NEXT GENERATION SEQUENCING OF PROTISTS 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 bacteria to process hydrocarbon derivates in the tailings ponds, very little is
known about the community of eukaryotic microorganisms 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
Archaea 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 DINOFLAGELLATES
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 Peridinium 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
protist 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 organelles, 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 organelle-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 eukaryotes, 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 Naegleria 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 Plant Biology, Michigan State University
Euglenophytes obtained their chloroplast from (a) secondary endosymbiotic
event(s) involving a heterotrophic euglenid 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 chloroplasts 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 euglenids.
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 flagellates such as the
ancyromonads, mantamonads, and collodictyonids. Previous works have variously proposed that
they are somehow related to the Amoebozoa or Obazoa (i.e., Opisthokonta, Breviatea, and
Apusomonadida). They have further been placed into the recently proposed ‘megagroup’
Amorphea. 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 Ancyromonas, Fabomonas,
Mantamonas, and Diphylleia as well as the small thecate amoeba 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”
flagellate Malawimonas, 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 + Collodictyon, 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 ANIMAL
EVOLUTION
Nicholas J. Butterfield1
1. Department of Earth Sciences, University of Cambridge
Animals are a monophyletic group of multicellular heterotrophic opisthokonts
which dominate and define the modern biosphere. The oldest direct evidence for
animals 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 coevolutionary 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: Dientamoeba fragilis,
Pentatrichomonas hominis, Trichomonas 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
ENDOSYMBIONT
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 Perkinsela. 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-eukaryote endosymbiosis that does not involve
photosynthesis as a possible selective factor in the persistence of the endosymbiont.
Dyková I., Fiala I., Lom J., Lukeš J. (2003). Perkinsiella amoebae-like endosymbionts 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:
Tubulinea) 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
mitochondrion-related organelles, presumably hydrogenosomes, closely associated with
symbiotic prokaryotes, 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 bikont 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 ciliates, 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
PLASTID 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 Rhizaria.
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 Algae
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 plants (embryophytes) dominate the terrestrial environment, but are
phylogenetically a lineage of charophyte green algae. 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 ethylenesignaling 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
1
Department 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 (pseudopodia), 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 fermentation 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 domain 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 (EXCAVATA) 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
Institute of Parasitology, Biology Centre ASCR, České Budějovice, Czech Republic
Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic
3
Station Biologique de Roscoff, Roscoff, France
4
Genoscope, CEA, Évry, France
1
2
Diplonemea remains an obscure group of Euglenozoa (Excavata), represented by few benthic
bacteriovorous or parasitic species living in marine or freshwater habitats (Diplonema, 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 Dinophyceae 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 510x 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 EUGLENA
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
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 Microbiology of the Czech Academy of Sciences, Třeboň, Czech Republic
5
University of South Bohemia, Faculty of Science, České Budějovice, Czech Republic
1
Euglenida comprise a diversity of species living in various environmental niches. While
Euglena gracilis 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 plastidtargeted 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 Trypanosoma 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 plastids 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 MARINE PROTISTS 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 Alveolate 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 ciliate Nyctotherus ovalis, and the stramenopile Blastocystis hominis. In each case,
the HPM retains mtDNA, a partial TCA cycle, and an incomplete ETC that lacks CIIICV, 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
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
2
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 OPISTHOKONT 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 metamonad
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
NAEGLERIA FOWLERI
Emily K. Herman1, Alex L. Greninger2, Govinda S. Visvesvara3, Francine MarcianoCabral4, 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 (Cercozoa, 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 model organism
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 heterotetramerprotocoatomer coat to its multiple manifestations in modern organisms.
WHENCE THE APICOPLAST? PHYLOGENETIC ANALYSES OF
PHOTOSYNTHETIC GENES GIVE US A HINT
Aleš Horák1, Heather J. Esson1, Petra Dufková1 and Miroslav Oborník1,2
Institute of Parasitology, Academy of Sciences of the Czech Republic. Branišovská 31, České
Budějovice, Czech Republic
2
Faculty of Science,University of South Bohemia. Branišovská 31, České Budějovice, Czech
Republic
1
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 red algae. Interestingly, our
analysis recovered a sister relationship between the plastids of chromerids (comprised of
Chromera velia and Vitrella brassicaformis) 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 PLASMODIUM 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 posttranscriptional 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 ZOOSPORE SETTLEMENT, WHICH CHARACTERIZED
THE LABYRINTHULOMYCETES (STRAMENOPILES)
Izumi IWATA1 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, contractile vacuole, 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 bilateria 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 CHOANOFLAGELLATES 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 flagellum 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 lorica – Acanthoecida (loricates) and Craspedida (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 Salpingoecidae 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 taxonomy 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 MESODINIUM 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 KARENIA 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 Protistology, 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
‘haptophyte-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
(dinoflagellate) 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 Toxoplasma gondii 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
oxymonads 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 taxonrich 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,
Archaeplastida 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 glaucophytes 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.
