomes affords an opportunity to better understand these modifications, in terms of whether they are pre-adaptive, or in fact represent specific adaptations within the Apicomplexa. Utilizing homology searching and phylogenetic algorithms, we searched for homologs of the ESCRT, Adaptor Protein, and Multi-Subunit Tethering Complex protein families in both genomes. Comparing these results to those found in several Apicomplexa and close outgroup taxa revealed patterns of retention and loss. Strikingly, the phylogenetic position of these taxa suggests that absences in the ciliates, perkinsids, and apicomplexans most parsimoniously explained by ancestral losses before, must now be attributed to multiple independent events.

INVESTIGATION OF CONTAMINATION LEVELS IN THE MARINE MICROBIAL EUKARYOTE TRANSCRIPTOME SEQUENCING PROJECT DATA

Martin Kolisko1, Fabien Burki1 and Patrick J. Keeling1

1. Department of Botany, University of British Columbia

The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) is a new and uniquely large dataset of the transcriptomes of ~500 marine single cell eukaryotes. The data were generated using Illumina technology, which provides extremely deep sequence coverage. However, a consequence of this technology is that even miniscule amounts of contamination in the starting material will often be present in the resulting data. This issue can be exacerbated by the high throughput character of the sequencing methodology because several samples may be processed at one time or could be multiplexed into one sequencing run. Therefore, contamination issues are a common problem for most current Illumina datasets. We have analyzed the MMETSP dataset to investigate whether and to which extent cross-contamination issues affect this dataset. We have used a combination of blast and phylogenetic analyses to identify and quantify potential cross-contaminations between particular datasets. We have also used read coverage information to generate a pre-cleaned version of the dataset. We will show that the MMETSP dataset indeed is affected by contamination issues but only to a small degree, which would be expected in this type of data. USING NETWORKS TO FASCILITATE TRANSCRIPTOME ASSEMBLY AND ANALYSIS

Christopher Lane1

1Department of Biological Sciences, University of Rhode Island, USA

Transcriptome data have rapidly become the most efficient way to characterize the core coding capacity of non-model organisms. These data often have to be assembled de novo because a comparative reference genome is usually unavailable. Dozens of programs, algorithms and settings can be used to assemble the raw reads, but traditional metrics of assembly quality (contig number, length, N50, etc.) do not represent quantitative comparisons. Sequence Comparative Analysis using Networks (SCAN) is software developed to provide statistical evaluation of multiple assemblies, using a reference proteome. Once the assembly that best represents a known proteome is chosen, network analysis can also provide rapid and visual comparative framework in which to examine the evolutionary dynamics of protein families across and within taxa. Several examples of the utility of networks to uncover evolutionary trends in proteome adaptation to lifestyle transitions, will be discussed.

CHANGES IN AN INTERTIDAL MICROBIAL COMMUNITY DURING CHRONIC EXPOSURE TO PETROLEUM HYDROCARBONS AT A BIOREMEDIATION SITE, PRUDENCE ISLAND, NARRAGANSETT BAY

Gaytha A. Langlois1, Cameron Larson1, and Ethan Beise1, Bryant University, Smithfield, RI, USA.

1. Bryant University, Smithfield, RI, USA

The release of petroleum byproducts into shallow coastal bays and estuaries alters ecosystem dynamics, including reduced biodiversity and selective loss of species. The damaging effects of the residual toxic organic compounds are especially evident in soft mud sediments. This study of an intertidal microbial community at a contamination site in Narragansett Bay, located at the south end of Prudence Island, characterizes some of the ecosystem changes resulting from chronic release of gasoline and diesel fuel residues from previously installed underground fuel storage tanks at a naval fuel depot, which were later removed as part of cleanup and mitigation efforts on the site. Changes in microbial population dynamics, trophic relationships, species composition, and predation patterns have been observed in field samples collected over an 8-year period, and then compared to a control site and to microcosm studies at the Marine Ecosystems Laboratory (MERL), Graduate School of Oceanography at the University of Rhode Island. Observations were made using light and fluorescence microscopy, SEM and video imaging, followed by DNA extraction and analysis, in order to characterize the protistan and micro- invertebrate populations in both communities, and to assess the coping mechanisms utilized by ciliates for survival in the oiled environment. Seasonal variation in populations was also evaluated. These comparisons confirm that the response of a marine microbial community to petroleum hydrocarbons shows a shift to an altered, but stable, ecological community during low-level, chronic oil exposure.

THE MOLECULAR INTERPLAY BETWEEN A CILIATE AND ITS STOLEN ORGANELLES Erica Lasek-Nesselquist1 and Matthew Johnson2 1. Department of Biology, University of Scranton 2. Department of Biology, Woods Hole Oceanographic Institution

Kleptoplastidic organisms steal plastids from their algal prey and exploit these organelles for their own metabolic purposes. Despite the ecological importance of these systems, where stolen plastids often confer competitive advantages to their hosts, little is known about the molecular interactions between host cells and sequestered organelles. is a ciliate that requires stolen plastids from Geminigera cryophila for survival. M. rubrum also retains other prey organelles, such as the nucleus and mitochondrion. We analyzed expression differences between M. rubrum and Mesodinium pulex – a close relative that is strictly heterotrophic - to elucidate the molecular interactions between the ciliate and its sequestered organelles. Additionally, we analyzed expression differences between sequestered prey organelles and free-living G. cryophila. Our results indicate that M. rubrum overexpresses proteins related to genetic information and cellular processes in comparison to M. pulex but shows a significant decrease in metabolic proteins. G. cryophila organelles display the opposite pattern with an underrepresentation of proteins involved in genetic information and cellular processes and an overrepresentation of metabolic proteins in comparison to free-living G. cryophila. Looking at more specific pathways reveals that sequestered organelles significantly upregulate genes related to photosynthesis, including those coding for components of the antenna complex. Overall, M. rubrum and its stolen organelles appear to interact in a largely compensatory manner. EFFECTS OF EGT ON DEEP PHYLOGENETIC PATTERNS IN PLASTID CONTAINING ORGANISMS AND THEIR CLOSE RELATIVES

H. Dail Laughinghouse IV1,*, Jessica Grant1, Laura A. Katz1,2 1. Department of Biological Sciences, Smith College, Northampton, Massachusetts 01063, USA 2. Program in Organismic and Evolutionary Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA *[email protected]

Endosymbiotic gene transfer (EGT) is a process that increases the complexity within the nucleus after transfer of DNA from organelles. EGT plays a crucial role in genome innovation and evolution, especially of plastid containing photosynthetic organisms (and their close relatives) where EGT from the plastid (organelle derived from cyanobacterial primary endosymbiosis) has shaped nuclear DNA. Though several studies have been published on effects of EGT on the deep phylogenetic patterns of photosynthetic organisms, few focus on patterns across all photosynthesizing groups. Here we test the hypothesis that analysis of genes originating from EGT lead to a different interpretation of the evolution of photosynthetic eukaryotes as compared to those evolved vertically. For example, we examine estimated relationships among the three lineages within Archaeplastida: Glaucophyta, Rhodophyta, and . We also include organisms resultant of secondary and tertiary endosymbiotic events in the analysis. We started with over 12000 nuclear genes from over 900 taxa sampled from Bacteria, Archaea, and Eukaryota to determine the inflow of genes into the nucleus due to EGT. Through custom python scripts and visual analysis, we are assessing the impact of EGT on the evolution of the plastid containing lineages. Our preliminary results indicate that EGT plays a more substantial role in the evolution of these organisms than previously appreciated.

IMPROVED UNDERSTANDING OF THE EVOLUTIONARY HISTORY AND DIVERSITY OF PHAGOTROPHIC EUGLENIDS.

Won Je Lee1 and Alastair GB Simpson2

1. Department of Urban Environmental Engineering, Kyungnam University 2. Canadian Institute for Advanced Research, Program in Integrated Microbial Diversity, and Department of Biology, Dalhousie University

Euglenids are an exemplar lineage for studying the evolutionary history of complex unicells. Phagotrophic euglenids are especially important because they represent most of the major-lineage-level diversity within Euglenida, and gave rise to photosynthetic forms (via secondary endosymbiosis), osmotrophs, and probably the anaerobic Symbiontida. However, our current understanding of phagotrophic euglenids is fragmentary, partly due to the limited availability of cultures. As a result, reconstructions of euglenid evolution are, at best, plausible rather than well-supported. We have cultured two phagotrophic euglenids that represent clades or genera for which there were no combined molecular and ultrastructural data. Neometanema parovale is a skidding heteronemid. SSUrRNA gene phylogenies confirm that Neometanema is the sister group to primary osmotrophs. In addition to sharing a predominantly swimming locomotion, Neometanema has a similar number and arrangement of pellicular strips to basal primary osmotrophs, including a distinctive arrangement of the subpellicular . This data allows a more detailed estimate of the ancestry of primary osmotrophs. Notosolenus urceolatus belongs to Petalomonadida, Petalomonads are perhaps the most unusual major group of phagotrophic euglenids, but the available data are peculiarly disjointed. The fine structure of the mitochondria, flagella and feeding apparatus of N. urceolatus are unusual amongst phagotrophic euglenids but consistent with most fragmentary accounts of other petalomonads. Petalomonads appear to have replaced the ancestral euglenozoan tubular with a non- homologous globular extrusome. Nonetheless there is currently limited-to-nil evidence from molecular phylogenies or ultrastructural data that petalomonads represent the deepest branch among euglenids, despite this being routinely assumed.

