Major Eukaryotic Groups Research Articles

Peroxisome diversity and evolution

Peroxisomes are organelles bounded by a single membrane that can be found in all major groups of eukaryotes. A single evolutionary origin of this cellular compartment is supported by the presence, in diverse organisms, of a common set of proteins implicated in peroxisome biogenesis and maintenance. Their enzymatic content, however, can vary substantially across species, indicating a high level of evolutionary plasticity. Proteomic analyses have greatly expanded our knowledge on peroxisomes in some model organisms, including plants, mammals and yeasts. However, we still have a limited knowledge about the distribution and functionalities of peroxisomes in the vast majority of groups of microbial eukaryotes. Here, I review recent advances in our understanding of peroxisome diversity and evolution, with a special emphasis on peroxisomes in microbial eukaryotes.

A hitchhiker's guide to the MADS world of plants

Plant life critically depends on the function of MADS-box genes encoding MADS-domain transcription factors, which are present to a limited extent in nearly all major eukaryotic groups, but constitute a large gene family in land plants. There are two types of MADS-box genes, termed type I and type II, and in plants these groups are distinguished by exon-intron and domain structure, rates of evolution, developmental function and degree of functional redundancy. The type I genes are further subdivided into three groups - Mα, Mβ and Mγ - while the type II genes are subdivided into the MIKCC and MIKC* groups. The functional diversification of MIKCC genes is closely linked to the origin of developmental and morphological novelties in the sporophytic (usually diploid) generation of seed plants, most spectacularly the floral organs and fruits of angiosperms. Functional studies suggest different specializations for the different classes of genes; whereas type I genes may preferentially contribute to female gametophyte, embryo and seed development and MIKC*-group genes to male gametophyte development, the MIKCC-group genes became essential for diverse aspects of sporophyte development. Beyond the usual transcriptional regulation, including feedback and feed-forward loops, various specialized mechanisms have evolved to control the expression of MADS-box genes, such as epigenetic control and regulation by small RNAs. In future, more data from genome projects and reverse genetic studies will allow us to understand the birth, functional diversification and death of members of this dynamic and important family of transcription factors in much more detail.

Eukaryotic diversity and phylogeny using small‐ and large‐subunit ribosomal RNA genes from environmental samples

The recent introduction of molecular techniques in eukaryotic microbial diversity studies, in particular those based in the amplification and sequencing of small-subunit ribosomal DNA (SSU rDNA), has revealed the existence of an unexpected variety of new phylotypes. The taxonomic ascription of the organisms bearing those sequences is generally deduced from phylogenetic analysis. Unfortunately, the SSU rDNA sequence alone has often not enough phylogenetic information to resolve the phylogeny of fast-evolving or very divergent sequences, leading to their misclassification. To address this problem, we tried to increase the phylogenetic signal by amplifying the complete eukaryotic rDNA cluster [i.e. the SSU rDNA, the internal transcribed spacers, the 5.8S rDNA and the large-subunit (LSU) rDNA] from environmental samples, and sequencing the SSU and LSU rDNA part of the clones. Using marine planktonic samples, we showed that surveys based on either SSU or SSU + LSU rDNA retrieved comparable diversity patterns. In addition, phylogenetic trees based on the concatenated SSU + LSU rDNA sequences showed better resolution, yielding good support for major eukaryotic groups such as the Opisthokonta, Rhizaria and Excavata. Finally, highly divergent SSU rDNA sequences, whose phylogenetic position was impossible to determine with the SSU rDNA data alone, could be placed correctly with the SSU + LSU rDNA approach. These results suggest that this method can be useful, in particular for the analysis of eukaryotic microbial communities rich in phylotypes of difficult phylogenetic ascription.

