Comprehensive multigene phylogenies of excavate protists reveal the evolutionary positions of "primitive" eukaryotes

Many of the protists thought to represent the deepest branches on the eukaryotic tree are assigned to a loose assemblage called the "excavates." This includes the mitochondrion-lacking diplomonads and parabasalids (e.g., Giardia and Trichomonas) and the jakobids (e.g., Reclinomonas). We report the first multigene phylogenetic analyses to include a comprehensive sampling of excavate groups (six nuclear-encoded protein-coding genes, nine of the 10 recognized excavate groups). Excavates coalesce into three clades with relatively strong maximum likelihood bootstrap support. Only the phylogenetic position of Malawimonas is uncertain. Diplomonads, parabasalids, and the free-living amitochondriate protist Carpediemonas are closely related to each other. Two other amitochondriate excavates, oxymonads and Trimastix, form the second monophyletic group. The third group is comprised of Euglenozoa (e.g., trypanosomes), Heterolobosea, and jakobids. Unexpectedly, jakobids appear to be specifically related to Heterolobosea. This tree topology calls into question the concept of Discicristata as a supergroup of eukaryotes united by discoidal mitochondrial cristae and makes it implausible that jakobids represent an independent early-diverging eukaryotic lineage. The close jakobids-Heterolobosea-Euglenozoa connection demands complex evolutionary scenarios to explain the transition between the presumed ancestral bacterial-type mitochondrial RNA polymerase found in jakobids and the phage-type protein in other eukaryotic lineages, including Euglenozoa and Heterolobosea.     

How oxymonads lost their groove: an ultrastructural comparison of Monocercomonoides and excavate taxa

Despite being amongst the more familiar groups of heterotrophic flagellates, the evolutionary affinities of oxymonads remain poorly understood. A re-interpretation of the cytoskeleton of the oxymonad Monocercomonoides hausmanni suggests that this organism has a similar ultrastructural organisation to members of the informal assemblage 'excavate taxa'. The preaxostyle, 'R1' root, and 'R2' root of M. hausmanni are proposed to be homologous to the right, left, and anterior roots respectively of excavate taxa. The 'paracrystalline' portion of the preaxostyle, previously treated as unique to oxymonads, is proposed to be homologous to the I fibre of excavate taxa. Other non-microtubular fibres are identified that have both positional and substructural similarity to the distinctive B and C fibres of excavate taxa. A homologue to the 'singlet root', otherwise distinctive for excavate taxa, is also proposed. The preaxostyle and C fibre homologue in Monocercomonoides are most similar to the homologous structures in Trimastix. suggesting a particularly close relationship. This supports and extends recent molecular phylogenetic findings that Trimastix and oxymonads form a clade. We conclude that oxymonads have an excavate ancestry, and that the 'excavate taxa' sensu stricto form a paraphyletic assemblage.     

Molecular phylogeny of three oxymonad genera: Pyrsonympha,Dinenympha and Oxymonas

Oxymonads are a morphologically well-characterized and highly diverse lineage of protists. They are, however, under sampled at a molecular level. It has recently been demonstrated that a genus of oxymonads, Pyrsonympha, is phylogenetically related to the excavate taxon Trimastix. Here, we addressed issues of internal oxymonad evolution. Pyrsonympha and Dinenympha are shown, by fluorescent in situ hybridization and phylogenetic evidence, to be separate genera and not morphotypes of the same organism. We demonstrated that three genera of oxymonads, Dinenympha, Pyrsonympha, and Oxymonas are each monophyletic and together form a clade which excludes other known eukaryotes. We have presented a taxonomic scheme of oxymonads taking into account their sisterhood with Trimastix and speculated on morphological evolution of oxymonads, particularly of their attachment apparatuses. Our biogeographical analysis with Japanese and Canadian Pyrsonympha and Dinenympha suggests that these genera diverged before the separation of termites that inhabit Eastern Asia and Western North America.     

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.     