ADAPTATIONS TO ANAEROBIOSIS AND ANCIENT MITOCHONDRIAL FEATURES IN THE MITOCHONDRION- RELATED ORGANELLES OF A FREE-LIVING

Leger, ML.1, Eme, L1., Hug, LA1,2. and Roger, AJ1. 1Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Canada. 2Present address: Department of Earth & Planetary Sciences, University of California, Berkeley, Berkeley, California, USA.

Mitochondrion-related organelles (MROs) have arisen independently in a wide range of anaerobic protist lineages. Only a few of these organelles and their functions have been investigated in detail, and most of what is known about MROs comes from studies of parasitic organisms such as the parasitic . Here, we describe the MRO of a free-living anaerobic jakobid excavate, incarcerata. We report an RNAseq-based reconstruction of the MRO proteome of Andalucia, with an associated biochemical map of the pathways predicted to be present in this organelle. The pyruvate metabolism and oxidative stress response pathways are strikingly similar to those found in the MROs of other anaerobic protists, such as Trichomonas. This elegant example of convergent evolution is suggestive of an anaerobic biochemical ‘module’ of prokaryotic origins that has been laterally transferred among eukaryotes, enabling them to adapt rapidly to anaerobiosis. We identified genes corresponding to a variety of mitochondrial processes not found in T. vaginalis including intermembrane space components of the mitochondrial protein import apparatus, and enzymes involved in amino acid metabolism and cardiolipin biosynthesis. Of particular interest, we report here a suite of mitochondrial division proteins that have never been described in MROs, and include dynamin-related protein and mitochondrial FtsZ homologs.

SPONGES, COMPLEX TRAITS, AND THE EVOLUTION OF MULTICELLULARITY

Sally P Leys1

1University of Alberta

The emergence of complex traits such as epithelia allowed animals to regulate homeostasis, develop complex signalling pathways, set up regionalized body plans, and coordinate responses to environmental stimuli. (Porifera), considered the first multicellular animals descended from a common ancestor shared with choanoflagellates, retain a simple body plan in comparison to other metazoans. What was required for their evolution from a colonial state, and what distinguishes them as metazoans? Sponges have 16-20 cell types, which arise during development of the embryo into a polarized swimming larva. Metamorphosis of the larva gives rise to an adult with regionalized tissues and optimal organization of choanocyte chambers for filter feeding. By studying transcriptomes and genomes of sponges and their closest animal relatives we find that many genes involved in the expression of complex traits evolved before the origin of metazoans. We identified representatives of most metazoan signalling pathways in sponges, some of which, however, such as Wnt, are uniquely metazoan and are involved in development of the polarized body plan. Proteins involved in adhesion and in sensory and contractile organelles are known across unicellular eukaryotes, but in sponges these are regionalized into structures that function as complex organs. While the evolution of complex cell types allows a high level of function in unicellular and colonial animals, the study of sponges suggests the regionalization of complex traits into complex structures was key to the evolution of the first metazoans. COMMUNITY PATTERNS IN MARINE PROTISTS

Ramiro Logares1, Stéphane Audic2,3, BioMarKs and TARAoceans consortiums, Sarah Romac2,3, Thomas A. Richards4, Colomban de Vargas2,3, Ramon Massana1

1. Institute of Marine Sciences (ICM), CSIC, Barcelona, Catalonia, Spain 2. ADMM UMR 7144, UPMC Paris 06, Station Biologique de Roscoff, Roscoff FR- 29682, France 3. ADMM UMR 7144, CNRS, Station Biologique de Roscoff, Roscoff FR-29682, France 4. Biosciences, Geoffrey Pope Building, University of Exeter, Exeter EX4 4QD, United Kingdom

Community ecology is a classic field in animal and plant biology. Yet, in microbes, community ecology is still in its infancy. In general, all biological communities are composed of a few abundant and many rare species. Previous High- Throughput sequencing studies have shown that this pattern is particularly prominent in microbial communities, where most constituent taxa are usually extremely rare. In this talk, I will present results from our research on abundant and rare sub-communities of marine protists. We surveyed surface waters of six separated coastal locations in Europe, considering independently the pico-, nano- and micro/meso- plankton organismal size fractions. Deep Illumina sequencing of the 18S rRNA indicated that the abundant regional community was mostly structured by organismal size fraction, while the rare regional community was mainly structured by geographic origin. Yet, some abundant and rare taxa presented similar biogeography, pointing to spatiotemporal structure in the rare protistan biosphere. Abundant and rare sub-communities presented regular proportions across samples, indicating similar species-abundance distributions despite taxonomic compositional variation. Several taxa were abundant in one location and rare in others, suggesting large oscillations in abundance. The substantial amount of metabolically active lineages found in the rare biosphere suggests that this sub- community constitutes a diversity reservoir that can respond rapidly to environmental change. Interestingly, we also found that several of the features observed in European marine coastal assemblages were also characteristic of communities from the global open ocean (TARAoceans expedition), pointing to consistent and generalized patterns of community structuring in marine protists.

DIVERSITY AND TEMPORAL DYNAMICS OF SMALL PLANKTONIC PROTISTS FROM SHALLOW FRESHWATER SYSTEMS

Purificación López-García1, Marianne Simon1, Philippe Deschamps1, David Moreira1, Gwendal Restoux1, Paola Bertolino1 and Ludwig Jardillier1

1Unité d'Ecologie, Systématique et Evolution, Centre National de la Recherche Scientifique – CNRS & Université Paris-Sud, 91405 Orsay, France

Although inland water bodies are more heterogeneous and sensitive to environmental variation than oceans, the diversity of small protists in these ecosystems is much less well- known. Some molecular surveys of lakes exist, but little information is available from smaller, shallower and often ephemeral freshwater systems, despite their global distribution and ecological importance. We carried out a comparative study based on massive 454- pyrosequencing of amplified 18S rRNA gene fragments of protists in the 0.2-5 μm-size range in one brook and four shallow ponds with different trophic status located in the Natural Regional Park of the Chevreuse Valley, France. Our study revealed a wide protist diversity, with 812 stringently defined operational taxonomic units (OTUs) belonging to the recognized eukaryotic supergroups (SAR −Stramenopiles, Alveolata, Rhizaria−, Archaeplastida, Excavata, Amoebozoa, Opisthokonta), to groups of unresolved phylogenetic position (Cryptophyta, Haptophyta, Centrohelida, Katablepharida, Telonemida, ) or to deep-branching lineages. In addition to MAST-2 and MAST-12 clades, already detected in freshwater, we also identified several lineages previously thought to be exclusively marine, including MAST-3 and possibly MAST-6. Protist community structures were different in the five ecosystems. These differences did not correlate with geographical distances, but seemed to be influenced by environmental parameters. To explore this possibility, we carried out a 2-year survey of protist communities in the five ecosystems on a monthly basis combined with multivariate statistical analyses including several physico-chemical parameters. Interestingly, protist diversity does not seem to be strongly influenced by the measured physico-chemical parameters, suggesting that other, possibly biological, factors influence community composition and dynamics. LARGE-SCALE PHYLOGENOMIC ANALYSIS REVEALS THE PHYLOGENETIC POSITION OF THE PROBLEMATIC TAXON PROTOCRUZIA AND UNRAVELS THE DEEP PHYLOGENETIC AFFINITIES OF THE CILIATE LINEAGES

Denis H. Lynn1 and Eleni Gentekaki2

1. Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 2. Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2

There is a consensus that there may be a dozen or more class-level clades in the phylum Ciliophora, based on morphology at both light and electron microscopic levels. Morphologists have also suggested some groupings of these clades into subphylum-level clades, based on ultrastructure (e.g. POSTCILIODESMA- TOPHORA) and on morphogenetic patterns (e.g. Lamellicorticata). There are a significant number of phylum-level analyses of the ciliated based on small subunit rRNA gene analyses and a handful of analyses based on multiple genes. Some of these confirm and others reject these groupings. The genus Protocruzia in particular seems to be flexibly affiliated near the base of the ciliate tree. To provide a more robust test of these deep relationships, Gentekaki et al. (2014. Mol. Phylogen. Evol. DOI: 10.1016/j.ympev.2014.04.020) sequenced EST libraries for 11 ciliate species, and added these data to genome alignments for five other ciliate species. The phylogenomic analyses were based on 158 genes, providing 42,158 characters, and used four dinoflagellates and nine apicomplexans as out-groups. We conclude the following: 1) the subdivision of the phylum into two major clades – POSTCILIODESMA-TOPHORA and ; 2) the Lamellicorticata to include the and ; 3) the SAL clade to include the Spirotrichea and Lamellicorticata; 4) the removal of Protocruzia from the Spirotrichea as in the phylum; and 5) initial support for the CONTHREEP supergroup.

PROBING THE EVOLUTION OF ENDOCYTOSIS USING PROTEOMICS

Paul Manna1, Cordula Boehm1 and Mark C. Field1

1. Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, Scotland, DD1 5EH.