Evolution of the RNA-dependent RNA polymerase (RdRP) genes: Duplications and possible losses before and after the divergence of major eukaryotic groups

Eukaryotic RNA-dependent RNA polymerases (RdRPs, encoded by RDR genes) play critical roles in developmental regulation, maintenance of genome integrity, and defense against foreign nucleic acids. However, the phylogenetic relationship of RDRs remains unclear. From available genome sequences, we identified 161 putative RDR genes from 56 eukaryotes, ranging from protists to multicellular organisms, including plants, fungi and invertebrate animals, such as nematodes, lancelet and sea anemone. On the other hand, we did not detect RDR homologs in vertebrates and insects, even though RNA interference functions in these organisms. Our phylogenetic analysis of the RDR genes suggests that the eukaryotic ancestor might have had three copies, i.e. RDRα, RDRβ and RDRγ. These three ancient copies were also supported by the patterns of protein sequence motifs. Further duplication events after the divergence of major eukaryotic groups were supported by the phylogenetic analyses, including some that likely occurred before the separation of subgroups within each kingdom. We present a model for a possible evolutionary history of RDR genes in eukaryotes.

The Phosphoproteome of Bloodstream Form Trypanosoma brucei, Causative Agent of African Sleeping Sickness

The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that phosphorylation-based signaling is a general and fundamental regulatory process that extends to this highly divergent lower eukaryote.

Phylogenetic analysis of 73 060 taxa corroborates major eukaryotic groups.

Obtaining a well supported schema of phylogenetic relationships among the major groups of living organisms requires considering as much taxonomic diversity as possible, but the computational cost of calculating large phylogenies has so far been a major obstacle. We show here that the parsimony algorithms implemented in TNT can successfully process the largest phylogenetic data set ever analysed, consisting of molecular sequences and morphology for 73 060 eukaryotic taxa. The trees resulting from molecules alone display a high degree of congruence with the major taxonomic groups, with a small proportion of misplaced species; the combined data set retrieves these groups with even higher congruence. This shows that tree-calculation algorithms effectively retrieve phylogenetic history for very large data sets, and at the same time provides strong corroboration for the major eukaryotic lineages long recognized by taxonomists.

A universal DNA mini-barcode for biodiversity analysis

BackgroundThe goal of DNA barcoding is to develop a species-specific sequence library for all eukaryotes. A 650 bp fragment of the cytochrome c oxidase 1 (CO1) gene has been used successfully for species-level identification in several animal groups. It may be difficult in practice, however, to retrieve a 650 bp fragment from archival specimens, (because of DNA degradation) or from environmental samples (where universal primers are needed).ResultsWe used a bioinformatics analysis using all CO1 barcode sequences from GenBank and calculated the probability of having species-specific barcodes for varied size fragments. This analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. We then developed a universal primer set for the amplification of mini-barcodes. We further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens.ConclusionIn this study we address the important issue of minimum amount of sequence information required for identifying species in DNA barcoding. We establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. This approach will significantly broaden the application of DNA barcoding in biodiversity studies.

An overview of the phylogeny and diversity of eukaryotes

Our understanding of eukaryote biology is dominated by the study of land plants, animals and fungi. However, these are only three isolated fragments of the full diversity of extant eukaryotes. The majority of eukaryotes, in terms of major taxa and probably also sheer numbers of cells, consists of exclusively or predominantly unicellular lineages. A surprising number of these lineages are poorly characterized. Nonetheless, they are fundamental to our understanding of eukaryote biology and the underlying forces that shaped it. This article consists of an overview of the current state of our understanding of the eukaryote tree. This includes the identity of the major groups of eukaryotes, some of their important, defining or simply interesting features and the proposed relationships of these groups to each other.

