Aleš Horák

Eukaryotic plankton diversity in the sunlit ocean

Marine plankton support global biological and geochemical processes. Surveys of their biodiversity have hitherto been geographically restricted and have not accounted for the full range of plankton size. We assessed eukaryotic diversity from 334 size-fractionated photic-zone plankton communities collected across tropical and temperate oceans during the circumglobal Tara Oceans expedition. We analyzed 18S ribosomal DNA sequences across the intermediate plankton-size spectrum from the smallest unicellular eukaryotes (protists, >0.8 micrometers) to small animals of a few millimeters. Eukaryotic ribosomal diversity saturated at ~150,000 operational taxonomic units, about one-third of which could not be assigned to known eukaryotic groups. Diversity emerged at all taxonomic levels, both within the groups comprising the ~11,200 cataloged morphospecies of eukaryotic plankton and among twice as many other deep-branching lineages of unappreciated importance in plankton ecology studies. Most eukaryotic plankton biodiversity belonged to heterotrophic protistan groups, particularly those known to be parasites or symbiotic hosts.

A common red algal origin of the apicomplexan, dinoflagellate, and heterokont plastids

The discovery of a nonphotosynthetic plastid in malaria and other apicomplexan parasites has sparked a contentious debate about its evolutionary origin. Molecular data have led to conflicting conclusions supporting either its green algal origin or red algal origin, perhaps in common with the plastid of related dinoflagellates. This distinction is critical to our understanding of apicomplexan evolution and the evolutionary history of endosymbiosis and photosynthesis; however, the two plastids are nearly impossible to compare due to their nonoverlapping information content. Here we describe the complete plastid genome sequences and plastid-associated data from two independent photosynthetic lineages represented by Chromera velia and an undescribed alga CCMP3155 that we show are closely related to apicomplexans. These plastids contain a suite of features retained in either apicomplexan (four plastid membranes, the ribosomal superoperon, conserved gene order) or dinoflagellate plastids (form II Rubisco acquired by horizontal transfer, transcript polyuridylylation, thylakoids stacked in triplets) and encode a full collective complement of their reduced gene sets. Together with whole plastid genome phylogenies, these characteristics provide multiple lines of evidence that the extant plastids of apicomplexans and dinoflagellates were inherited by linear descent from a common red algal endosymbiont. Our phylogenetic analyses also support their close relationship to plastids of heterokont algae, indicating they all derive from the same endosymbiosis. Altogether, these findings support a relatively simple path of linear descent for the evolution of photosynthesis in a large proportion of algae and emphasize plastid loss in several lineages (e.g., ciliates, Cryptosporidium, and Phytophthora).

Evolution and metabolic significance of the urea cycle in photosynthetic diatoms.

Diatoms dominate the biomass of phytoplankton in nutrient-rich conditions and form the basis of some of the world’s most productive marine food webs. The diatom nuclear genome contains genes with bacterial and plastid origins as well as genes of the secondary endosymbiotic host (the exosymbiont), yet little is known about the relative contribution of each gene group to diatom metabolism. Here we show that the exosymbiont-derived ornithine-urea cycle, which is similar to that of metazoans but is absent in green algae and plants, facilitates rapid recovery from prolonged nitrogen limitation. RNA-interference-mediated knockdown of a mitochondrial carbamoyl phosphate synthase impairs the response of nitrogen-limited diatoms to nitrogen addition. Metabolomic analyses indicate that intermediates in the ornithine-urea cycle are particularly depleted and that both the tricarboxylic acid cycle and the glutamine synthetase/glutamate synthase cycles are linked directly with the ornithine-urea cycle. Several other depleted metabolites are generated from ornithine-urea cycle intermediates by the products of genes laterally acquired from bacteria. This metabolic coupling of bacterial- and exosymbiont-derived proteins seems to be fundamental to diatom physiology because the compounds affected include the major diatom osmolyte proline and the precursors for long-chain polyamines required for silica precipitation during cell wall formation. So far, the ornithine-urea cycle is only known for its essential role in the removal of fixed nitrogen in metazoans. In diatoms, this cycle serves as a distribution and repackaging hub for inorganic carbon and nitrogen and contributes significantly to the metabolic response of diatoms to episodic nitrogen availability. The diatom ornithine-urea cycle therefore represents a key pathway for anaplerotic carbon fixation into nitrogenous compounds that are essential for diatom growth and for the contribution of diatoms to marine productivity.

Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites

The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga. DOI: http://dx.doi.org/10.7554/eLife.06974.001

Updating algal evolutionary relationships through plastid genome sequencing: did alveolate plastids emerge through endosymbiosis of an ochrophyte?

Algae with secondary plastids of a red algal origin, such as ochrophytes (photosynthetic stramenopiles), are diverse and ecologically important, yet their evolutionary history remains controversial. We sequenced plastid genomes of two ochrophytes, Ochromonas sp. CCMP1393 (Chrysophyceae) and Trachydiscus minutus (Eustigmatophyceae). A shared split of the clpC gene as well as phylogenomic analyses of concatenated protein sequences demonstrated that chrysophytes and eustigmatophytes form a clade, the Limnista, exhibiting an unexpectedly elevated rate of plastid gene evolution. Our analyses also indicate that the root of the ochrophyte phylogeny falls between the recently redefined Khakista and Phaeista assemblages. Taking advantage of the expanded sampling of plastid genome sequences, we revisited the phylogenetic position of the plastid of Vitrella brassicaformis, a member of Alveolata with the least derived plastid genome known for the whole group. The results varied depending on the dataset and phylogenetic method employed, but suggested that the Vitrella plastids emerged from a deep ochrophyte lineage rather than being derived vertically from a hypothetical plastid-bearing common ancestor of alveolates and stramenopiles. Thus, we hypothesize that the plastid in Vitrella, and potentially in other alveolates, may have been acquired by an endosymbiosis of an early ochrophyte.

Cosmopolitan distribution of a trypanosomatid Leptomonas pyrrhocoris.

A trypanosomatid species, designated as Typing Unit 1 (TU1) by sequences of SL RNA gene repeats, has been found in the intestine of pyrrhocorids (Insecta: Heteroptera) in Europe, Mediterranean, Central America and some parts of Asia and Africa. Phylogenetic analysis of the SL repeat sequences has shown that the isolates group in the tree according to their geographic origin. The maximal sequence divergence was observed in parasites from Neotropics suggesting the origin within and subsequent migrations from this region. The global distribution of the parasite could have been facilitated by ubiquity of its hosts that include several genera of the family Pyrrhocoridae. In Europe the TU1 flagellates frequently occur in Pyrrhocoris apterus, the host of Leptomonas pyrrhocorisZotta, 1912, a species that had been insufficiently defined by host and light microscopy level morphology. Herein, the Zottas species description has been amended to include the TU1 SL RNA repeat, SSU rRNA, glycosomal GAPDH gene sequences, as well as ultrastructure. In addition, Leptomonas scantii n. sp. with an overlapping host range has been described. Moreover, 10 typing units of trypanosomatids found in the pyrrhocorid hosts demonstrate the extent of variability of trypanosomatids occurring in one host family.

Novel Insights into the Genetic Diversity of Balantidium and Balantidium-like Cyst-forming Ciliates

Balantidiasis is considered a neglected zoonotic disease with pigs serving as reservoir hosts. However, Balantidium coli has been recorded in many other mammalian species, including primates. Here, we evaluated the genetic diversity of B. coli in non-human primates using two gene markers (SSrDNA and ITS1-5.8SDNA-ITS2). We analyzed 49 isolates of ciliates from fecal samples originating from 11 species of captive and wild primates, domestic pigs and wild boar. The phylogenetic trees were computed using Bayesian inference and Maximum likelihood. Balantidium entozoon from edible frog and Buxtonella sulcata from cattle were included in the analyses as the closest relatives of B. coli, as well as reference sequences of vestibuliferids. The SSrDNA tree showed the same phylogenetic diversification of B. coli at genus level as the tree constructed based on the ITS region. Based on the polymorphism of SSrDNA sequences, the type species of the genus, namely B. entozoon, appeared to be phylogenetically distinct from B. coli. Thus, we propose a new genus Neobalantidium for the homeothermic clade. Moreover, several isolates from both captive and wild primates (excluding great apes) clustered with B. sulcata with high support, suggesting the existence of a new species within this genus. The cysts of Buxtonella and Neobalantidium are morphologically indistinguishable and the presence of Buxtonella-like ciliates in primates opens the question about possible occurrence of these pathogens in humans.