Internal membranes, once considered a feature unique to eukaryotes, are now known to also be widely present in bacterial species. Attempts to understand the origins of these systems, and their relatedness, are difficult due to sequence diversity of the gene products concerned, as well as the absence of detailed sampling in a broad range of taxa. This latter issue, which we have termed asymmetry, means that the identification of truly novel components that are lineage-specific is, by definition, not amenable to in silico analysis. However, such studies can indicate where possible novelty lies within specific lineages, providing clues as to where to focus investigations and also the strategies employed in adaptation to particular life styles. We have exploited the trypanosomatids as a novel, potentially early branching eukaryotic clade for the detailed dissection of endocytic pathways. An approach, fusing in silico analysis, study of distant orthologs and new methods for the identification of true novelty will be presented. Our data facilitate reconstruction of interaction networks in trypanosomes that likely subtend the endocytic apparatus, and which implicate high levels of non- conserved proteins operating in these organisms, highlighting those systems and mechanisms that are potentially most susceptible to evolutionary pressure. The methods and data are also generalisable, and provide routes towards the identification of novelty in a wide range of taxa, as well as targets for chemical manipulation of pathogenic and agriculturally important organisms.

EVOLUTION AND DIVERSIFICATION OF LIGHT HARVESTING COMPLEX DRIVEN BY GENE DUPLICATION

Shinichiro Maruyama1, Eiichi Shoguchi2, Nori Satoh2, and Jun Minagawa1 1. Division of Environmental Photobiology, National Institute for Basic Biology 2. Marine Genomics Unit, Okinawa Institute of Science and Technology Promotion Corporation

Light harvesting complex (LHC) is an essential component in light energy capture and transduction toward downstream photosynthetic reactions in plant and algal plastids. Previous studies suggested that genes encoding the three-helix LHCs were derived form a protein containing a single transmembrane helix, and that gene duplication had played a key role in the functional diversification of LHCs in the evolution of plastids. Although the dinoflagellate minutum, an endosymbiont alga of cnidarian animals including corals and sea anemone, is known to possess a unique LHC gene family called chlorophyll a-chlorophyll c2-peridinin protein complex (acpPC) but not any of stress responsive LHCs for excess light energy dissipation, the diversity and evolutionary trajectories of the gene family have not been fully investigated. Our phylogenetic data reveal that many of the acpPCs are encoded in extensively duplicated nuclear genes with the multi-unit structures in the nuclear genome of S. minutum, suggesting that the diversity of the LHC genes have been forged through multiple rounds of intra- and intergenic gene-unit duplication events in symbiotic dinoflagellates. This mode of gene duplication has been reported in other LHC subfamilies in Euglena, which may represent an example of convergent evolution between the two distantly related photosynthetic lineages, dinoflagellates and euglenophytes.

Nuclear Architecture of the ciliate correlates with patterns of molecular evolution

Xyrus X. Maurer-Alcala1,2 and Laura. A. Katz1,2 1. Department of Biological Sciences, Smith College, Northampton, Massachusetts 2. Program in Organismic and Evolutionary Biology, UMass-Amherst, Amherst, Massachusetts

The heteromeric of the ciliate Chilodonella uncinata (Cl: ) provides a model to assess the impact of nuclear architecture on patterns of gene expression and . The influence of of nuclear architecture on gene expression is well documented in plants and animals where variation in territories underlies the distribution of euchromatin and hence transcription. The nucleus of C. uncinata is divided into two areas, with a DNA rich orthomere positioned towards the nuclear membrane and a DNA poor, central paramere. Previous work using qPCR demonstrated that 1) MAC chromosome copy numbers can be as high as 67,000 for protein coding genes and 2) that levels of gene expression do not correlate with mac chromosome copy number (Bellec and Katz 2012; Huang and Katz 2014). Building on these observations, we used fluorescent microscopy to examine the: 1) location of nascent transcription; 2) position of highly amplified gene-sized and 3) relationship between mac chromosomes location and patterns of molecular evolution (e.g. GC content and codon bias). Together these data reveal that chromosome position is related to transcriptional activity and strength of selection.

DRUG RESISTANCE IN PARASITES, NOT AS CLEVER AS WE THOUGHT THEY WERE.

Geoffrey Ian McFadden Botany School, University of Melbourne, VIC 3010, Australia

Malaria is a major global health issue. Parasite resistance to chloroquine spread globally over two decades and rendered the drug useless. Ominously, resistance to artemisinin (our current front line antimalarial) is already spreading. Atovaquone, developed as an antimalarial in 1990, mimics the mitochondrial electron transport intermediate ubiquinol and binds to mitochondrial protein cytochrome b to perturb electron transport essential for pyrimidine biosynthesis when parasites are in the human host. Parasite resistance to atovaquone emerged remarkably rapidly and the drug was deprioritised for use. Atovaquone resistance occurs through mutations to the parasite’s mitochondrial cytochrome b gene. Mutations prevent binding of the drug to cytochrome b protein.

We explored the transmissibility of atovaquone resistant parasites through mosquitoes in our malaria life cycle facility. None of five different atovaquone resistant mutants tested were transmissible, all failing to produce sporozoites and being unable to infect naive mice. Why? Aerobic respiration is only necessary in the insect phase of the parasite and the cytochrome b mutations are apparently tolerated in the blood phase but not the insect phase. This means that atovaquone resistance cannot be transmitted via the vector. We traced the transmission failure to a defect in female gametes, through which the mitochondrion is inherited. We crossed the atovaquone resistant (female sterile) line with a male sterile line, which restored fertility via complementation. Progeny inherited atovaquone sensitive mitochondria, further demonstrating that drug resistance cannot be transmitted. Non-transmissible resistance makes the drug vastly more useful than was initially thought.

MOLECULAR PHYLOGENY AND ULTRASTRUCTURE OF APHELIDIUM AFF. MELOSIRAE (, ) AND THE DIVERSITY OF FRESHWATER APHELIDS

David Moreira1, Sergey A. Karpov2,3, Maria A. Mamkaeva2,3, Karim Benzerara4, Marianne Simon1, Philippe Deschamps1, Ludwig Jardillier1 and Purificación López-García1

1Unité d’Ecologie, Systématique et Evolution, UMR CNRS 8079, Université Paris-Sud. 91405 Orsay cedex, France 2Zoological Institute, Russian Academy of Sciences, St. Petersburg 199034, Russian Federation 3St. Petersburg State University, St. Petersburg 199034, Russian Federation 4Institut de Minéralogie et de Physique des Milieux Condensés, Université Pierre et Marie Curie et CNRS, 4 place Jussieu. 75252 Paris cedex 05, France

Aphelids are a poorly known group of algal parasitoids that have raised considerable interest due to their pivotal phylogenetic position. Together with Cryptomycota () and the highly derived Microsporidia, they have recently been reclassified as the Opisthosporidia, which constitutes the sister group to the (true) fungi within the . Despite their phylogenetic interest and their huge diversity revealed by molecular environmental studies, only three genera have been described (Aphelidium, Amoeboaphelidium, and Pseudaphelidium) and 18S rRNA gene sequence information is available only for Amoeboaphelidium. We have studied the ultrastructure of Aphelidium aff. melosirae, its life cycle, and provided the first 18S rRNA gene sequence for this genus. Aphelidium parasitoids encyst and penetrate their host, the alga Tribonema gayanum, through an infection tube. This cyst germination leads to a young trophont that phagocytes the algal cell content and progressively develops a plasmodium, which becomes a zoospore-producing sporangium. Aphelidium are amoeboflagellated, have tubular/lamellar mitochondrial cristae, a metazoan type of centrosome, and closed orthomitosis with intranuclear spindle. These features together with trophont distinguish Aphelidium from fungi and support the erection of the new superphylum Opisthosporidia as sister to fungi. 18S rDNA molecular phylogeny analysis indicates that Aphelidium is very distantly related to Amoebaphelidium, highlighting the wide genetic diversity of aphelids and making possible to ascribe a large variety of environmental sequences to this group. Analysis of massive 18S rDNA data from freshwater ecosystems shows that different aphelid species coexist in the same environments and have different seasonal dynamics.

NEPHROMYCES, A MUTUALISTIC ENDOSYMBIONT REPRESENTING A NEW MAJOR BRANCH OF THE APICOMPLEXAN TREE Sergio A. Muñoz-Gómez1, Mary B. Saffo2, Chris E. Lane2, Chris Paight2, and Claudio H. Slamovits1 1 Department of Biochemistry and Molecular Biology, Centre for Comparative Genomics and Evolutionary Bioinformatics, Dalhousie University 2 Department of Biological Sciences, University of Rhode Island represents a largely unexplored eukaryotic lineage that only recently found a home among apicomplexans. This endosymbiont of molgulid tunicates inhabits a peculiar habitat, the renal sac of its host, where it lives forming a heterogeneous population of morphologically diverse cell types. Nephromyces emerges as a divergent apicomplexan considering its unusual morphology, life-cycle, presence of cytoplasmic bacterial endosymbionts, and a presumably mutualistic association with its animal host. We aimed to gain a better understanding of this tri-partite (bacteria-Nephromyces-tunicate) mutualistic relationship and its evolutionary implications by generating genomic and trascriptomic data from the contents of the tunicate renal sac. Our initial surveys of the genomic data allowed us to assemble 10 different contigs, each representing almost entire plastid (apicoplast) genomes. Phylogenetic analyses of 27 apicoplast proteins clarified the phylogenetic position of Nephromyces as sister to ‘core’ apicomplexans (Hematozoa+), and highlighted the significant intra-clade divergence among these . We furthermore were able to identify several bacterial contigs, which presumably represent Nephromyces’ endosymbionts. These preliminary results suggest that molgulid renal sacs are complex ecosystems inhabited by a diverse community of different Nephromyces lineages, each with its own repertoire of bacterial endosymbionts. Future efforts will focus on elucidating the metabolic contribution of each partner to the dynamics of this complex symbiotic system.