Morphology and molecular phylogeny of a marine interstitial tetraflagellate with putative endosymbionts: Auranticordis quadriverberis n. gen. et sp. (Cercozoa)

BackgroundComparative morphological studies and environmental sequencing surveys indicate that marine benthic environments contain a diverse assortment of microorganisms that are just beginning to be explored and characterized. The most conspicuous predatory flagellates in these habitats range from about 20–150 μm in size and fall into three major groups of eukaryotes that are very distantly related to one another: dinoflagellates, euglenids and cercozoans. The Cercozoa is a diverse group of amoeboflagellates that cluster together in molecular phylogenies inferred mainly from ribosomal gene sequences. These molecular phylogenetic studies have demonstrated that several enigmatic taxa, previously treated as Eukaryota insertae sedis, fall within the Cercozoa, and suggest that the actual diversity of this group is largely unknown. Improved knowledge of cercozoan diversity is expected to help resolve major branches in the tree of eukaryotes and demonstrate important cellular innovations for understanding eukaryote evolution.ResultsA rare tetraflagellate, Auranticordis quadriverberis n. gen. et sp., was isolated from marine sand samples. Uncultured cells were in low abundance and were individually prepared for electron microscopy and DNA sequencing. These flagellates possessed several novel features, such as (1) gliding motility associated with four bundled recurrent flagella, (2) heart-shaped cells about 35–75 μm in diam., and (3) bright orange coloration caused by linear arrays of muciferous bodies. Each cell also possessed about 2–30 pale orange bodies (usually 4–5 μm in diam.) that were enveloped by two membranes and sac-like vesicles. The innermost membrane invaginated to form unstacked thylakoids that extended towards a central pyrenoid containing tailed viral particles. Although to our knowledge, these bodies have never been described in any other eukaryote, the ultrastructure was most consistent with photosynthetic endosymbionts of cyanobacterial origin. This combination of morphological features did not allow us to assign A. quadriverberis to any known eukaryotic supergroup. Thus, we sequenced the small subunit rDNA sequence from two different isolates and demonstrated that this lineage evolved from within the Cercozoa.ConclusionOur discovery and characterization of A. quadriverberis underscores how poorly we understand the diversity of cercozoans and, potentially, represents one of the few independent cases of primary endosymbiosis within the Cercozoa and beyond.

Evolutionary history of histone demethylase families: distinct evolutionary patterns suggest functional divergence

BackgroundHistone methylation can dramatically affect chromatin structure and gene expression and was considered irreversible until recent discoveries of two families of histone demethylases, the KDM1 (previously LSD1) and JmjC domain-containing proteins. These two types of proteins have different functional domains and distinct substrate specificities. Although more and more KDM1 and JmjC proteins have been shown to have histone demethylase activity, our knowledge about their evolution history is limited.ResultsWe performed systematic phylogenetic analysis of these histone demethylase families and uncovered different evolutionary patterns. The KDM1 genes have been maintained with a stable low copy number in most organisms except for a few duplication events in flowering plants. In contrast, multiple genes for JmjC proteins with distinct domain architectures were present before the split of major eukaryotic groups, and experienced subsequent birth-and-death evolution. In addition, distinct evolutionary patterns can also be observed between animal and plant histone demethylases in both families. Furthermore, our results showed that some JmjC subfamilies contain only animal genes with specific demethylase activities, but do not have plant members.ConclusionOur study improves the understanding about the evolutionary history of KDM1 and JmjC genes and provides valuable insights into their functions. Based on the phylogenetic relationship, we discussed possible histone demethylase activities for several plant JmjC proteins. Finally, we proposed that the observed differences in evolutionary pattern imply functional divergence between animal and plant histone demethylases.

Toward Resolving the Eukaryotic Tree: The Phylogenetic Positions of Jakobids and Cercozoans

Resolving the global phylogeny of eukaryotes has proven to be challenging. Among the eukaryotic groups of uncertain phylogenetic position are jakobids, a group of bacterivorous flagellates that possess the most bacteria-like mitochondrial genomes known. Jakobids share several ultrastructural features with malawimonads and an assemblage of anaerobic protists (e.g., diplomonads and oxymonads). These lineages together with Euglenozoa and Heterolobosea have collectively been designated "excavates". However, published molecular phylogenies based on the sequences of nuclear rRNAs and up to six nucleus-encoded proteins do not provide convincing support for the monophyly of excavates, nor do they uncover their relationship to other major eukaryotic groups. Here, we report the first large-scale eukaryotic phylogeny, inferred from 143 nucleus-encoded proteins comprising 31,604 amino acid positions, that includes jakobids, malawimonads and cercozoans. We obtain compelling support for the monophyly of jakobids, Euglenozoa plus Heterolobosea (JEH group), and for the association of cercozoans with stramenopiles plus alveolates. Furthermore, we observe a sister-group relationship between the JEH group and malawimonads after removing fast-evolving species from the dataset. We discuss the implications of these results for the concept of "excavates" and for the elucidation of eukaryotic phylogeny in general.