The phylogeny of diphyllobothriid tapeworms (Cestoda: Pseudophyllidea) based on ITS-2 rDNA sequences

Phylogenetic analysis of sequences of the ITS-2 rRNA genes of 20 samples of pseudophyllidean cestodes of the family Diphyllobothriidae (Ligula, Digramma, Diphyllobothrium, and Schistocephalus) from different hosts and geographical regions revealed that: (1) the inclusion of ligulids (previously family Ligulidae) to the Diphyllobothriidae is correct; (2) Schistocephalus appears as the most basal taxon of the Diphyllobothriidae, well separated from Ligula and Digramma, thus supporting the validity of Schistocephalinae Dubinina, 1962; (3) Digramma belonged with samples of Ligula, thus suggesting its invalidity as a genus; and (4) isolates of Ligula, presumably belonging to Ligula intestinalis, are paraphyletic, indicating that this species may represent a complex of separate taxa. Our results indicate the necessity for a taxonomic revision of the family Diphyllobothriidae.

Environmental distribution of coral-associated relatives of apicomplexan parasites.

A lineage of plastid-bearing eukaryotic microbes that is closely related to apicomplexan parasites was recently found in a specific association with coral reefs (apicomplexan-related lineage-V, or ARL-V). Here, we address the possible nature of this association using plastid ‘contamination’ in fine-scale bacterial sequence surveys. In a transect between corals and associated macroalgae, ARL-V is specifically associated with the coral, in contrast to all microalgal types (including diatoms, haptophytes, pelagophytes and photosynthetic apicomplexan relatives, Chromera and Vitrella), which are associated with macroalgae. ARL-V is associated with at least 20 species of symbiotic corals through extended time periods and large geographic distances. It is significantly enriched in healthy coral tissue and shallow reef depths. Altogether, the evidence points to a specific relationship between ARL-V and corals, and is suggestive of symbiosis, perhaps based on photosynthesis.

Description of Two Species of Early Branching Dinoflagellates, Psammosa pacifica n. g., n. sp. and P. atlantica n. sp

In alveolate evolution, dinoflagellates have developed many unique features, including the cell that has epicone and hypocone, the undulating transverse flagellum. However, it remains unclear how these features evolved. The early branching dinoflagellates so far investigated such as Hematodinium, Amoebophrya and Oxyrrhis marina differ in many ways from of core dinoflagellates, or dinokaryotes. Except those handful of well studied taxa, the vast majority of early branching dinoflagellates are known only by environmental sequences, and remain enigmatic. In this study we describe two new species of the early branching dinoflagellates, Psammosa pacifica n. g., n. sp. and P. atlantica n. sp. from marine intertidal sandy beach. Molecular phylogeny of the small subunit (SSU) ribosomal RNA and Hsp90 gene places Psammosa spp. as an early branch among the dinoflagellates. Morphologically (1) they lack the typical dinoflagellate epicone–hypocone structure, and (2) undulation in either flagella. Instead they display a mosaïc of dinokaryotes traits, i.e. (3) presence of bi-partite trychocysts; Oxyrrhis marina–like traits, i.e. (4) presence of flagellar hairs, (5) presence of two-dimensional cobweb scales ornamenting both flagella (6) transversal cell division; a trait shared with some syndineansand Parvilucifera spp. i.e. (7) a nucleus with a conspicuous nucleolus and condensed chromatin distributed beneath the nuclear envelope; as well as Perkinsus marinus -like features i.e. (8) separate ventral grooves where flagella emerge and (9) lacking dinoflagellate-type undulating flagellum. Notably Psammosa retains an apical complex structure, which is shared between perkinsids, colpodellids, chromerids and apicomplexans, but is not found in dinokaryotic dinoflagellates.