“GREEN GENES” IN NOVEL GREEN COLORED DINOFLAGELLATES: SIGNS OF THE GENOMES

Takuro Nakayama1, Goro Tanifuji2, Ryoma Kamikawa3, Eriko Matsuo4, Chihiro Sarai5, Kazuya Takahashi5, Ken-ichiro Ishida4, Mitsunori Iwataki6 and Yuji Inagaki1,4 1. Center of Computational Sciences, University of Tsukuba, 2. Faculty of life and environmental sciences, University of Tsukuba, 3. Graduate School of Human and Environmental studies, Kyoto University, 4. Graduate School of Life and Environmental Sciences, University of Tsukuba, 5. Graduate School of Science and Engineering, Yamagata University, 6. Asian Natural Environmental Science Center, University of Tokyo

Plastid acquisitions by heterotrophic eukaryotes through endosymbioses with red and green algae (secondary endosymbioses) gave rise to multiple lineages with complex plastids, and have expanded the diversity of eukaryotes. During secondary endosymbioses, both genome of a host and that of an endosymbiont were rearranged. In particular, the nuclear genome of the endosymbionts has been severely reduced or completely lost in many cases. are the remnant nuclei of the photosynthetic endosymbionts, which can be seen only in cryptophytes and chlorarachniophytes. Despite different origins of the plastids, the genomes of nucleomorphs both in cryptophytes and chlorarachniophytes are similar in size and structure suggesting a striking convergent evolution. Recently we discovered that two dinoflagellate strains with green alga derived-plastids possess nucleomorphs within the periplastidal compartments. If those structures contain active genomes, comparison between the newly found and previously sequenced nucleomorph genomes would provide insights into the reductive evolution of eukaryotic genome and genome rearrangement in secondary endosymbioses. We here obtained transcriptomic data from the two newly found dinoflagellate isolates to survey for genes acquired from the green algal endosymbiont. Both dinoflagellate isolates were found to express “green” genes for translation, transcription and intron splicing in addition to dinoflagellate homologs, suggesting the presence of transcriptionally active nucleomorph genomes. A certain portion of the “green” transcripts, including housekeeping genes, was found to possess relatively high AT-ratio compared to the majority of dinoflagellate transcripts. From the transcriptomic data of the two dinoflagellate isolates, we will discuss the blueprint of the novel nucleomorph genomes. PAULINELLA CHROMATOPHORA RETAINS TWO EVOLUTIONARILY DISTINCT PATHWAYS FOR TETRAPYRROLE BIOSYNTHESIS.

Yuki Nishimura1, 2, Mami Nomura1, Takuro Nakayama3, Shin-ya Miyagishima4, Yukihiro Kabeya4, Junichi Obokata5, Ken-ichiro Ishida1, Tetsuo Hashimoto1,3, Yuji Inagaki1,3

1. Graduate school of Life and Environmental Sciences, University of Tsukuba, 2. Graduate school of Systems and Information Engineering, University of Tsukuba, 3. Center for Computational Science, University of Tsukuba, 4. National Institute of Genetics, 5. Graduate School of Environmental Life Science, Kyoto Prefectural University

Synthesis of tetrapyrrole (TP) is critical for all organisms. The TP biosynthetic pathway in the plastid-bearing eukaryotes was established by replacement of the ancestral (heterotrophic) genes with those acquired from the cyanobacterial endosymbiont that gave rise to the first plastid. However, we have no precise knowledge regarding the evolution of the TP biosynthetic pathway in a testate amoeba Palinella chromatophora (Pc), which acquired an obligate cyanobacterial endosymbiont for photosynthesis (so-called cyanelle) after separating from a closely related but heterotrophic relative in the same genus. We generated the RNA-seq data of Pc strain MYN1, and successfully identified the genes in the heterotrophic-type TP biosynthesis pathway. The genome data of the cyanelle in strain MYN1 confirmed that the endosymbiont still retains the cyanobactrium-type pathway. These results strongly suggest that Pc possesses two evolutionarily distinct pathways for TP biosynthesis, corresponding to a putative early stage of plastid acquisition, in which the ancestral (heterotrophic-type) pathway has yet to be replaced with the cyanobacterial homologues. Intriguingly, two enzymes in the heterotrophic-type pathway in Pc displayed phylogenetic affinities exclusively to the homologues in plastid-bearing eukaryotes, implying that Pc had some ‘evolutionary contact’ with plastid-bearing eukaryotes prior to the acquisition of the cyanelle.

MITOCHONDRIAL ORGANELLE OF PYRIFORMIS

Lukáš Novák1, Zuzana Zubáčová1, Ondřej Brzoň1, Anna Karnkowska1 and Vladimír Hampl1

1. Department of Parasitology, Faculty of Science, Charles University in Prague

Preaxostyla (Metamonada, Excavata) is a fascinating and understudied clade of anaerobic flagellates. It comprises two distinct groups: free-living genus Trimastix with typical excavate morphology and a diverse and divergent group called Oxymonadida which exclusively inhabits a gut of various metazoans. Oxymonads are the largest remaining group of eukaryotes without any hint of mitochondrion and a very real possibility of being secondarily amitochondrial. On the other hand, there have been suspicious double-membrane bound organelles resembling hydrogenosomes observed in the cytoplasm of Trimastix. Preaxostyla therefore have the potential of becoming a model-group for study of mitochondrion reduction and subsequent loss. We have shown the presence of a complete glycine cleavage system, an exclusively mitochondrial pathway, in the suspicious organelle of Trimastix pyriformis, as well as 3 paralogues of Hydrogenase, Maturase of Hydrogenase, Mitochondrial Processing Peptidase, CPN60 and MCF carrier. The cytosolic localization of PFO and Malic Enzyme is also interesting for inferring the function of the organelle. Among other proteins localized in the cytosol are Aconitase, SHMT and OTC. These results show that Trimastix pyriformis contains a mitochondrion-related organelle with a function similar to a . Comparative genetics on evolution of protein in

Noriko Okamoto, Fabien Burki, Patrick J. Keeling Department of Botany, University of British Columbia

Myzozoa is the lineage that includes all the descendants of the common ancestor of the dinoflagellates and the apicomplexans. Myzozoans are diverse in trophic strategies. In particular, there are a few distinct parasitic lineages. The key subcellular structure to understand the frequent occurrence of parasitism is the apical complex. The apical complex is originally described as an invasion machinery in the apicomplexan parasites. It was revealed that the apical complex is wide spread among Myzozoa, not only in parasitic lineages but also among the predators. Based on morphological resemblance, the homology of the apical complex is hypothesized, though has not been tested at the genetic level. The protein components of the apical complex are well studied among the apicomplexans. Aiming to demonstrate the homology of proteins from structures of the apical complex, we searched for conserved domains known from the apicomplexans against the available genomic and transcriptomic data of other myzozoans, including the dinoflagellate (Psammosa pacifica, Symbiodinium sp., and marina), the perkinsids ( marinus), and the colpodellids ( pontica). The conserved domains that are shared among the apicomplexans, namely, thrombospondin-1 (TSP1) domain, von Willebrand factor type A (VWA) domain, and epidermal growth factor (EGF) domain are also conserved among myzozoans.

CRENEIDAE FAM. NOV. – NOVEL ANAEROBIC LINEAGE OF HETEROLOBOSEA (EXCAVATA) WITH UNIQUE CELL STRUCTURE AND PECULIAR MULTIFLAGELLATE STAGE WITHIN THE LIFE CYCLE

Tomáš Pánek1, Alastair G. B. Simpson2,3, Vladimír Hampl4, Miluše Hroudová5, Čestmír Vlček5, and Ivan Čepička1

1 Department of Zoology, Charles University in Prague, Prague, Czech Republic 2 Department of Parasitology, Charles University in Prague, Prague, Czech Republic 3 Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada 4 Canadian Institute for Advanced Research, program in Integrated Microbial Diversity 5 Department of Genomics and Bioinformatics, Institute of Molecular Genetics ASCR, v.v.i, Prague, Czech Republic

Despite extensive adoption of the PCR-based methodologies in microbiology, culture- based approach still reveals novel species or even deep-branching eukaryotic lineages previously non-detected in environmental clone libraries. Here, we report isolation and subsequent characterization of one such lineage, Creneis carolina gen. et sp. nov., obtained from marine anoxic sediments. C. carolina is a heterotrophic, obligatory anaerobic amoeboid flagellate whose main trophic stage superficially resembles some pelobionts (Amoebozoa) or (Obazoa: Breviatea) by possessing a single anterior flagellum closely associated with the nucleus and by the anaerobic lifestyle. However, its life cycle contains also multiflagellate cells with peculiar morphology that is not particularly similar to any other eukaryote. Unexpectedly, phylogenetic analyses based on SSU rDNA and protein-coding genes convincingly showed that C. carolina is a member of Heterolobosea and specifically belongs to Tetramitia. Phylogenetic analysis based on six protein-coding genes even indicates an independent origin of the anaerobiosis in Psalteriomonadidae Cavalier-Smith, 1993 and Creneidae fam. nov. Furthermore, the flagellar apparatus of C. carolina contains elements that are probably homologous to certain structures typical for the Excavata and Heterolobosea, but its organization is highly unusual and unprecedented at the eukaryotic level. DEFINING THE ‘NORMAL’ HUMAN EUKARYOTIC MICROBIOTA

Laura Wegener Parfrey1,2

1. Departments of Botany and Zoology, University of British Columbia 2. Integrated Microbial Diversity Program, Canadian Institute for Advanced Research

Humans, like all animals, have been continuously exposed to a broad spectrum of intestinal microbes including microbial eukaryotes over millions of years of evolution. The bacterial communities found in the mammalian gut have been well characterized in recent years with high-throughput sequencing tools, and it is clear that they influence myriad processes such as immune system development, behavior and disease susceptibility. Though the eukaryotic component of the microbiota is sparsely studied at the community level, parasitologists have cataloged many amoebae, flagellates, and worms that reside in the mammalian gut, and these are likely part of our co-evolved microbial community. We use comparisons to remote human populations, other mammals, and ancient human samples to show that eukaryotic communities are much less diverse in populations with Western lifestyles. Similar levels of biodiversity loss in Westernized populations have also been reported for bacteria and helminths. One of the consequences of the depauperate microbiota appears to be greater incidence of inflammatory and autoimmune disease, which become much more prevalent as populations adopt Western lifestyles around the globe. Intriguingly, treatments to re-introduce components of the microbiota represent promising therapies for inflammatory disease.

