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Abstract Sterols are key components of eukaryotic cellular membranes that are synthesized by multi-enzyme pathways that require molecular oxygen. Because prokaryotes fundamentally lack sterols, it is unclear how the vast diversity of bacterivorous eukaryotes that inhabit hypoxic environments obtain, or synthesize, sterols. Here we show that tetrahymanol, a triterpenoid that does not require molecular oxygen for its biosynthesis, likely functions as a surrogate of sterol in eukaryotes inhabiting oxygen-poor environments. Genes encoding the tetrahymanol synthesizing enzyme squalene-tetrahymanol cyclase were found from several phylogenetically diverged eukaryotes that live in oxygen-poor environments and appear to have been laterally transferred among such eukaryotes. Reviewers This article was reviewed by Eric Bapteste and Eugene Koonin.

Abstract Background The covarion hypothesis of molecular evolution holds that selective pressures on a given amino acid or nucleotide site are dependent on the identity of other sites in the molecule that change throughout time, resulting in changes of evolutionary rates of sites along the branches of a phylogenetic tree. At the sequence level, covarion-like evolution at a site manifests as conservation of nucleotide or amino acid states among some homologs where the states are not conserved in other homologs (or groups of homologs). Covarion-like evolution has been shown to relate to changes in functions at sites in different clades, and, if ignored, can adversely affect the accuracy of phylogenetic inference. Results PROCOV (protein covarion analysis) is a software tool that implements a number of previously proposed covarion models of protein evolution for phylogenetic inference in a maximum likelihood framework. Several algorithmic and implementation improvements in this tool over previous versions make computationally expensive tree searches with covarion models more efficient and analyses of large phylogenomic data sets tractable. PROCOV can be used to identify covarion sites by comparing the site likelihoods under the covarion process to the corresponding site likelihoods under a rates-across-sites (RAS) process. Those sites with the greatest log-likelihood difference between a 'covarion' and an RAS process were found to be of functional or structural significance in a dataset of bacterial and eukaryotic elongation factors. Conclusion Covarion models implemented in PROCOV may be especially useful for phylogenetic estimation when ancient divergences between sequences have occurred and rates of evolution at sites are likely to have changed over the tree. It can also be used to study lineage-specific functional shifts in protein families that result in changes in the patterns of site variability among subtrees.

Abstract Background Widely used substitution models for proteins, such as the Jones-Taylor-Thornton (JTT) or Whelan and Goldman (WAG) models, are based on empirical amino acid interchange matrices estimated from databases of protein alignments that incorporate the average amino acid frequencies of the data set under examination (e.g JTT + F). Variation in the evolutionary process between sites is typically modelled by a rates-across-sites distribution such as the gamma (Γ) distribution. However, sites in proteins also vary in the kinds of amino acid interchanges that are favoured, a feature that is ignored by standard empirical substitution matrices. Here we examine the degree to which the pattern of evolution at sites differs from that expected based on empirical amino acid substitution models and evaluate the impact of these deviations on phylogenetic estimation. Results We analyzed 21 large protein alignments with two statistical tests designed to detect deviation of site-specific amino acid distributions from data simulated under the standard empirical substitution model: JTT+ F + Γ. We found that the number of states at a given site is, on average, smaller and the frequencies of these states are less uniform than expected based on a JTT + F + Γ substitution model. With a four-taxon example, we show that phylogenetic estimation under the JTT + F + Γ model is seriously biased by a long-branch attraction artefact if the data are simulated under a model utilizing the observed site-specific amino acid frequencies from an alignment. Principal components analyses indicate the existence of at least four major site-specific frequency classes in these 21 protein alignments. Using a mixture model with these four separate classes of site-specific state frequencies plus a fifth class of global frequencies (the JTT + cF + Γ model), significant improvements in model fit for real data sets can be achieved. This simple mixture model also reduces the long-branch attraction problem, as shown by simulations and analyses of a real phylogenomic data set. Conclusion Protein families display site-specific evolutionary dynamics that are ignored by standard protein phylogenetic models. Accurate estimation of protein phylogenies requires models that accommodate the heterogeneity in the evolutionary process across sites. To this end, we have implemented a class frequency mixture model (cF) in a freely available program called QmmRAxML for phylogenetic estimation.

