Microbiomes of microscopic marine invertebrates do not reveal signatures of phylosymbiosis.

Animals and microorganisms often establish close ecological relationships. However, much of our knowledge about animal microbiomes comes from two deeply studied groups: vertebrates and arthropods. To understand interactions on a broader scale of diversity, we characterized the bacterial microbiomes of close to 1,000 microscopic marine invertebrates from 21 phyla, spanning most of the remaining tree of metazoans. Samples were collected from five temperate and tropical locations covering three marine habitats (sediment, water column and intertidal macroalgae) and bacterial microbiomes were characterized using 16S ribosomal RNA gene sequencing. Our data show that, despite their size, these animals harbour bacterial communities that differ from those in the surrounding environment. Distantly related but coexisting invertebrates tend to share many of the same bacteria, suggesting that guilds of microorganisms preferentially associated with animals, but not tied to any specific host lineage, are the main drivers of the ecological relationship. Host identity is a minor factor shaping these microbiomes, which do not show the same correlation with host phylogeny, or 'phylosymbiosis', observed in many large animals. Hence, the current debate on the varying strength of phylosymbiosis within selected lineages should be reframed to account for the possibility that such a pattern might be the exception rather than the rule.

Multigene phylogenetics of euglenids based on single-cell transcriptomics of diverse phagotrophs.

Euglenids are a well-known group of single-celled eukaryotes, with phototrophic, osmotrophic and phagotrophic members. Phagotrophs represent most of the phylogenetic diversity of euglenids, and gave rise to the phototrophs and osmotrophs, but their evolutionary relationships are poorly understood. Symbiontids, in contrast, are anaerobes that are alternatively inferred to be derived euglenids, or a separate euglenozoan group. Most phylogenetic studies of euglenids have examined the SSU rDNA only, which is often highly divergent. Also, many phagotrophic euglenids (and symbiontids) are uncultured, restricting collection of other molecular data. We generated transcriptome data for 28 taxa, mostly using a single-cell approach, and conducted the first multigene phylogenetic analyses of euglenids to include phagotrophs and symbiontids. Euglenids are recovered as monophyletic, with symbiontids forming an independent branch within Euglenozoa. Spirocuta, the clade of flexible euglenids that contains both the phototrophs (Euglenophyceae) and osmotrophs (Aphagea), is robustly resolved, with the ploeotid Olkasia as its sister group, forming the new taxon Olkaspira. Ploeotids are paraphyletic, although Ploeotiidae (represented by Ploeotia spp.), Lentomonas, and Keelungia form a robust clade (new taxon Alistosa). Petalomonadida branches robustly as sister to other euglenids in outgroup-rooted analyses. Within Spirocuta, Euglenophyceae is a robust clade that includes Rapaza, and Anisonemia is a well-supported monophyletic group containing Anisonemidae (Anisonema and Dinema spp.), 'Heteronema II' (represented by H. vittatum), and a clade of Neometanema plus Aphagea. Among 'peranemid' phagotrophs, Chasmostoma branches with included Urceolus, and Peranema with the undescribed 'Jenningsia II', while other relationships are weakly supported and consequently the closest sister group to Euglenophyceae remains unresolved. Our results are inconsistent with recent inferences that Entosiphon is the evolutionarily pivotal sister either to other euglenids, or to Spirocuta. At least three transitions between posterior and anterior flagellar gliding occurred in euglenids, with the phylogenetic positions and directions of those transitions remaining ambiguous.

Microsporidia - Emergent Pathogens in the Global Food Chain.

Intensification of food production has the potential to drive increased disease prevalence in food plants and animals. Microsporidia are diversely distributed, opportunistic, and density-dependent parasites infecting hosts from almost all known animal taxa. They are frequent in highly managed aquatic and terrestrial hosts, many of which are vulnerable to epizootics, and all of which are crucial for the stability of the animal-human food chain. Mass rearing and changes in global climate may exacerbate disease and more efficient transmission of parasites in stressed or immune-deficient hosts. Further, human microsporidiosis appears to be adventitious and primarily associated with an increasing community of immune-deficient individuals. Taken together, strong evidence exists for an increasing prevalence of microsporidiosis in animals and humans, and for sharing of pathogens across hosts and biomes.

The origins of malaria: there are more things in heaven and earth .