DIVERSITY AND SPARTIAL VARIATION OF PROTOZOA IN GEOGRAPHICALLY DISPARATE HYPERSALINE ENVIRONMENTS

Jong Soo Park1 and Alastair GB Simpson2

1. Department of Oceanography, Kyungpook National University 2. Canadian Institute for Advanced Research, Program in Integrated Microbial Diversity, and Department of Biology, Dalhousie University

Free-living protozoa are a diverse but understudied component of the biota of extreme hypersaline environments. There are <10 species of truly halophilic protozoa for which there are both light microscopy and gene sequence data, usually from just one or two isolates. Consequently, there are little data on the molecular diversity within species of halophiles, and almost nothing known of their biogeographic distribution. We have garnered SSU rRNA gene sequences for several clades of halophilic protozoa from enrichments from waters of >12.5% salinity from Australia, North America, and Europe (6 geographic sites, 21 different water masses sampled). The small stramenopile Halocafeteria was found at all sites by microscopy and sequencing, with no evident phylogenetic clustering of sequences by site or continent. The ciliate Trimeyma was recorded by microscopy and sequencing from 6 non-european samples. Phylogenies confirmed a monophyletic halophilic Trimeyma group that included western Australian, south-east Australian, and North American clusters distinct from sequences from the Mediterranean and Korea. Several halophilic Heterolobosea were detected, demonstrating that the Pleurostumum clade contains at least three molecular species clusters, and increasing known continental ranges for Tulamoeba peronaphora and Euplaesiobystra hypersalinica. The unclassified flagellate Palustrimonas was identified from one Australian sample and shown to be a novel deep-branch within . Our survey is consistent with a global distribution of halophilic protozoa, but also with a biogeography for larger forms at least. The molecular detection/characterization of halophilic protozoa is clearly a long way from saturated at both the clade level and the 'species level'.

MULTIPLE HEMOSPORIDIAN LINEAGES PARASITIZING A POPULATION OF MIGRATORY COLUMBIDS

Andrew Peters1,2, Shane R. Raidal1,2, Tania Areori3, Daniel Okena3, Heather Taitibe3, and Wallace Takendu4

1. Graham Centre for Agricultural Innovation, Wagga Wagga, NSW, Australia 2. School of Animal & Veterinary Sciences, Charles Sturt University, NSW, Australia 3. PNG Institute for Biological Research, Goroka, Papua New Guinea 4. Wildlife Conservation Society, Goroka, Papua New Guinea

Epidemiological theory suggests that migratory, colony-breeding tropical birds are likely to maintain increased abundance and diversity of hemoparasites. This is a result of life history events for these vertebrates that are likely to be of significance to the maintenance and transmission of other pathogenic infectious organisms. A study examining the distribution and phylogenetic identity of hemosporidians in pigeons and doves (columbids) across Australia and Papua New Guinea was carried out over four years (2009-2013). Blood smears of more than 500 individuals belonging to seventeen species of columbids with diverse ecological traits were examined for the presence of intraerythrocytic hemoparasites. PCR amplification specific for hemosporidian genes was performed on birds with observable hemoparasites. The densely sampled migratory, frugivorous pied imperial-pigeon (Ducula bicolor), was found to maintain three lineages of hemosporidans at relatively high prevalence, including a sp. related to H. columbae and two lineages of Parahaemoproteus spp. Such diversity was not found in sedentary columbid species, though these populations were not sampled as densely. This appears to support current theory regarding the association between life history traits and hemoparasitism, and further investigation into protozoal diversity in the ecologically diverse Australasian columbids may demonstrate a broader relationship between infection dynamics and behaviors such migration or colony breeding. GENOME-WIDE TRANSCRIPT PROFILING REVEALS THE COEVOLUTION OF PLASTID GENE SEQUENCES AND TRANSCRIPT PROCESSING PATHWAYS IN THE FUCOXANTHIN DINOFLAGELLATE VENEFICUM

Elisabeth Richardson1, Richard G. Dorrell1 and Christopher J. Howe1

1. Department of Biochemistry, University of Cambridge, Building O, Downing Site, Tennis Court Road, Cambridge, CB2 1QW UK

The fucoxanthin dinoflagellates are a group of algae that have undergone serial endosymbiosis, where the original peridinin-containing chloroplast has been replaced with a fucoxanthin-containing chloroplast derived from a haptophyte. The ancestral and replacement chloroplasts differ in genome structure and content. The peridinin dinoflagellates have extremely reduced chloroplast genomes located on minicircles. The chloroplasts of fucoxanthin dinoflagellates contain a single circular genome containing the majority of genes. However, this genome is extremely divergent to the chloroplast genomes of free-living , having undergone extensive gene loss, genome recombination, and fragmentation, with certain genes located on episomal elements. Peridinin dinoflagellates also have two RNA processing pathways, polyuridylylation and RNA editing, which have come to be applied to the genome of the replacement chloroplast in fucoxanthin dinoflagellates. We have created a genome-wide profile of RNA processing in the chloroplasts of the fucoxanthin dinoflagellate Karlodinium veneficum, and have demonstrated that addition of poly(U) tails and editing of transcripts are likely to constrain the phenotypic effects of rapid evolution of the chloroplast genome. We have also identified RNA processing events that have coevolved with the extremely divergent regions of the genome, including the first complete evidence for minicircles in a fucoxanthin chloroplast. This suggests that application of ancestral RNA processing pathways to the genome of the K. veneficum replacement chloroplast may be evolutionarily dynamic, responding to changes in the underlying genome sequence. Uncovering evolution in 3D: Architecture of the nuclear pore complex reveals conserved features of the eukaryotic endomembrane system across a billion years

Rout, Michael1; Obado, Samson1; Fernandez-Martinez, Javi1; Sampathkumar, Parthasarathy3; Sali, Andrej4; Kim, Seung Joong4; Chait, Brian1; Field, Mark2.

1. Rockefeller University, New York, NY, USA 2. Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, Scotland, United Kingdom 3. Department of Biochemistry, Albert Einstein College of Medicine, New York NY, USA 4. California Institute for Quantitative Biosciences, University of California, San Francisco, CA, USA

Distinctive internal membrane systems define major subcellular compartments in eukaryotic cells, most of which appear to have evolved autogenously through internalization, as suggested by the endomembrane hypothesis. Comparative genomic and phylogenetic analyses suggest that many of the components of these membrane systems evolved from common ancestors in an ancient “proto-eukaryote” by gene duplication and subsequent functional divergence. One of the most prominent examples of the evolutionary connections between internal membrane systems comes from the comparison of the architecture between different kinds of coated vesicles (CVs) and between CVs and nuclear pore complex (NPC). Composed of nucleoporins (nups), nuclear pore complexes (NPCs) mediate bi-directional trafficking between the nucleoplasm and cytoplasm across the double-membraned nuclear envelope, acting as a dynamic barrier to control access to the nucleus. We use an integrative approach to determine the structure and molecular organization of the entire NPC. We see an arrangement of coaxial rings forming an elaborate scaffold that defines a ~30 nm diameter passageway between the nucleus and cytoplasm. We have also found that there is modularity in the architecture of the NPC; moreover, the presence of shared components, fold types and arrangements, overall architecture and functions in membrane curvature are the key elements that support the idea that CVs and NPC are evolved from a common protocoatomer ancestor. Our work on the architecture of the NPC from the divergent eukaryote, , has revealed that despite divergent peripheral features, elaborate core structures of the NPC and even NE may have been established well prior to the radiation of the eukarya from a common ancestor. We are now mapping the morphology, connectivity and functionality of the nups and nup complexes constituting the NPC. One such study generated high-resolution data for the inner ring component, Nup192. Comparative modeling analyses indicated a structural similarity between Nup192 and -catenins, clathrins, and adaptins, and suggested a hypothesis whereby NPCs, vesicle coating and tethering complexes are descended from an elaboratekaryopherins, common β membrane-coating ancestral complex. RED ALGAE AS A MODEL FOR EARLY GENOMIC IMPACTS OF PARASITE EVOLUTION