Did an ancient chlamydial endosymbiosis facilitate the establishment of primary plastids?

Phylogenomic analyses of the red alga Cyanidioschyzon merolae shows that at least 21 genes were transferred between chlamydiae and primary photosynthetic eukaryotes, suggesting an ancient chlamydial endosymbiosis with the ancestral primary photosynthetic eukaryote.

Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata

Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of this huge eukaryotic group. Multiple protein sequences will be needed fully to resolve basal bikont phylogeny. Nonetheless, our results suggest that combined SSU+LSU rDNA phylogenies can help to resolve several ambiguous regions of the eukaryotic tree and identify key taxa for subsequent multi-gene analyses.

Evolutionary Relationships of Apusomonads Inferred from Taxon-Rich Analyses of 6 Nuclear Encoded Genes

The phylogenetic relationships of the biflagellate protist group Apusomonadidae have been unclear despite the availability of some molecular data. We analyzed sequences from 6 nuclear encoded genes-small-subunit rRNA, large-subunit rRNA, alpha-tubulin, beta-tubulin, actin, and heat shock protein 90-to infer the phylogenetic position of Apusomonas proboscidea Aléxéieff 1924. To increase the taxon richness of the study, we also obtained new sequences from representatives of several other major eukaryotic groups: Chrysochromulina sp. National Institute for Environmental Studies 1333 (Haptophyta), Cyanophora paradoxa (Glaucophyta), Goniomonas truncata (Cryptophyceae), Leucocryptos marina (Kathablepharidae), Mesostigma viride (Streptophyta, Viridiplantae), Peridinium limbatum (Alveolata), Pterosperma cristatum (Prasinophytae, Viridiplantae), Synura sphagnicola (Stramenopiles), and Thaumatomonas sp. (Rhizaria). In most individual gene phylogenies, Apusomonas branched close to either of the 2 related taxa-Opisthokonta (including animals, fungi, and choanoflagellates) or Amoebozoa. Combined analyses of all 4 protein-coding genes or all 6 studied genes strongly supported the hypothesis that Apusomonadidae is closely related to Opisthokonta (or to all other eukaryotic groups except Opisthokonta, depending on the position of the eukaryotic root). Alternative hypotheses were rejected in approximately unbiased tests at the 5% level. However, the strong phylogenetic signal supporting a specific affiliation between Apusomonadidae and Opisthokonta largely originated from the alpha-tubulin data. If alpha-tubulin is not considered, topologies in which Apusomonadidae is sister to Opisthokonta or is sister to Amoebozoa were more or less equally supported. One current model for deep eukaryotic evolution holds that eukaryotes are divided into primary "unikont" and "bikont" clades and are descended from a "uniflagellate" common ancestor. Together with other information, our data suggest instead that unikonts (=Opisthokonta and Amoebozoa) are not strictly monophyletic and are descended from biflagellate ancestors.

Genetic Diversity of Microbial Eukaryotes in Anoxic Sediment of the Saline Meromictic Lake Namako-ike (Japan): On the Detection of Anaerobic or Anoxic-tolerant Lineages of Eukaryotes