SUMMARY Malaria remains one of the most significant global public health burdens, with nearly half of the world's population at risk of infection. Malaria is not however a monolithic disease - it can be caused by multiple different parasite species of the Plasmodium genus, each of which can induce different symptoms and pathology, and which pose quite different challenges for control. Furthermore, malaria is in no way restricted to humans. There are Plasmodium species that have adapted to infect most warm-blooded vertebrate species, and the genus as a whole is both highly successful and highly diverse. How, where and when human malaria parasites originated from within this diversity has long been a subject of fascination and sometimes also controversy. The past decade has seen the publication of a number of important discoveries about malaria parasite origins, all based on the application of molecular diagnostic tools to new sources of samples. This review summarizes some of those recent discoveries and discusses their implication for our current understanding of the origin and evolution of the Plasmodium genus. The nature of these discoveries and the manner in which they are made are then used to lay out a series of opportunities and challenges for the next wave of parasite hunters.

Molecular phylogeny and surface morphology of marine aseptate gregarines (Apicomplexa): Selenidium spp. and Lecudina spp.

Many aseptate gregarines from marine invertebrate hosts are thought to have retained several plesiomorphic characteristics and are instrumental in understanding the early evolution of intracellular parasitism in apicomplexans and the phylogenetic position of cryptosporidians. We sequenced the small-subunit (SSU) ribosomal RNA genes from 2 archigregarines, Selenidium terebellae and Selenidium vivax, and 2 morphotypes of the marine eugregarine Lecudina polymorpha. We also used scanning electron microscopy to investigate the surface morphology of trophozoites from Lecudina tuzetae, Monocystis agilis, the 2 species of Selenidium, and the 2 morphotypes of L. polymorpha. The SSU ribosomal DNA sequences from S. vivax and L. polymorpha had long branch lengths characteristic of other gregarine sequences. However, the sequence from S. terebellae was not exceptionally divergent and consistently emerged as 1 of the earliest 'true' gregarines in phylogenetic analyses. Statistical support for the sister relationship between Cryptosporidium spp. and gregarines was significantly bolstered in analyses including the sequence from S. terebellae but excluding the longest branches in the alignment. Eugregarines formed a monophyletic group with the neogregarine Ophryocystis, suggesting that trophozoites with elaborate cortex folds and gliding motility evolved only once. The trophozoites from the 2 species of Selenidium shared novel transverse striations but differed from one another in overall cell morphologies and writhing behavior.

Diplonemid glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and prokaryote-to-eukaryote lateral gene transfer.

Lateral gene transfer refers to the movement of genetic information from one genome to another, and the integration of that foreign DNA into its new genetic environment. There are currently only a few well-supported cases of prokaryote-to-eukaryote transfer known that do not involve mitochondria or plastids, but it is not clear whether this reflects a lack of such transfer events, or poor sampling of diverse eukaryotes. One gene where this process is apparently active is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), where lateral transfer has been implicated in the origin of euglenoid and kinetoplastid genes. We have characterised GAPDH genes from diplonemids, heterotrophic flagellates that are closely related to kinetoplastids and euglenoids. Two distinct classes of diplonemid GAPDH genes were found in diplonemids, however, neither class is closely related to any other euglenozoan GAPDH. One diplonemid GAPDH is related to the cytosolic gapC of eukaryotes, although not to either euglenoids or kinetoplastids, and the second is related to cyanobacterial and proteobacterial gap3. The bacterial gap3 gene in diplonemids provides one of the most well-supported examples of lateral gene transfer from a bacterium to a eukaryote characterised to date, and may indicate that diplonemids have acquired a novel biochemical capacity through lateral transfer.

Alpha and beta subunits of pyruvate dehydrogenase E1 from the microsporidian Nosema locustae: mitochondrion-derived carbon metabolism in microsporidia.