Eric Salomaki1 & Chris Lane1

1. Dept. of Biological Sciences, University of Rhode Island, Kingston, RI, USA

Parasitism is a life strategy that has independently evolved countless times throughout the eukaryotic tree of life, however most parasitic lineages are distantly related from a free-living taxon making comparative studies difficult. Rhodophytes have an evolutionary relationship between parasites and their free-living hosts, where the parasite and host evolved from a recent common ancestor. This relationship provides an ideal framework to study the early genomic consequences as an organism shifts from a free-living to a parasitic life strategy. Additionally, red algal parasites do not maintain their own plastid and instead hijack and maintain a photosynthetically inactive plastid from the host. However the purpose of parasite maintaining a non-photosynthetic plastid remains unclear. In typical eukaryotic parasites non-essential genes are lost, as they rely on a host for energy and nutrition. Red algal parasites have never been examined at the genome level and mechanisms driving their lifestyle change are unknown. To gain insight into the evolution of parasitism we are conducting a genomic survey of a parasite and host pair from the Rhodomelaceae. Sequencing and annotation of the organellar genomes from the parasite Choreocolax polysiphoniae and its host Vertebrata lanosa are completed and nuclear genome sequencing and assembly is ongoing. Genomic consequences and the implications for the evolution of parasitism in red algae will be discussed. WHAT CAN TRANSCRIPTOME DATA TELL US ABOUT MIXOTROPHY IN A MARINE PLANKTONIC CILIATE? Luciana F. Santoferrara1, Stephanie Guida2, Huan Zhang1, George B. McManus1 1. Department of Marine Sciences, University of Connecticut, Groton, CT, USA 2. The National Center for Genome Resources, Santa Fe, NM, USA

In the context of MMETSP, we investigated the transcriptomes of two marine planktonic ciliates, the mixotrophic Strombidium rassoulzadegani and the heterotrophic Strombidinopsis sp., and their respective algal foods. Our aim was to characterize the transcriptomes of these contrasting ciliates and to identify genes potentially involved in mixotrophy. We obtained approximately 10,000 and 7,600 transcripts for S. rassoulzadegani and Strombidinopsis sp., respectively, and about half of them had significant hits (BLASTP, E-value < 10-6) against known sequences, mostly from model ciliates. Transcriptomes from both the and the heterotroph provided similar annotations for GO terms and KEGG pathways. Most of the identified genes were related to housekeeping activity and pathways such as the metabolism of carbohydrates, lipids, amino acids, and nucleotides. Although S. rassoulzadegani can keep and use chloroplasts from its prey, we did not find genes clearly linked to chloroplast maintenance or functioning in the transcriptome of this ciliate. Also, while chloroplasts are known sources of reactive oxygen species (ROS), we found evidence for the same antioxidant pathways in both ciliates. The only exception was one enzyme possibly linked to ascorbic acid recycling found exclusively in the mixotroph. Contrary to our expectations, we did not find qualitative differences in genes potentially related to mixotrophy. However, these transcriptomes will help to establish a basis for the evaluation of differential gene expression in oligotrichs and choreotrichs and experimental investigation of the costs and benefits of mixotrophy. NEW TAXONOMY OF UBIQUITOUS GENUS PARAPHYSOMONAS (CHRYSOPHYCEAE) USING LIGHT AND ELECTRON MICROSCOPY AND 18S rDNA SEQUENCES TO MAKE 26 NEW SPECIES AND A NEW GENUS, CLATHROMONAS.

Josephine M Scoble, and Tom Cavalier-Smith

Department of Zoology, University of Oxford. Oxford. UK.

The common scaly protist genus, Paraphysomonas, had become a repository for an excessively heterogeneous set of colourless scaly chrysophytes making it morphologically too diverse to constitute a single genus. Published sequence data, although including very few named species, suggested that some must have been misidentified, especially those assigned to the type species, P. vestita, (Stokes, 1885). We sampled soil, freshwater and marine environments and established 75 putatively Paraphysomonas clonal cultures and obtained 59 genetically distinct and highly divergent 18S rDNA sequences, and discovered that virtually all previous sequences were wrongly named. Light and electron microscopy data revealed that these sequences grouped into four morphologically distinct simple spine scale types that we describe as subgenera containing 26 new species; Paraphysomonas, Hebetomonas, Acrospina, Brevispina. All of our isolates, except one would have been identified as either P. vestita or P.imperforata. We make the genus more homogeneous by removing all species that do not have simple nail-like spine scale species and create a new genus for the basket-scaled species, Clathromonas n. gen. of which we cultured one representative. Other genera are being made for yet other scale types

HOW ANIMALS EMERGED? A GENOMICS AND CELL BIOLOGY PERSPECTIVE

Arnau Sebé-Pedrós1, Hiroshi Suga1,2, Guifré Torruella1, Alex de Mendoza & Iñaki Ruiz Trillo1,3,4

1. Institut de Biologia Evolutiva (CSIC-UPF), Barcelona, Spain. 2. Department of Life Sciences Faculty of Life and Environmental Science, Hiroshima Prefectural University, Hiroshima, Japan 3. Departament de Genètica, Universitat de Barcelona, Spain. 4. Institut de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.

How animals or metazoans emerged from their single-celled ancestors remains a major question in biology. Recent genome data from close unicellular relatives of Metazoa has shown that the unicellular ancestor that gave rise to Metazoa was genetically much more complex than previously thought. Thus, the unicellular ancestor of animals already had a good repertoire of genes involved in cell adhesion, cell signaling and transcriptional regulation. This suggests that co-option and an increase in gene regulation may have played an important role into the origin of animals. All this data provides insights both into the origin of animal multicellularity and the emergence of the different animal cell types. MORPHOLOGY AND ULTRASTRUCTURE OF VENTRIFISSURA SPP., A DEEPEST LINEAGE OF CERCOZOAN CLASS THECOFILOSEA.

Takashi Shiratori1, Ken-ichiro Ishida1

1. Graduate School of Life and Environmental Sciences, University of Tsukuba

Ventrifissura is a poorly studied genus of heterotrophic and gliding flagellates belonging to the phylum Cercozoa. Ventrifissura consists of two uncultured species that were described in Chantangsi and Leander (2010) based on light microscopy and SSU rDNA barcodes. Although phylogenetic position of Ventrifissura remain unclear, Howe et al. (2011) regards the genus as one of the basal groups of class Thecofilosea on the basis of the morphological affinities with other thecofilosean flagellates. Culture-based detailed morphological and ultrastructural investigations are needed to confirm the taxonomic position of Ventrifissura. We established two strains of Ventrifissura from marine samples in

Japan, and performed light and electron microscopic observations and a molecular phylogenetic analysis. Our microscopic observations showed that the cultures share several ultrastructural characteristics with other thecofilosean flagellates such as extracellular organic and extremely slender . On the basis of the morphological and ultrastructural information provided in this study, we will discuss the taxonomic position of

Ventrifissura and the character evolution of Cercozoan flagellates. PAPATRYPANOSOMA CONFUSUM – AN UNEXPECTED EARLY-BRANCHING TRYPANOSOMATID

Tomáš Skalický1,2, Pavel Flegontov1,3, Dagmar Jirsová1,4, Jan Votýpka1,5, Eva Dobáková1,6, Vyacheslav Yurchenko1,3 & Julius Lukeš1,2

1 Institute of Parasitology, Biology Centre, České Budějovice, Czech Republic 2 Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic 3 Life Science Research Centre, University of Ostrava, Ostrava, Czech Republic 4 University of Veterinary and Pharmaceutical Science, Brno, Czech Republic 5 Faculty of Science, Charles University, Prague, Czech Republic 6 Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia

Trypanosomatids are widespread and important parasites, with many species causing devastating diseases. Recently we have isolated a new species named Paratrypanosoma confusum from the gut of pipiens. From draft genome sequence data we have identified 114 protein genes shared between P. confusum, 15 other trypanosomatids, Bodo saltans, the early-branching kinetoplastid Perkinsela sp. and Naegleria gruberi. Single protein phylogenies together with analysis of concatenated alignments using complex phylogenetic models show that P. confusum branches at the base of the family Trypanosomatidae, between free-living B. saltans and obligatory parasitic trypanosomatids. P. confusum forms two different life stages in axenic culture: a motile promastigote-like stage and a sessile stage with entirely different and unique morphology. Under certain conditions, the motile stage transforms into the sessile stage, which is attached to the surface by a very sticky pad. We believe that comparative analyses of the P. confusum genome and transcriptomic data of the motile and sessile stages will provide important insight into the emergence of parasitism in trypanosomatids. GENE TRANSFER ACCOMPANYING THE SECONDARY ENDOSYMBIOSIS OF EUGLENID PLASTID

Petr Soukal 1, Štěpánka Hrdá1, Anna Karnkowska1, Miluše Hroudová2, Čestmir Vlček 2 and Vladimír Hampl 1

1. Department of Parasitology, Faculty of Science, Charles University in Prague 2. Institute of Molecular Genetics of the ASCR

Euglenozoa consist of four groups (, Diplonemida, Symbiontida and Euglenida) and use various trophic strategies including autotrophy. The autotrophic euglenids contain secondary green plastids derived from a prasinophyte green alga. The fact that the plastid is specific for one clade supports the plastid-late hypothesis postulating that the plastid was gained by the ancestor of the autotrophic clade but hypothetical scenario that the plastid acquisition happened much earlier cannot be ruled out. Because the process of organelle acquisition should be accompanied by transfer of genes from endosymbiont to host (EGT), the presence of such genes provides an indication of past endosymbiosis. We are analysing transcriptomes of primary osmotroph Rhabdomonas and autotrophic Eutreptiella for the amount of EGT derived genes. Using semiautomatic pipeline, we have selected transcripts of genes putatively related to algae. The selection was based on BLASTing against local database followed by maximum likelihood phylogenetic analysis of euglenid gene together with its homologues from the local database. The phylogenetic position of selected candidates was verified by re-analysis using enriched data set and bootstraping. In case of Rhabdomonas and Eutreptiella, 63 and 7508 candidates, respectively, were produced by the first round of selection which represents 0.9 % and 10 % of transcripts. In case of Rhabdomonas, only 11 genes were found robustly (BS>75%) related to algae after the re-analyses. Out of these, only a single gene was related specifically to green algae. The re-analysis of Eutreptiella candidates is in progress. The preliminary results support the plastid-late hypothesis for euglenid plastid origin. Was LECA a Landlubber?