Diversification of the Microtubule System in the Early Stage of Eukaryote Evolution: Elongation Factor 1α and α-Tubulin Protein Phylogeny of Termite Symbiotic Oxymonad and Hypermastigote Protists

The symbiotic protists of the lower termite have been regarded as a model of early-branched eukaryotes because of their simple cellular systems and morphological features. However, cultivation of these symbiotic protists is very difficult. For this reason, these interesting protists have not been well characterized in terms of their molecular biology. In research on these organisms which have not yet been cultivated, we developed a method for retrieving specific genes from a small number of cells, through micromanipulation without axenic cultivation, and we obtained EF-1 alpha and alpha-tubulin genes from members of the Hypermastigida--the parabasalid protist Trichonympha agilis and the oxymonad protists Pyrsonympha grandis and Dinenympha exilis--from the termite Reticulitermes speratus gut community. Results of phylogenetic analysis of the amino acid sequences of both proteins, EF-1 alpha and alpha-tubulin, indicate that the hypermastigid, parabasalid, and oxymonad protists do not share a close common ancestor. In addition, although the EF-1 alpha phylogeny indicates that these two groups of protists branched at an early stage of eukaryotic evolution, the alpha-tubulin phylogeny indicates that these protists can be assigned to two diversified clades. As shown in a recent investigation of alpha-tubulin phylogeny, eukaryotic organisms can be divided into three classes: an animal--parabasalids clade, a plant--protists clade, and the diplomonads. In this study, we show that parabasalids, including hypermastigids, can be classified as belonging to the animal--parabasalids clade and the early-branching eukaryote oxymonads can be classified as belonging to the plant--protists clade. Our findings suggest that these protists have a cellular microtubule system that has diverged considerably, and it seems that such divergence of the microtubule system occurred in the earliest stage of eukaryotic evolution.     

The new phylogeny of eukaryotes

Molecular phylogeny has been regarded as the ultimate tool for the reconstruction of relationships among eukaryotes-especially the different protist groups-given the difficulty in interpreting morphological data from an evolutionary point of view. In fact, the use of ribosomal RNA as a marker has provided the first well resolved eukaryotic phylogenies, leading to several important evolutionary hypotheses. The most significant is that several early-emerging, amitochondriate lineages, are living relics from the early times of eukaryotic evolution. The use of alternative protein markers and the recognition of several molecular phylogeny reconstruction artefacts, however, have strongly challenged these ideas. The putative early emerging lineages have been demonstrated as late-emerging ones, artefactually misplaced to the base of the tree. The present state of eukaryotic evolution is best described by a multifurcation, in agreement with the 'big bang' hypothesis that assumes a rapid diversification of the major eukaryotic phyla. For further resolution, the analysis of genomic data through improved phylogenetic methods will be required.     

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.     

Ancient phylogenetic relationships

Traditional views on deep evolutionary events have been seriously challenged over the last few years, following the identification of major pitfalls affecting molecular phylogeny reconstruction. Here we describe the principally encountered artifacts, notably long branch attraction, and their causes (i.e., difference in evolutionary rates, mutational saturation, compositional biases). Additional difficulties due to phenomena of biological nature (i.e., lateral gene transfer, recombination, hidden paralogy) are also discussed. Moreover, contrary to common beliefs, we show that the use of rare genomic events can also be misleading and should be treated with the same caution as standard molecular phylogeny. The universal tree of life, as described in most textbooks, is partly affected by tree reconstruction artifacts, e.g. (i) the bacterial rooting of the universal tree of life; (ii) the early emergence of amitochondriate lineages in eukaryotic phylogenies; and (iii) the position of hyperthermophilic taxa in bacterial phylogenies. We present an alternative view of this tree, based on recent evidence obtained from reanalyses of ancient data sets and from novel analyses of large combination of genes.     

Phylogenetic positions of several amitochondriate protozoa

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. diplo-monads, 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 poly-phyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.     

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.     

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.     

Early-branching or fast-evolving eukaryotes? An answer based on slowly evolving positions.

The current paradigm of eukaryotic evolution is based primarily on comparative analysis of ribosomal RNA sequences. It shows several early-emerging lineages, mostly amitochondriate, which might be living relics of a progressive assembly of the eukaryotic cell. However, the analysis of slow-evolving positions, carried out with the newly developed slow-fast method, reveals that these lineages are, in terms of nucleotide substitution, fast-evolving ones, misplaced at the base of the tree by a long branch attraction artefact. Since the fast-evolving groups are not always the same, depending on which macromolecule is used as a marker, this explains most of the observed incongruent phylogenies. The current paradigm of eukaryotic evolution thus has to be seriously re-examined as the eukaryotic phylogeny is presently best summarized by a multifurcation. This is consistent with the Big Bang hypothesis that all extant eukaryotic lineages are the result of multiple cladogeneses within a relatively brief period, although insufficiency of data is also a possible explanation for the lack of resolution. For further resolution, rare evolutionary events such as shared insertions and/or deletions or gene fusions might be helpful.     