C ONT R A ST I NG OUT C OM E S OF T H E E V OL UT I ONA R Y T R A NSI T I ON
T O PA R A SI T I SM : M I K R OC Y T OS A ND H E L I C OSPOR I DI UM
Patr ick K eeling 1, J ean-F r ançois Pomber t 1, F abien B ur ki 1, C hr is L ane 2, C athyr n A bbott 3,
Dr ion B oucias 4
1
Botany Department, University of British Columbia,Vancouver, BC, V6T 1Z4, Canada
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
2
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 “mitosome”-like organelle functionally restricted to ironsulfur cluster assembly, and so evolved in parallel with mitosomes in other lineages (e.g.
microsporidia). 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, Kingdom 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 Apicomplexa, 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 micronemes (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 (rhoptries 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
1
Department 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 microinvertebrate 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.
Mesodinium rubrum 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 Chlorophyta. 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 microtubules. 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 extrusome with a nonhomologous 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 MITOCHONDRIONRELATED ORGANELLES OF A FREE-LIVING JAKOBID
Leger, ML.1, Eme, L1., Hug, LA1,2. and Roger, AJ1.
1
Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of
Biochemistry and Molecular Biology, Dalhousie University, Halifax, Canada.
2
Present 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 parabasalid Trichomonas vaginalis.
Here, we describe the MRO of a free-living anaerobic jakobid excavate, Andalucia
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
1
University 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. Sponges (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 FR29682, 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 HighThroughput 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 subcommunity 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
1
Unité 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 wellknown. 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 454pyrosequencing 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, Apusozoa) 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. POSTCILIODESMATOPHORA) and on morphogenetic patterns (e.g. Lamellicorticata). There are a
significant number of phylum-level analyses of the ciliated protozoa 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 INTRAMACRONUCLEATA; 2) the
Lamellicorticata to include the Armophorea and Litostomatea; 3) the SAL clade to
include the Spirotrichea and Lamellicorticata; 4) the removal of Protocruzia from the
Spirotrichea as incertae sedis 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 nonconserved 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 Symbiodinium 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 Chilodonella uncinata 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 macronucleus of the ciliate Chilodonella uncinata (Cl:
Phyllopharyngea) provides a model to assess the impact of nuclear architecture on
patterns of gene expression and genome evolution. The influence of of nuclear
architecture on gene expression is well documented in plants and animals where variation
in chromosome 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
chromosomes 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 MALARIA 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 (APHELIDA, OPISTHOSPORIDIA) 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
1
Unité d’Ecologie, Systématique et Evolution, UMR CNRS 8079, Université Paris-Sud. 91405
Orsay cedex, France
2
Zoological Institute, Russian Academy of Sciences, St. Petersburg 199034, Russian Federation
3
St. Petersburg State University, St. Petersburg 199034, Russian Federation
4
Institut 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 (Rozellida) and the highly
derived Microsporidia, they have recently been reclassified as the Opisthosporidia, which
constitutes the sister group to the (true) fungi within the Holomycota. 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 zoospores are amoeboflagellated, have
tubular/lamellar mitochondrial cristae, a metazoan type of centrosome, and closed orthomitosis
with intranuclear spindle. These features together with trophont phagocytosis 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
Nephromyces 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+Coccidia), and highlighted the significant intra-clade
divergence among these apicoplasts. 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 NUCLEOMORPH 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.
Nucleomorphs 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 TRIMASTIX 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 hydrogenosome.
Comparative genetics on evolution of microneme protein in
Myzozoa
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 Oxyrrhis marina), the
perkinsids (Perkinsus marinus), and the colpodellids (Voromonas 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
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
2
Despite extensive adoption of the PCR-based methodologies in microbiology, culturebased 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 Breviata (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 alveolates. 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 Haemoproteus 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 KARLODINIUM 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 haptophytes, 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, Trypanosoma brucei, 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
karyopherins, β-catenins, clathrins, and adaptins, and suggested a hypothesis
whereby NPCs, vesicle coating and tethering complexes are descended from an
elaborate common 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 oligotrich Strombidium rassoulzadegani and the heterotrophic
choreotrich 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
mixotroph 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 theca and extremely slender extrusomes. 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
Institute of Parasitology, Biology Centre, České Budějovice, Czech Republic
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
1
2
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 Culex 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 (Kinetoplastida, 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
1
Department 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
parabasalids, 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, Colponema, 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 trichomoniasis, 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., Carpediemonas) and parasitic lineages (e.g. Giardia and Retortamonas) 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
cytoskeleton (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 amoebas
(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).