Available sequence data on eukaryotic small-subunit ribosomal DNA (SSU rDNA) directly retrieved from various environments have increased recently, and the diversity of microbial eukaryotes (protists) has been shown to be much greater than previously expected. However, the molecular information accumulated to date does still not thoroughly reveal ecological distribution patterns of microbial eukaryotes. In the ongoing challenge to detect anaerobic or anoxic-tolerant lineages of eukaryotes, we directly extracted DNA from the anoxic sediment of a saline meromictic lake, constructed genetic libraries of PCR-amplified SSU rDNA, and performed phylogenetic analyses with the cloned SSU rDNA sequences. Although a few sequences could not be confidently assigned to any major eukaryotic groups in the analyses and are debatable regarding their taxonomic positions, most sequences obtained have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, Stramenopiles, and Opisthokonta). Among these sequences, some branched with lineages predominantly composed of uncultured environmental clones retrieved from other anoxic environments, while others were closely related to those of eukaryotic parasites (e.g. Phytomyxea of Cercozoa, Gregarinea of Alveolata, and Ichthyosporea of Opisthokonta).

Phylogeny of the Centrohelida Inferred from SSU rRNA, Tubulins, and Actin Genes

Amoeboid protists are major targets of recent molecular phylogeny in connection with reconstruction of global phylogeny of eukaryotes as well as the search for the root of eukaryotes. The Centrohelida are one of the major groups of Heliozoa, classified in the Actinopodida, whose evolutionary position is not well understood. To clarify the relationships between the Centrohelida and other eukaryotes, we sequenced SSU rRNA, alpha-tubulin, and beta-tubulin genes from a centroheliozoan protist, Raphidiophrys contractilis. The SSU rRNA phylogeny showed that the Centrohelida are not closely related to other heliozoan groups, Actinophryida, Desmothoracida, or Taxopodida. Maximum likelihood analyses of the combined phylogeny using a concatenate model for an alpha- + beta-tubulin + actin data set, and a separate model for SSU rRNA, alpha- and beta-tubulin, and actin gene data sets revealed the best tree, in which the Centrohelida have a closer relationship to Rhodophyta than to other major eukaryotic groups. However, both weighted Shimodaira-Hasegawa and approximately unbiased tests for the concatenate protein phylogeny did not reject alternative trees in which Centrohelida were constrained to be sisters to the Amoebozoa. Moreover, alternative trees in which Centrohelida were placed at the node branching before and after Amoebozoa or Viridiplantae were not rejected by the WSH tests. These results narrowed the possibilities for the position of Centrohelida to a sister to the Rhodophyta, to the Amoebozoa, or to an independent branch between the branchings of Amoebozoa and Rhodophyta (or possibly Plantae) at the basal position within the bikonts clade in the eukaryotic tree.

Inference of the Phylogenetic Position of Oxymonads Based on Nine Genes: Support for Metamonada and Excavata

Circumscribing major eukaryote groups and resolving higher order relationships between them are among the most challenging tasks facing molecular evolutionists. Recently, evidence suggesting a new supergroup (the Excavata) comprising a wide array of flagellates has been collected. This group consists of diplomonads, retortamonads, Carpediemonas, heteroloboseans, Trimastix, jakobids, and Malawimonas, all of which possess a particular type of ventral feeding groove that is proposed to be homologous. Euglenozoans, parabasalids, and oxymonads have also been associated with Excavata as their relationships to one or more core excavate taxa were demonstrated. However, the main barrier to the general acceptance of Excavata is that its existence is founded primarily on cytoskeletal similarities, without consistent support from molecular phylogenetics. In gene trees, Excavata are typically not recovered together. In this paper, we present an analysis of the phylogenetic position of oxymonads (genus Monocercomonoides) based on concatenation of eight protein sequences (alpha-tubulin, beta-tubulin, gamma-tubulin, EF-1alpha, EF-2, cytosolic (cyt) HSP70, HSP90, and ubiquitin) and 18S rRNA. We demonstrate that the genes are in conflict regarding the position of oxymonads. Concatenation of alpha- and beta-tubulin placed oxymonads in the plant-chromist part of the tree, while the concatenation of other genes recovered a well-supported group of Metamonada (oxymonads, diplomonads, and parabasalids) that branched weakly with euglenozoans--connecting all four excavates included in the analyses and thus providing conditional support for the existence of Excavata.