Microsporidia are highly adapted eukaryotic intracellular parasites that infect a variety of animals. Microsporidia contain no recognisable mitochondrion, but recently have been shown to have evolved from fungi and to possess heat shock protein genes derived from mitochondria. These findings make it clear that microsporidian ancestors were mitochondrial, yet it remains unknown whether they still contain the organelle, and if so what its role in microsporidian metabolism might be. Here we have characterised genes encoding the alpha and beta subunits of pyruvate dehydrogenase complex E1 (PDH, EC 1.2.4.1) from the microsporidian Nosema locustae. All other amitochondriate eukaryotes studied to date have lost the PDH complex and replaced it with pyruvate:ferredoxin oxidoreductase (PFOR). Nevertheless, molecular phylogeny shows that these Nosema enzymes are most closely related to mitochondrial PDH from other eukaryotes, demonstrating that elements of mitochondrial metabolism have been retained in microsporidia, and that PDH has not been wholly lost. However, there is still no evidence for a mitochondrion in microsporidia, and neither PDH subunit is predicted to encode an amino terminal leader sequence that could function as a mitochondrion-targeting transit peptide, raising questions as to whether these proteins function in a relic organelle or in the cytosol. Moreover, it is also unclear whether these proteins remain part of the PDH complex, or whether they have been retained for another purpose. We propose that microsporidia may utilise a unique pyruvate decarboxylation pathway involving PDH, demonstrating once again the diversity of core metabolism in amitochondriate eukaryotes.

Dinoflagellate nuclear SSU rRNA phylogeny suggests multiple plastid losses and replacements.

Dinoflagellates are a trophically diverse group of protists with photosynthetic and non-photosynthetic members that appears to incorporate and lose endosymbionts relatively easily. To trace the gain and loss of plastids in dinoflagellates, we have sequenced the nuclear small subunit rRNA gene of 28 photosynthetic and four non-photosynthetic species, and produced phylogenetic trees with a total of 81 dinoflagellate sequences. Patterns of plastid gain, loss, and replacement were plotted onto this phylogeny. With the exception of the apparently early-diverging Syndiniales and Noctilucales, all non-photosynthetic dinoflagellates are very likely to have had photosynthetic ancestors with peridinin-containing plastids. The same is true for all dinoflagellates with plastids other than the peridinin-containing plastid: their ancestors have replaced one type of plastid for another, in some cases most likely through a non-photosynthetic intermediate. Eight independent instances of plastid loss and three of replacement can be inferred from existing data, but as more non-photosynthetic lineages are characterized these numbers will surely grow.

Lateral transfer at the gene and subgenic levels in the evolution of eukaryotic enolase.

Enolase genes from land plants and apicomplexa (intracellular parasites, including the malarial parasite, Plasmodium) share two short insertions. This observation has led to the suggestion that the apicomplexan enolase is the product of a lateral transfer event involving the algal endosymbiont from which the apicomplexan plastid is derived. We have examined enolases from a wide variety of algae, as well as ciliates (close relatives of apicomplexa), to determine whether lateral transfer can account for the origin of the apicomplexan enolase. We find that lateral gene transfer, likely occurring intracellularly between endosymbiont and host nucleus, does account for the evolution of cryptomonad and chlorarachniophyte algal enolases but fails to explain the apicomplexan enolase. This failure is because the phylogenetic distribution of the insertions--which we find in apicomplexa, ciliates, land plants, and charophyte green algae--directly conflicts with the phylogeny of the gene itself. Protein insertions have traditionally been treated as reliable markers of evolutionary events; however, these enolase insertions do not seem to reflect accurately the evolutionary history of the molecule. The lack of congruence between insertions and phylogeny could be because of the parallel loss of both insertions in two or more lineages, or what is more likely, because the insertions were transmitted between distantly related genes by lateral transfer and fine-scale recombination, resulting in a mosaic gene. This latter process would be difficult to detect without such insertions to act as markers, and such mosaic genes could blur the "tree of life" beyond the extent to which whole-gene lateral transfer is already known to confound evolutionary reconstruction.

Foraminifera and Cercozoa are related in actin phylogeny: two orphans find a home?

In recent years, the increased sampling of protein-coding genes from diverse eukaryotes has revealed that many aspects of each gene tree are at odds with other phylogenies. This has led to the belief that each gene tree has unique strengths and weaknesses, suggesting that an accurate picture of eukaryotic relationships will be achieved only through comparative phylogeny using several different genes. To this end, actin genes were characterized from two genera of chlorarachniophytes, Chlorarachnion and Lotharella, and three species of the cercomonad flagellate Cercomonas: Phylogenetic trees including these new actin genes confirm the recently proposed relationship between chlorarachniophytes and cercomonads (Cercozoa) and, more importantly, also show a close relationship between Cercozoa and Foraminifera. Both of these are major eukaryotic groups encompassing extremely diverse organisms, yet there is no strong evidence for the evolutionary position of either from morphological or molecular data. The union of Cercozoa and Foraminifera suggested by actin phylogeny represents a novel step in the long process of determining the broad relationships between all major eukaryotic groups.