Abstract Background Fornicata is a relatively recently established group of protists that includes the diplokaryotic diplomonads (which have two similar nuclei per cell), and the monokaryotic enteromonads, retortamonads and Carpediemonas, with the more typical one nucleus per cell. The monophyly of the group was confirmed by molecular phylogenetic studies, but neither the internal phylogeny nor its position on the eukaryotic tree has been clearly resolved. Results Here we have introduced data for three genes (SSU rRNA, α-tubulin and HSP90) with a wide taxonomic sampling of Fornicata, including ten isolates of enteromonads, representing the genera Trimitus and Enteromonas, and a new undescribed enteromonad genus. The diplomonad sequences formed two main clades in individual gene and combined gene analyses, with Giardia (and Octomitus) on one side of the basal divergence and Spironucleus, Hexamita and Trepomonas on the other. Contrary to earlier evolutionary scenarios, none of the studied enteromonads appeared basal to diplokaryotic diplomonads. Instead, the enteromonad isolates were all robustly situated within the second of the two diplomonad clades. Furthermore, our analyses suggested that enteromonads do not constitute a monophyletic group, and enteromonad monophyly was statistically rejected in 'approximately unbiased' tests of the combined gene data. Conclusion We suggest that all higher taxa intended to unite multiple enteromonad genera be abandoned, that Trimitus and Enteromonas be considered as part of Hexamitinae, and that the term 'enteromonads' be used in a strictly utilitarian sense. Our result suggests either that the diplokaryotic condition characteristic of diplomonads arose several times independently, or that the monokaryotic cell of enteromonads originated several times independently by secondary reduction from the diplokaryotic state. Both scenarios are evolutionarily complex. More comparative data on the similarity of the genomes of the two nuclei of diplomonads will be necessary to resolve which evolutionary scenario is more probable.

Abstract Background Comparative genomic studies of the mitochondrion-lacking protist group Diplomonadida (diplomonads) has been lacking, although Giardia lamblia has been intensively studied. We have performed a sequence survey project resulting in 2341 expressed sequence tags (EST) corresponding to 853 unique clones, 5275 genome survey sequences (GSS), and eleven finished contigs from the diplomonad fish parasite Spironucleus salmonicida (previously described as S. barkhanus). Results The analyses revealed a compact genome with few, if any, introns and very short 3' untranslated regions. Strikingly different patterns of codon usage were observed in genes corresponding to frequently sampled ESTs versus genes poorly sampled, indicating that translational selection is influencing the codon usage of highly expressed genes. Rigorous phylogenomic analyses identified 84 genes – mostly encoding metabolic proteins – that have been acquired by diplomonads or their relatively close ancestors via lateral gene transfer (LGT). Although most acquisitions were from prokaryotes, more than a dozen represent likely transfers of genes between eukaryotic lineages. Many genes that provide novel insights into the genetic basis of the biology and pathogenicity of this parasitic protist were identified including 149 that putatively encode variant-surface cysteine-rich proteins which are candidate virulence factors. A number of genomic properties that distinguish S. salmonicida from its human parasitic relative G. lamblia were identified such as nineteen putative lineage-specific gene acquisitions, distinct mutational biases and codon usage and distinct polyadenylation signals. Conclusion Our results highlight the power of comparative genomic studies to yield insights into the biology of parasitic protists and the evolution of their genomes, and suggest that genetic exchange between distantly-related protist lineages may be occurring at an appreciable rate in eukaryote genome evolution.

Abstract Background Glycolysis and subsequent fermentation is the main energy source for many anaerobic organisms. The glycolytic pathway consists of ten enzymatic steps which appear to be universal amongst eukaryotes. However, it has been shown that the origins of these enzymes in specific eukaryote lineages can differ, and sometimes involve lateral gene transfer events. We have conducted an expressed sequence tag (EST) survey of the anaerobic flagellate Trimastix pyriformis to investigate the nature of the evolutionary origins of the glycolytic enzymes in this relatively unstudied organism. Results We have found genes in the Trimastix EST data that encode enzymes potentially catalyzing nine of the ten steps of the glycolytic conversion of glucose to pyruvate. Furthermore, we have found two different enzymes that in principle could catalyze the conversion of phosphoenol pyruvate (PEP) to pyruvate (or the reverse reaction) as part of the last step in glycolysis. Our phylogenetic analyses of all of these enzymes revealed at least four cases where the relationship of the Trimastix genes to homologs from other species is at odds with accepted organismal relationships. Although lateral gene transfer events likely account for these anomalies, with the data at hand we were not able to establish with confidence the bacterial donor lineage that gave rise to the respective Trimastix enzymes. Conclusion A number of the glycolytic enzymes of Trimastix have been transferred laterally from bacteria instead of being inherited from the last common eukaryotic ancestor. Thus, despite widespread conservation of the glycolytic biochemical pathway across eukaryote diversity, in a number of protist lineages the enzymatic components of the pathway have been replaced by lateral gene transfer from disparate evolutionary sources. It remains unclear if these replacements result from selectively advantageous properties of the introduced enzymes or if they are neutral outcomes of a gene transfer 'ratchet' from food or endosymbiotic organisms or a combination of both processes.