Frederick W. Spiegel

Department of Biological Sciences, SCEN 601, University of Arkansas, Fayetteville, AR 72701, USA

The idea that living things “come from the sea” is an idea that often seems to be taken by biologists as common knowledge. Certainly, it is at least the case that most biologists assume that living things moved from aquatic environments (as defined from the human scale) onto the inhospitable land relatively late in biological history. The existence of terrestrial communities is often considered to have begun with the origin of the embryophytes. However, there is a lot of evidence for the existence of terrestrial microbial mat/crust communities going back well over 2.5GA. Thus, terrestrial communities were present adequately early for them to predate the origin of the Last Eukaryote Common Ancestor (LECA). Using discussion points with respect to the diaphoretickan lineage, Archaeplastida, and the amorphean lineage, Amoebozoa, I will build a case for considering the hypothesis that the last common ancestors of both these groups were members of the terrestrial microbial mat/crust communities. I will then extend the hypothesis to suggest that LECA, as well, was a terrestrial organism. EVOLUTION OF RHODOQUINONE BIOSYNTHESIS IN THE MITOCHONDRION RELATED ORGANELLES OF PROTISTS

Courtney W. Stairs, Laura Eme and Andrew J. Roger.

Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Canada.

Many protists that live in low-oxygen environments have independently evolved mitochondrion-related organelles (MROs). Curiously, many of these protists that encode only one mitochondrial respiratory complex (succinate dehydrogenase, CII) also encode a putative rhodoquinone (RQ) biosynthesis protein (RQUA). RQ is an electron-transporting molecule that participates in anaerobic fumarate reduction in photoheterotrophic bacteria (e.g., Rhodospirillium rubrum), and in the mitochondria of Euglena gracilis and parasitic helminths (e.g., Ascaris suum). Until the recent discovery of RQUA, the RQ biosynthesis pathway was a mystery. Here, we identified RquA homologs from protists with mitochondria (E. gracilis and Monosiga ovata) or MROs (Blastocystis sp., Mastigamoeba balamuthi, and Pygsuia biforma). Our phylogenetic analyses show that RQUA branches within the ubiquinone methyltransferase family and appears to have been transferred at least twice between eukaryotes and bacteria. Most of the protistan homologs have a predicted mitochondrial targeting signal and, using immunofluorescence confocal microscopy, we confirmed that RquA localized to the MRO of Pygsuia. To test if the protistan RquA homologs can function in RQ biosynthesis, we are establishing a heterologous complementation system in R. rubrum rqua mutants. Our findings allow us to hypothesize that the identification of RQUA in protists could be used as a proxy for predicting quinone composition and whether CII functions as a fumarate reductase in newly-discovered organisms. This work highlights how lateral gene transfer contributes to the metabolic remodeling of organelles in response to anaerobiosis forging ‘mosaic’ pathways integrating both ancestral (i.e., CII) and new (i.e., RQUA) features.

CAN MICROBES IN THE HINDGUT COMMUNITIES OF TERMITES AND WOOD-EATING COCKROACHES BE USED TO STUDY SPECIATION?

Vera Tai1 and Patrick J. Keeling1 1Department of Botany, University of British Columbia, Vancouver, BC, Canada

The speciation of eukaryotic microbes in natural communities is poorly understood in part because population level diversity and variation are rarely known. The microbial communities in the hindguts of sister species of the dampwood termite Zootermopsis and of the wood-eating cockroach Cryptocercus were examined for evidence of microbial speciation. These insects harbour a complex community of , oxymonads, bacteria, and archaea that have co-evolved with their hosts and can be used as model systems to examine the role of selection, adaptation, and genetic drift in the diversification of microorganisms. Morphology-based identifications and 18S rRNA sequences indicate that the same parabasalid species reside in sister species of Zootermopsis and of the Cryptocercus punctulatus species complex, but using high throughput sequencing of the internal transcribed spacer region, we show that the symbionts occurring in different host species are genetically distinct. We also show that the bacterial community is more variable in composition than the eukaryotes, indicating that the importance of factors regulating community structure, such as dispersal and niche specificity, for these two fundamental groups of microorganisms is different.

THE DISCOVERY OF NOVEL NUCLEOMORPH-BEARING ALGAE

Goro Tanifuji1, Chihiro Sarai2, Ryoma Kamikawa3, Kazuya Takahashi2, Takuro Nakayama4, Konosuke Morita5, Ken-Ichiro Ishida5, Tetsuo Hashimoto5, Mitsunori Iwataki6, Yuji Inagaki4

1 Faculty of life and environmental sciences, University of Tsukuba, 2Graduate School of Science and Engineering, Yamagata University, 3Graduate School of Human and Environmental studies, Kyoto University, 4Center of Computational Sciences, University of Tsukuba, 5Graduate School of Life and Environmental Sciences, University of Tsukuba, 6 Asian Natural Environmental Science Center, University of Tokyo

Endosymbiosis has been one of the major driving forces in eukaryotic evolution. Independent endosymbiotic events gave rise to diverse photosynthetic eukaryotes bearing different plastid origins. During these events, dynamic reorganizations of both host and endosymbiont genomes are necessary for integration into one cell. Nucleomorphs, the vestige nuclei found in cryptophytes and chlorarachniophytes, have been a model for investigation of gene/genome evolution in eukaryote-eukaryote endosymbioses (secondary endosymbioses). Although cryptophyte and chlorarachniophyte plastids were established through two separate endosymbioses, the nucleomorph genomes in both lineages show similar genomic features (e.g., gene contents). To explain the parallel trend between the independent nucleomorph genomes, it is attractive to hypothesize common driving forces on the endosymbiont genomes during secondary endosymbioses. Nevertheless, to infer the general aspects of endosymbiont genome evolution in secondary endosymbioses, additional nucleomorph genomes separated from those in cryptophytes and chlorarachniophytes, are necessary. Here, we report novel nucleomorphs in two green colored dinoflagellate strains for the first time in 30 years. TEM observation clearly showed that (1) their plastids are surrounded by four membranes, and (2) the double membrane-bound nuclei in the periplastidal compartment (PPC), which corresponds to the cytosol of the engulfed endosymbiont. Ribosome-like structures were also observed, but no mitochondrion was found in the PPC. While the two dinoflagellates are placed in distant positions in the host phylogeny, the plastid lineages are related to one particular green algal group, Pedinomonadales. Our data suggested that the nucleomorphs in these dinoflagellates are vestiges of green algal nuclei. In this presentation, the definition of nucleomorph will be discussed as well.

FREE-LIVING PREDATORY FLAGELLATES AS BASAL BRANCHES OF EUKARYOTIC SUPERGROUPS Denis V. Tikhonenkov1,2, Jan Janouškovec3, Fabien Burki1, Alexander P. Mylnikov2, and Patrick J. Keeling1 1. Canadian Institute for Advanced Research, Botany Department, University of British Columbia 2. Institute for Biology of Inland Waters, Russian Academy of Sciences 3. Department of Biology, San Diego State University

The evolutionary and ecological importance of predatory flagellates are too often overlooked. This is not only a gap in our understanding of microbial diversity, but also impacts how we interpret their better-studied relatives. Here we report the establishment of multiple cultures of free-living predatory flagellates belonging to several eukaryotic supergroups. The basal or intermediate evolutionary positions occupied by these organisms make them particularly important for elucidating the origin and evolution of some important and well-studied lineages and groups. A prime example of this problem is found in the alveolates. We reported the first cultivation and molecular analysis of several colponemid-like organisms representing two novel clades in molecular trees. The first, , is the sistergroup of all alveolates. The second lineage, Acavomonas, is the closest known sister to myzozoans (apicomplexans and dinoflagellates). We provide ultrastructural analysis and formal species descriptions for both new species, Colponema vietnamica n. sp. and Acavomonas peruviana n. gen. n. sp. Morphological characteristics concur with molecular data that both species are distinct members of alveolates. Based on ultrastructure and molecular phylogenies, which both provide concrete rationale for a taxonomic reclassification of Alveolata, we establish the new phyla Colponemidia nom. nov. for the genus Colponema and its close relatives, and Acavomonidia nom. nov. for the genus Acavomonas and its close relatives. The morphological data presented here suggests that colponemids are central to our understanding of early alveolate evolution, and suggest they also retain features of the common ancestor of all eukaryotes. MITOCHONDRIOMICS IN NAEGLERIA SPECIES

Anastasios D. Tsaousis1, Christopher N. Miller1, Eva Nývltová2, Emily Herman3, Charles Chiu4, Alexander L. Greninger4,5, Joel B. Dacks3, Jan Tachezy2, Mark van der Giezen6