Nuclear-encoded, plastid-targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids.

The phylum Apicomplexa encompasses a large number of intracellular protozoan parasites, including the causative agents of malaria (Plasmodium), toxoplasmosis (Toxoplasma), and many other human and animal diseases. Apicomplexa have recently been found to contain a relic, nonphotosynthetic plastid that has attracted considerable interest as a possible target for therapeutics. This plastid is known to have been acquired by secondary endosymbiosis, but when this occurred and from which type of alga it was acquired remain uncertain. Based on the molecular phylogeny of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, we provide evidence that the apicomplexan plastid is homologous to plastids found in dinoflagellates-close relatives of apicomplexa that contain secondary plastids of red algal origin. Surprisingly, apicomplexan and dinoflagellate plastid-targeted GAPDH sequences were also found to be closely related to the plastid-targeted GAPDH genes of heterokonts and cryptomonads, two other groups that contain secondary plastids of red algal origin. These results address several outstanding issues: (1) apicomplexan and dinoflagellate plastids appear to be the result of a single endosymbiotic event which occurred relatively early in eukaryotic evolution, also giving rise to the plastids of heterokonts and perhaps cryptomonads; (2) apicomplexan plastids are derived from a red algal ancestor; and (3) the ancestral state of apicomplexan parasites was photosynthetic.

An evaluation of Hsp90 as a mediator of cortical patterning in Tetrahymena.

This study asks two questions: 1) whether Hsp90 is involved in the regulation of cortical patterning in Tetrahymena, and 2) if it is, whether specific defects in this regulation can be attributed to functional insufficiency of the Hsp90 molecule. To address question 1, we compared the effects of a specific inhibitor of Hsp90, geldanamycin, on population growth and on development of the oral apparatus in two Tetrahymena species, T. pyriformis and T. thermophila. We observed that geldanamycin inhibits population growth in both species at very low concentrations, and that it has far more severe effects on oral patterning in T. pyriformis than in T. thermophila. These effects are parallel to those of high temperature in the same two species, and provide a tentative affirmative answer to the first question. To address question 2, we ascertained the base sequence of the genes that encode the Hsp90 molecules which are induced at high temperatures in both Tetrahymena species, as well as corresponding sequences in Paramecium tetraurelia. Extensive comparative analyses of the deduced amino acid sequences of the Hsp90 molecules of the two Tetrahymena species indicate that on the basis of what we currently know about Hsp90 both proteins are equally likely to be functional. Phylogenetic analyses of Hsp90 amino acid sequences indicate that the two Tetrahymena Hsp90 molecules have undergone a similar number of amino acid substitutions from their most recent common ancestor, with none of these corresponding to any known functionally critical region of the molecule. Thus there is no evidence that the Hsp90 molecule of T. pyriformis is functionally impaired; the flaw in the control of cortical patterning is more likely to be caused by defects in mechanism(s) that mediate the response to Hsp90, as would be expected from the "Hsp90 capacitor" model of Rutherford and Lindquist.

Symbiotic origin of a novel actin gene in the cryptophyte Pyrenomonas helgolandii.

Cryptophytes are photosynthetic protists that have acquired their plastids through the secondary symbiotic uptake of a red alga. A remarkable feature of cryptophytes is that they maintain a reduced form of the red algal nucleus, the nucleomorph, between the second and third plastid membranes (periplastidial compartment; PC). The nucleomorph is thought to be a transition state in the evolution of secondary plastids, with this genome ultimately being lost when photosynthesis comes under full control of the "host" nucleus (e.g., as in heterokonts, haptophytes, and euglenophytes). Genes presently found in the nucleomorph seem to be restricted to those involved in its own maintenance and to that of the plastid; other genes were lost as the endosymbiont was progressively reduced to its present state. Surprisingly, we found that the cryptophyte Pyrenomonas helgolandii possesses a novel type of actin gene that originated from the nucleomorph genome of the symbiont. Our results demonstrate for the first time that secondary symbionts can contribute genes to the host lineage which are unrelated to plastid function. These genes are akin to the products of gene duplication or lateral transfer and provide a source of evolutionary novelty that can significantly increase the genetic diversity of the host lineage. We postulate that this may be a common phenomenon in algae containing secondary plastids that has yet to be fully appreciated due to a dearth of evolutionary studies of nuclear genes in these taxa.