Phylogenetic positions of several amitochondriate protozoa——Evidence from phylogenetic analysis of DNA topoisomerase II

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group--Archezoa. The main evidence for this is their 'lacking mitochondria' and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of 'long-branch attraction' appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplomonads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be polyphyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.

Root of the Eukaryota Tree as Inferred from Combined Maximum Likelihood Analyses of Multiple Molecular Sequence Data

Extensive studies aiming to establish the structure and root of the Eukaryota tree by phylogenetic analyses of molecular sequences have thus far not resulted in a generally accepted tree. To re-examine the eukaryotic phylogeny using alternative genes, and to obtain a more robust inference for the root of the tree as well as the relationship among major eukaryotic groups, we sequenced the genes encoding isoleucyl-tRNA and valyl-tRNA synthetases, cytosolic-type heat shock protein 90, and the largest subunit of RNA polymerase II from several protists. Combined maximum likelihood analyses of 22 protein-coding genes including the above four genes clearly demonstrated that Diplomonadida and Parabasala shared a common ancestor in the rooted tree of Eukaryota, but only when the fast-evolving sites were excluded from the original data sets. The combined analyses, together with recent findings on the distribution of a fused dihydrofolate reductase-thymidylate synthetase gene, narrowed the possible position of the root of the Eukaryota tree on the branch leading to Opisthokonta or to the common ancestor of Diplomonadida/Parabasala. However, the analyses did not agree with the position of the root located on the common ancestor of Opisthokonta and Amoebozoa, which was argued by Stechmann and Cavalier-Smith [Curr. Biol. 13:R665-666, 2003] based on the presence or absence of a three-gene fusion of the pyrimidine biosynthetic pathway: carbamoyl-phosphate synthetase II, dihydroorotase, and aspartate carbamoyltransferase. The presence of the three-gene fusion recently found in the Cyanidioschyzon merolae (Rhodophyta) genome sequence data supported our analyses against the Stechmann and Cavalier-Smith-rooting in 2003.

Analyses of RNA Polymerase II Genes from Free-Living Protists: Phylogeny, Long Branch Attraction, and the Eukaryotic Big Bang

The phylogenetic relationships among major eukaryotic protist lineages are largely uncertain. Two significant obstacles in reconstructing eukaryotic phylogeny are long-branch attraction (LBA) effects and poor taxon sampling of free-living protists. We have obtained and analyzed gene sequences encoding the largest subunit of RNA Polymerase II (RPB1) from Naegleria gruberi (a heterolobosean), Cercomonas ATCC 50319 (a cercozoan), and Ochromonas danica (a heterokont); we have also analyzed the RPB1 gene from the nucleomorph (nm) genome of Guillardia theta (a cryptomonad). Using a variety of phylogenetic methods our analysis shows that RPB1s from Giardia intestinalis and Trichomonas vaginalis are probably subject to intense LBA effects. Thus, the deep branching of these taxa on RPB1 trees is questionable and should not be interpreted as evidence favoring their early divergence. Similar effects are discernable, to a lesser extent, with the Mastigamoeba invertens RPB1 sequence. Upon removal of the outgroup and these problematic sequences, analyses of the remaining RPB1s indicate some resolution among major eukaryotic groups. The most robustly supported higher-level clades are the opisthokonts (animals plus fungi) and the red algae plus the cryptomonad nm-the latter result gives added support to the red algal origin of cryptomonad chloroplasts. Clades comprising Dictyostelium discoideum plus Acanthamoeba castellanii (Amoebozoa) and Ochromonas plus Plasmodium falciparum (chromalveolates) are consistently observed and moderately supported. The clades supported by our RPB1 analyses are congruent with other data, suggesting that bona fide phylogenetic relationships are being resolved. Thus, the RPB1 gene has apparently retained some phylogenetically meaningful signal, making it worthwhile to obtain sequences from more diverse protist taxa. Additional RPB1 data, especially in combination with other genes, should provide further resolution of branching orders among protist groups within the apparently rapid early divergence of eukaryotes.