Abstract Background Lateral gene transfer (LGT) in eukaryotes from non-organellar sources is a controversial subject in need of further study. Here we present gene distribution and phylogenetic analyses of the genes encoding the hybrid-cluster protein, A-type flavoprotein, glucosamine-6-phosphate isomerase, and alcohol dehydrogenase E. These four genes have a limited distribution among sequenced prokaryotic and eukaryotic genomes and were previously implicated in gene transfer events affecting eukaryotes. If our previous contention that these genes were introduced by LGT independently into the diplomonad and Entamoeba lineages were true, we expect that the number of putative transfers and the phylogenetic signal supporting LGT should be stable or increase, rather than decrease, when novel eukaryotic and prokaryotic homologs are added to the analyses. Results The addition of homologs from phagotrophic protists, including several Entamoeba species, the pelobiont Mastigamoeba balamuthi, and the parabasalid Trichomonas vaginalis, and a large quantity of sequences from genome projects resulted in an apparent increase in the number of putative transfer events affecting all three domains of life. Some of the eukaryotic transfers affect a wide range of protists, such as three divergent lineages of Amoebozoa, represented by Entamoeba, Mastigamoeba, and Dictyostelium, while other transfers only affect a limited diversity, for example only the Entamoeba lineage. These observations are consistent with a model where these genes have been introduced into protist genomes independently from various sources over a long evolutionary time. Conclusion Phylogenetic analyses of the updated datasets using more sophisticated phylogenetic methods, in combination with the gene distribution analyses, strengthened, rather than weakened, the support for LGT as an important mechanism affecting the evolution of these gene families. Thus, gene transfer seems to be an on-going evolutionary mechanism by which genes are spread between unrelated lineages of all three domains of life, further indicating the importance of LGT from non-organellar sources into eukaryotic genomes.

Background An increasing number of bioinformatics methods are considering the phylogenetic relationships between biological sequences. Implementing new methodologies using the maximum likelihood phylogenetic framework can be a time consuming task. Results The bioinformatics library libcov is a collection of C++ classes that provides a high and low-level interface to maximum likelihood phylogenetics, sequence analysis and a data structure for structural biological methods. libcov can be used to compute likelihoods, search tree topologies, estimate site rates, cluster sequences, manipulate tree structures and compare phylogenies for a broad selection of applications. Conclusion Using this library, it is possible to rapidly prototype applications that use the sophistication of phylogenetic likelihoods without getting involved in a major software engineering project. libcov is thus a potentially valuable building block to develop in-house methodologies in the field of protein phylogenetics.

Abstract Background Lateral gene transfer can introduce genes with novel functions into genomes or replace genes with functionally similar orthologs or paralogs. Here we present a study of the occurrence of the latter gene replacement phenomenon in the four gene families encoding different classes of glutamate dehydrogenase (GDH), to evaluate and compare the patterns and rates of lateral gene transfer (LGT) in prokaryotes and eukaryotes. Results We extend the taxon sampling of gdh genes with nine new eukaryotic sequences and examine the phylogenetic distribution pattern of the various GDH classes in combination with maximum likelihood phylogenetic analyses. The distribution pattern analyses indicate that LGT has played a significant role in the evolution of the four gdh gene families. Indeed, a number of gene transfer events are identified by phylogenetic analyses, including numerous prokaryotic intra-domain transfers, some prokaryotic inter-domain transfers and several inter-domain transfers between prokaryotes and microbial eukaryotes (protists). Conclusion LGT has apparently affected eukaryotes and prokaryotes to a similar extent within the gdh gene families. In the absence of indications that the evolution of the gdh gene families is radically different from other families, these results suggest that gene transfer might be an important evolutionary mechanism in microbial eukaryote genome evolution.