Phylogenetic positions of several amitochondriate protozoa

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, En-tamoeba 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.     

Long-branch attraction and the rDNA model of early eukaryotic evolution

Phylogenetic analyses of ribosomal RNA genes have become widely accepted as a framework for understanding broad-scale eukaryotic evolution. Nevertheless, conflicts exist between the phylogenetic placement of certain taxa in rDNA trees and their expected position based on fossils, cytology, or protein-encoding gene sequences. For example, pelobiont amoebae appear to be an ancient group based on cytologic features, but they are not among the early eukaryotic brances in rDNA analyses. In this report, the derived position of pelobionts in rDNA trees is shown to be unreliable and likely due to long-branch attraction among more deeply branching sequences. All sequences that branch near the base of the tree suffer from relatively high apparent substitution rates and exhibit greater variation in ssu rDNA sequence length. Moreover, the order of the branches leading from the root of the eukaryotic tree to the base of the so-called "crown taxa" is consistent with a sequential attachment, due to "long-branch" effects, of sequences with increasing rates of evolution. These results suggest that the basal eurkaryotic topology drawn from rDNA analyses may be, in reality, an artifact of variation in the rate of molecular evolution among eukaryotic taxa.     

The chaperonin genes of jakobid and jakobid-like flagellates: implications for eukaryotic evolution

The jakobids are free-living mitochondriate protists that share ultrastructural features with certain amitochondriate groups and possess the most bacterial-like mitochondrial genomes described thus far. Jakobids belong to a diverse group of mitochondriate and amitochondriate eukaryotes, the excavate taxa. The relationships among the various excavate taxa and their relationships to other putative deep-branching protist groups are largely unknown. With the hope of clarifying these issues, we have isolated the cytosolic chaperonin CCTalpha gene from the jakobid Reclinomonas americana (strains 50394 and 50283), the jakobid-like malawimonad Malawimonas jakobiformis, two heteroloboseans (Acrasis rosea and Naegleria gruberi), a euglenozoan (Trypanosoma brucei), and a parabasalid (Monocercomonas sp.). We also amplified the CCTdelta gene from M. jakobiformis. The Reclinomonas and Malawimonas sequences presented here are among the first nuclear protein-coding genes to be described from these organisms. Unlike other putative early diverging protist lineages, a high density of spliceosomal introns was found in the jakobid and malawimonad CCTs-similar to that observed in vertebrate protein-coding genes. An analysis of intron positions in CCT genes from protists, plants, animals, and fungi suggests that many of the intron-sparse or intron-lacking protist lineages may not be primitively so but have lost spliceosomal introns during their evolutionary history. In phylogenetic trees constructed from CCTalpha protein sequences, R. americana (but not M. jakobiformis) shows a weak but consistent affinity for the Heterolobosea and Euglenozoa.     

Phylogenetic affinity of a Giardia lamblia cysteine desulfurase conforms to canonical pattern of mitochondrial ancestry

Among a few potential archezoan groups, only the Metamonada (diplomonads, retortamonads, and oxymonads) still retain the status of amitochondriate protists that diverged before the acquisition or retention of mitochondria. Indeed, finding that diplomonad genomes harbor a gene encoding a mitochondrial type chaperonin 60, the most compelling evidence for their secondarily amitochondriate nature, may be interpreted as an acquisition of this important general chaperone during some transient alpha-proteobacterial endosymbiosis. Recently published data on the cysteine desulfurase IscS demonstrated an alpha-proteobacterial origin of mitochondrial enzymes including a diplomonad Giardia lamblia homolog. An extended phylogenetic analysis of IscS is reported here that revealed a full canonical pattern of mitochondrial ancestry for the giardial enzyme. The above canonical pattern, a sister group relationship of mitochondria and rickettsiae exclusive of free-living alpha-proteobacteria, was robustly confirmed by a comprehensive analysis of Cob and Cox1 subunits of the respiratory chain encoded by resident mitochondrial genes. Given that Fe-S cluster assembly involving IscS represents an essential mitochondrial function, these data strongly suggest that diplomonads once harbored bona fide mitochondria.     