Predictive value of wavelet decomposition of the signal-averaged electrocardiogram in idiopathic dilated cardiomyopathy.

BACKGROUND: Wavelet decomposition of the signal-averaged electrocardiogram has been proposed as a method of detecting small and transient irregularities hidden within the QRS complex and of overcoming some of the limitations of time domain analysis of the signal-averaged electrocardiogram. AIM: This study evaluated the potential utility of wavelet decomposition analysis in the risk stratification of patients with idiopathic dilated cardiomyopathy. METHODS AND RESULTS: Both wavelet decomposition and time domain analysis were applied to the signal-averaged electrocardiogram recordings of 82 patients with idiopathic dilated cardiomyopathy (mean age 43 +/- 14 years, 60 men) and 72 normal controls (mean age 44 +/- 15 years, 48 men). Three conventional time domain indices and four wavelet decomposition analysis parameters (QRS length, maximum count, surface area, and relative length) were derived from each recording using a Del Mar CEWS system and an in-house software package, respectively. The results showed that (1) more patients with idiopathic dilated cardiomyopathy than without had late potentials, and that the filtered QRS duration was significantly longer in patients than in controls (P<0.001). Similarly, abnormal wavelet decomposition analysis was more common in patients and wavelet decomposition measurements were significantly different between patients and controls (P<0.01); (2) conventional time domain analysis did not distinguish between clinically stable patients and patients who developed progressive heart failure, or between patients with and without arrhythmic events; (3) wavelet decomposition analysis identified patients who went on to develop progressive heart failure but failed to distinguish patients with arrhythmic events from those without; (4) survival analyses of a mean follow-up of 23 months showed that patients with late potentials tended to develop progressive heart failure more frequently than others (P=0.06). Patients with an abnormal wavelet decomposition result more frequently developed progressive heart failure than those with a normal wavelet decomposition result (P=0.027); (5) in a univariate analysis (Cox model), wavelet decomposition measurements but not time domain indices significantly correlated with the development of progressive heart failure (P=0.01). Multivariate analysis showed that only left ventricular end-diastolic dimension and peak oxygen consumption during exercise remained significant predictors of progressive heart failure. CONCLUSION: Wavelet decomposition analysis of the signal-averaged electrocardiogram is superior to conventional time domain analysis for identifying patients with idiopathic dilated cardiomyopathy at increased risk of clinical deterioration. Wavelet decomposition analysis, however, is unlikely to prospectively distinguish patients at a high risk of arrhythmic events in idiopathic dilated cardiomyopathy in its present form.

Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi.

Microsporidia are obligate intracellular parasites that were thought to be an ancient eukaryotic lineage based on molecular phylogenies using ribosomal RNA and translation elongation factors. However, this ancient origin of microsporidia has been contested recently, as several other molecular phylogenies suggest that microsporidia are closely related to fungi. Most of the protein trees that place microsporidia with fungi are not well sampled, however, and it is impossible to resolve whether microsporidia evolved from a fungus or from a protistan relative of fungi. We have sequenced beta-tubulins from 3 microsporidia, 4 chytrid fungi, and 12 zygomycete fungi, expanding the representation of beta-tubulin to include all four fungal divisions and a wide diversity of microsporidia. In phylogenetic trees including these new sequences, the overall topology of the fungal beta-tubulins generally matched the expected relationships among the four fungal divisions, although the zygomycetes were polyphyletic in some analyses. The microsporidia consistently fell within this fungal diversification, and not as a sister group to fungi. Overall, beta-tubulin phylogeny suggests that microsporidia evolved from a fungus sometime after the divergence of chytrids. We also found that chytrid alpha- and beta-tubulins are much less divergent than are tubulins from other fungi or microsporidia. In trees in which the only fungal representatives were the chytrids, microsporidia still branched with fungi (i.e., with chytrids), suggesting that the affiliation between microsporidian and fungal tubulins is not an artifact of long-branch attraction.