1. Laboratory of Molecular and Evolutionary Parasitology, School of Biosciences, University of Kent, Canterbury, CT2 9EB, Kent, United Kingdom 2. Laboratory of Molecular and Biochemical Parasitology, Department of Parasitology, Faculty of Science, Charles University, Prague 2, 128 44, Czech Republic 3. Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada 4. UCSF-Abbott Viral Diagnostics and Discovery Center, University of California San Francisco, San Francisco, California, USA 5. Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, California, USA 6. Biocatalysis Centre, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK

Naegleria is a lineage of heterolobosean amoeboflagellate protist. The genus mainly consists of non-pathogenic free-living microorganisms found in water, mud or soil; N. gruberi is the most studied species from the genus. Its sister species, N. fowleri, is a facultative human parasite causing progressive amoebic meningoenecephalitis (PAM), a deadly disease if untreated. Recent analysis of N. gruberi’s genome demonstrated that it harbors canonical mitochondria bearing all enzymes involved in oxidative phosphorylation. Strikingly, the genome also encodes enzymes typically found in organisms with derived mitochondria (hydrogenosomes or mitosomes) including cytosolic malate dehydrogenase, cytochrome b5-containing nitrate reductase and most importantly [FeFe]-hydrogenase. Apart from these, protein alternatives to mitochondrial complexes I and IV have also been identified. Using a combination of bioinformatics and cell biology we have characterized components of the mitochondrial metabolism and physiology of N. gruberi. The predicted mitochondrial proteome of N. gruberi was also compared with the predicted mitochondrial proteome of N. fowleri in order to elucidate the adaptations of the pathogen versus the free-living organism at the mitochondrial level. Preliminarily data from these investigations will be discussed. This work is also going to provide traits found in the mitochondria of the last eukaryotic common ancestor in addition to clues for new possible therapeutic targets for treatment of PAM.

WHAT SHOULD BE THE MINIMUM REQUIREMENTS FOR ESTABLISHING NEW CILIATE SPECIES IN THE 21ST CENTURY?

Alan Warren

Department of Life Sciences, Natural History Museum, London, UK

The current requirements for establishing new species of heterotrophic protists are enshrined in the International Code of Zoological Nomenclature, and include the deposition in a collection of a specimen (or series of specimens) that constitutes the name-bearing type. In the case of ciliates and most other protists this is usually a microscope slide containing one or more individual organisms in which the name-bearing types are clearly indicated. For many years, when techniques for the examination of protists relied primarily on light microscopy, such specimens sufficed. However, with the advent of modern, especially molecular, techniques for investigating the taxonomy, systematics and biodiversity of protists, questions are increasingly asked as to the utility of microscope slide specimens alone as reference material. The International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC) was established in 2011 with joint USA–Chinese funding to promote research in the three dimensions of ciliate biodiversity: functional, genetic and taxonomic. At the forthcoming annual workshop of the IRCN-BC, 1-3 September 2014, Royal Holloway, University of London, UK, we will discuss what should be the minimum requirements for establishing new ciliate species in the 21st century (for further details see: http://ircn-bc.org/default.html). Topics will include: traditional specimens, morphological descriptions, DNA-barcoding, frozen cell and molecular collections, and preserved viable specimens. In this talk I will give a brief overview of each of these topics and how we plan to take the debate forwards. TRANSPOSABLE ELEMENTS AFFECT GENE EXPRESSION IN THE PARASITIC PROTIST TRICHOMONAS VAGINALIS

Sally Warring1, Martina Bradic1, Vivien Low1 and Jane Carlton1

1. Center for Genomics and Systems Biology, Department of Biology, New York University

Trichomonas vaginalis is the causative agent of , the most prevalent non-viral sexually transmitted infection world-wide. The parasite harbors an unusually large genome when compared to other parasitic protists (~160 Mb), partially due to the recent colonization and expansion of multiple transposable element (TE) families. These TEs account for at least one quarter of the T. vaginalis genome, the largest TE colonization observed in any parasitic protist. Additionally, these TE families appear to have been recently acquired by T. vaginalis, and many may still retain their transposition activity. To understand the biological consequences of harboring such a large load of active TEs in this asexual, haploid organism, we chose to investigate the effect of TE insertions on the expression of nearby T. vaginalis genes. We focused our investigations on one family of T. vaginalis TEs, the Tc1/mariner transposon family Tvmar1. We found that Tvmar1 insertions in or near T. vaginalis gene open reading frames are capable of ablating or decreasing the expression of these genes. This study provides the first example of TEs influencing gene expression in T. vaginalis. Further, our preliminary RNA-Seq data indicate that TE genes are not expressed routinely in T. vaginalis, suggesting that these repeats are targeted for silencing, possibly due to the negative consequences of transposition. Our results raise important questions as to the role TEs may be playing in shaping T. vaginalis gene expression and genome evolution, and the mechanisms employed by the parasite to counteract their effect. DIATOM-DERIVED CHLOROPLASTS IN DINOFLAGELLATES ORIGINATE FROM EIGHT DIFFERENT FREE-LIVING DIATOMS

Norico Yamada1, 4, Stuart D. Sym2, Horiguchi Takeo3

1. Graduate school of Science, Hokkaido University 2. Animal, Plant and Environmental Science Department, University of the Witwatersrand 3. Faculty of Science, Hokkaido University 4. JSPS research fellow

Most photosynthetic dinoflagellates typically possess red alga-derived chloroplasts, but some of them have chloroplasts derived from other microalgae. Eleven species of dinoflagellates are known to harbour diatom-derived chloroplasts and these dinoflagellates are called dinotoms. Phylogenetic analyses based on 18S rDNA indicate all the host dinoflagellates are monophyletic, while the endosymbiotic diatoms in dinotoms are derived from three species of free-living diatoms, belonging to the genera Discostella, Chaetoceros and Nitzschia. Recently we successfully established cultures of seven dinotoms, including three novel species. In addition to determining the phylogenetic position of the host dinoflagellates, those of the endosymbiont diatoms relative to the free-living diatoms were investigated based on the plastid-encoded rbcL gene and the 18S rDNA of the endosymbiont nucleus. As before, three endosymbiotic genera of diatoms were recognized, but, surprisingly, we found that they represented eight, rather than three, discrete species. Contrary to previous belief, the finding here is that most of the Nitzschia-type dinotoms possess a different species of Nitzschia, i.e. at least six species were recognized; Durinskia baltica from the United States possesses Nitzschia palea, D. capensis has a N. draveiliensis-like endosymbiont and Durinskia sp. nov. has N. cf. fonticola. The Nitzschia-type endosymbionts of D. baltica from Japan, various Galeidinium spp. and Kryptoperidinium foliaceum were recovered in three distinct clades with high bootstrap, although their exact identities could not be established. Our study suggests a complex evolutionary scenario of endosymbiont acquisition in dinotoms. COMPARATIVE ULTRASTRUCTURE OF FORNICATE EXCAVATES, INCLUDING A DESCRIPTION OF A NOVEL LINEAGE (CL2)

Naoji Yubuki1, Sam Huang1 and Brian S. Leander1

1. Departments of Botany and Zoology, University of British Columbia

The Fornicata (Excavata) is a group of microbial eukaryotes consisting of free-living lineages (e.g., ) and parasitic lineages (e.g. and ) that share several molecular and ultrastructural characteristics. Carpediemonas-like organisms (CLOs) are free-living lineages that diverged early within the Fornicata, making them important for inferring the early evolutionary history of the group. Molecular phylogenetic analyses of small subunit (SSU) rDNA sequences from free-living fornicates, including sequences from environmental PCR surveys, demonstrate that CLOs represent six different lineages. Representatives from five of these lineages have been studied at the ultrastractural level: Carpediemonas membranifera, Dysnectes brevis, Hicanonectes teleskopos, Ergobibamus cyprinoides and Kipferlia bialata. The sixth lineage has been labeled “CL2” but has yet to be described with ultrastructural data. Improved understanding of CL2 is expected to help elucidate character evolution within the Fornicata and beyond, including traits associated with highly modified mitochondria. Therefore, we isolated, cultivated and comprehensively characterized CL2 (NY0171) with TEM in order to understand the ultrastructural traits in this lineage, especially the organization of the microtubular (e.g., the flagellar apparatus). CL2 has several distinctive features, including one vane on the posterior flagellum and an ER network extending around the periphery of the cell. CL2 and its closest relative, Hicanonectes, shared several features, including a large mictrotubular root (R3) extending from the anterior basal body, a rotational mode of swimming and a curved feeding groove. The combination of ultrastructural traits in CL2 was distinctive among CLOs and provided additional insights into the evolutionary history of the Fornicata. GENOME ANALYSIS OF AN ANAEROBIC PROTIST MASTIGAMOEBA BALAMUTHI

Vojtech Zarsky1, Jan Paces2, Cestmir Vlcek2, Vladimir Klimes1 and Jan Tachezy1

1. Faculty of Science, Charles University in Prague 2. Institute of Molecular Genetics, Academy of Sciences of the Czech Republic

Mastigamoeba balamuthi is a free-living amoeboflagellate found in anoxic fresh waters and mud. We exploit genomics and transcriptomics data to assess the many peculiar features that distinguish it from its parasitic relatives (Entamoeba) and aerobic free-living (Dictyostelia), while we also observe similar/convergent patterns with unrelated anaerobic protists. We further focus on the description of eukaryotic organelles and predict an unusual metabolic compartmentalization.

The work of VZ is supported by the Grant Agency of Charles University in Prague (573112).