Phylogenetic and primary sequence characterization of cathepsin B cysteine proteases from the oxymonad flagellate Monocercomonoides

Cysteine proteases are crucial for general lysosomal function and for the pathogenic mechanisms of many protistan parasites. Cathepsin B cysteine proteases are currently defined by the presence of the "occluding loop" motif and have been best characterized from humans and their parasites. Though related to a variety of pathogenic excavate flagellates, oxymonads are themselves commensals. While studying this cell biologically aberrant protist lineage, we identified 11 different cathepsin B homologues. These were found to be expressed, at comparable levels to common house-keeping genes, such as elongation factor 1-alpha, alpha-tubulin, beta-tubulin, and glyceraldehyde phosphate dehydrogenase. Primary structure examination of the cathepsin B homologues identified putative signal peptide sequences, and the pre-, pro-, and mature domains of the protein. However, the occluding loop motif was either partially or entirely absent. Comparative genomics, sequence alignment, and phylogenetics of cathepsin sequences from across the diversity of eukaryotes demonstrated that absence of the occluding loop is not a feature exclusive to oxymonads, but is relatively common, suggesting that the "occluding loop" should no longer be used as the defining feature of the cathepsin B subfamily. Overall, this report identifies an abundant protein family in oxymonads, and provides insight both into the evolution and classification of cathepsin B cysteine proteases.     

Turning the crown upside down: gene tree parsimony roots the eukaryotic tree of life

The first analyses of gene sequence data indicated that the eukaryotic tree of life consisted of a long stem of microbial groups "topped" by a crown-containing plants, animals, and fungi and their microbial relatives. Although more recent multigene concatenated analyses have refined the relationships among the many branches of eukaryotes, the root of the eukaryotic tree of life has remained elusive. Inferring the root of extant eukaryotes is challenging because of the age of the group (～1.7-2.1 billion years old), tremendous heterogeneity in rates of evolution among lineages, and lack of obvious outgroups for many genes. Here, we reconstruct a rooted phylogeny of extant eukaryotes based on minimizing the number of duplications and losses among a collection of gene trees. This approach does not require outgroup sequences or assumptions of orthology among sequences. We also explore the impact of taxon and gene sampling and assess support for alternative hypotheses for the root. Using 20 gene trees from 84 diverse eukaryotic lineages, this approach recovers robust eukaryotic clades and reveals evidence for a eukaryotic root that lies between the Opisthokonta (animals, fungi and their microbial relatives) and all remaining eukaryotes.     

A Phylogenetic Perspective on Locomotory Strategies in Early Amniotes

Past approaches to understanding the evolution of locomotorystrategies among Paleozoic amniotes ("primitive reptiles" ofprevious parlance) have been influenced by preservational bias:early occurrences of some amniote taxa were used to polarizethe acquisition or development of locomotory structures amongthe earliest amniotes. Using a phylogeny representing the currentconsensus in the literature, we investigate the major locomotorystrategies that have been posited for Paleozoic amniotes (basalsynapsids on one hand and early reptiles on the other) by optimizingthe major locomotory styles identified for these taxa onto theconsensus tree, in order to present an overview of the patternof evolution of locomotory strategies inherited and adoptedby various amniote lineages.     

Evolutionary relationships of eukaryotic kingdoms

The evolutionary relationships of four eukaryotic kingdoms—Animalia, Plantae, Fungi, and Protista—remain unclear. In particular, statistical support for the closeness of animals to fungi rather than to plants is lacking, and a preferred branching order of these and other eukaryotic lineages is still controversial even though molecular sequences from diverse eukaryotic taxa have been analyzed. We report a statistical analysis of 214 sequences of nuclear small-subunit ribosomal RNA (srRNA) gene undertaken to clarify these evolutionary relationships. We have considered the variability of substitution rates and the nonindependence of nucleotide substitution across sites in the srRNA gene in testing alternative hypotheses regarding the branching patterns of eukaryote phylogeny. We find that the rates of evolution among sites in the srRNA sequences vary substantially and are approximately gamma distributed with size and shape parameter equal to 0.76. Our results suggest that (1) the animals and true fungi are indeed closer to each other than to any other crown group in the eukaryote tree, (2) red algae are the closest relatives of animals, true fungi, and green plants, and (3) the heterokonts and alveolates probably evolved prior to the divergence of red algae and animal-fungus-green-plant lineages. Furthermore, our analyses indicate that the branching order of the eukaryotic lineages that diverged prior to the evolution of alveolates may be generally difficult to resolve with the srRNA sequence data.