Metamonads are a large and exclusively anaerobic group of protists. Additionally, they are one of the three clades proposed to ancestrally possess an "excavate" cell morphology, with a conspicuous ventral groove accompanied by a posterior flagellum with a vane. Here, we cultivate and characterize four anaerobic bacterivorous flagellates from hypersaline and alkaline soda lake environments, which represent a novel clade. Small subunit ribosomal RNA (SSU rRNA) gene phylogenies support recent phylogenomic analyses in placing them as the sister of barthelonids, a group that is itself sister to or deeply branching within Fornicata (Metamonada). The new isolates have a distinctive morphology: the hunchbacked cell body is traversed by a narrow ventral groove ending in a large opening to a conspicuous recurrent cytopharynx. The right margin of the groove is defined by a thin "lip." The posterior flagellum bears a wide ventral‐facing vane. The narrow ventral groove and elongate cytopharynx are shared with barthelonids. We describe one isolate as Skoliomonas litria, gen. et sp. nov. Further investigation of their mitochondrial‐related organelles (MROs) and detailed ultrastructural studies would be important to understanding the adaptation to anaerobic conditions in Metamonads—especially fornicates—as well as the evolution of the "excavate" cell architecture. [ABSTRACT FROM AUTHOR]

Profile mixture models capture distinct biochemical constraints on the amino acid substitution process at different sites in proteins. These models feature a mixture of time-reversible models with a common matrix of exchangeabilities and distinct sets of equilibrium amino acid frequencies known as profiles. Combining the exchangeability matrix with each profile generates the matrix of instantaneous rates of amino acid exchange for that profile. Currently, empirically estimated exchangeability matrices (e.g. the LG matrix) are widely used for phylogenetic inference under profile mixture models. However, these were estimated using a single profile and are unlikely optimal for profile mixture models. Here, we describe the GTRpmix model that allows maximum likelihood estimation of a common exchangeability matrix under any profile mixture model. We show that exchangeability matrices estimated under profile mixture models differ from the LG matrix, dramatically improving model fit and topological estimation accuracy for empirical test cases. Because the GTRpmix model is computationally expensive, we provide two exchangeability matrices estimated from large concatenated phylogenomic-supermatrices to be used for phylogenetic analyses. One, called Eukaryotic Linked Mixture (ELM), is designed for phylogenetic analysis of proteins encoded by nuclear genomes of eukaryotes, and the other, Eukaryotic and Archaeal Linked mixture (EAL), for reconstructing relationships between eukaryotes and Archaea. These matrices, combined with profile mixture models, fit data better and have improved topology estimation relative to the LG matrix combined with the same mixture models. Starting with version 2.3.1, IQ-TREE2 allows users to estimate linked exchangeabilities (i.e. amino acid exchange rates) under profile mixture models. [ABSTRACT FROM AUTHOR]

Almost all eukaryote life forms have now been placed within one of five to eight supra-kingdom-level groups using molecular phylogenetics.sup.1-4. The 'phylum' Hemimastigophora is probably the most distinctive morphologically defined lineage that still awaits such a phylogenetic assignment. First observed in the nineteenth century, hemimastigotes are free-living predatory protists with two rows of flagella and a unique cell architecture.sup.5-7; to our knowledge, no molecular sequence data or cultures are currently available for this group. Here we report phylogenomic analyses based on high-coverage, cultivation-independent transcriptomics that place Hemimastigophora outside of all established eukaryote supergroups. They instead comprise an independent supra-kingdom-level lineage that most likely forms a sister clade to the 'Diaphoretickes' half of eukaryote diversity (that is, the 'stramenopiles, alveolates and Rhizaria' supergroup (Sar), Archaeplastida and Cryptista, as well as other major groups). The previous ranking of Hemimastigophora as a phylum understates the evolutionary distinctiveness of this group, which has considerable importance for investigations into the deep-level evolutionary history of eukaryotic life--ranging from understanding the origins of fundamental cell systems to placing the root of the tree. We have also established the first culture of a hemimastigote (Hemimastix kukwesjijk sp. nov.), which will facilitate future genomic and cell-biological investigations into eukaryote evolution and the last eukaryotic common ancestor. Phylogenetic analyses based on single-cell transcriptomic data from two hemimastigotes, a Spironema species and the newly described Hemimastix kukwesjijk, indicate that Hemimastigophora is a supra-kingdom-level lineage of eukaryotes., Author(s): Gordon Lax [sup.1] , Yana Eglit [sup.1] , Laura Eme [sup.2] [sup.3] , Erin M. Bertrand [sup.1] , Andrew J. Roger [sup.2] , Alastair G. B. Simpson [sup.1] Author [...]