Long-branch Attraction Artifact Research Articles

The network of life: genome beginnings and evolution

The genome sequence is an icon of early twenty-first century biology. Genomes of nearly 2000 cellular organisms, and from many thousands of organelles and viruses, are now in the public domain. For biological research in individual species, the genome sequence increasingly provides the common

Phylogenomic analyses support the monophyly of Excavata and resolve relationships among eukaryotic “supergroups”

Nearly all of eukaryotic diversity has been classified into 6 suprakingdom-level groups (supergroups) based on molecular and morphological/cell-biological evidence; these are Opisthokonta, Amoebozoa, Archaeplastida, Rhizaria, Chromalveolata, and Excavata. However, molecular phylogeny has not provided clear evidence that either Chromalveolata or Excavata is monophyletic, nor has it resolved the relationships among the supergroups. To establish the affinities of Excavata, which contains parasites of global importance and organisms regarded previously as primitive eukaryotes, we conducted a phylogenomic analysis of a dataset of 143 proteins and 48 taxa, including 19 excavates. Previous phylogenomic studies have not included all major subgroups of Excavata, and thus have not definitively addressed their interrelationships. The enigmatic flagellate Andalucia is sister to typical jakobids. Jakobids (including Andalucia), Euglenozoa and Heterolobosea form a major clade that we name Discoba. Analyses of the complete dataset group Discoba with the mitochondrion-lacking excavates or "metamonads" (diplomonads, parabasalids, and Preaxostyla), but not with the final excavate group, Malawimonas. This separation likely results from a long-branch attraction artifact. Gradual removal of rapidly-evolving taxa from the dataset leads to moderate bootstrap support (69%) for the monophyly of all Excavata, and 90% support once all metamonads are removed. Most importantly, Excavata robustly emerges between unikonts (Amoebozoa + Opisthokonta) and "megagrouping" of Archaeplastida, Rhizaria, and chromalveolates. Our analyses indicate that Excavata forms a monophyletic suprakingdom-level group that is one of the 3 primary divisions within eukaryotes, along with unikonts and a megagroup of Archaeplastida, Rhizaria, and the chromalveolate lineages.

Support Patterns from Different Outgroups Provide a Strong Phylogenetic Signal

It is known that the accuracy of phylogenetic reconstruction decreases when more distant outgroups are used. We quantify this phenomenon with a novel scoring method, the outgroup score pOG. This score expresses if the support for a particular branch of a tree decreases with increasingly distant outgroups. Large-scale simulations confirmed that the outgroup support follows this expectation and that the pOG score captures this pattern. The score often identifies the correct topology even when the primary reconstruction methods fail, particularly in the presence of model violations. In simulations of problematic phylogenetic scenarios such as rate variation among lineages (which can lead to long-branch attraction artifacts) and quartet-based reconstruction, the pOG analysis outperformed the primary reconstruction methods. Because the pOG method does not make any assumptions about the evolutionary model (besides the decreasing support from increasingly distant outgroups), it can detect cases of violations not treated by a specific model or too strong to be fully corrected. When used as an optimization criterion in the construction of a tree of 23 mammals, the outgroup signal confirmed many well-accepted mammalian orders and superorders. It supports Atlantogenata, a clade of Afrotheria and Xenarthra, and suggests an Artiodactyla-Chiroptera clade.

Evolution: Revisiting the Root of the Eukaryote Tree

A recent phylogenomic investigation shows that the enigmatic flagellate Breviata is a distinct anaerobic lineage within the eukaryote super-group Amoebozoa and challenges the unikont-bikont rooting of the tree of eukaryotes.

A class frequency mixture model that adjusts for site-specific amino acid frequencies and improves inference of protein phylogeny

BackgroundWidely 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.ResultsWe 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.ConclusionProtein 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.

Phylogenomic Analyses Support the Monophyly of Taphrinomycotina, including Schizosaccharomyces Fission Yeasts

Several morphologically dissimilar ascomycete fungi including Schizosaccharomyces, Taphrina, Saitoella, Pneumocystis, and Neolecta have been grouped into the taxon Taphrinomycotina (Archiascomycota or Archiascomycotina), originally based on rRNA phylogeny. These analyses lack statistically significant support for the monophyly of this grouping, and although confirmed by more recent multigene analyses, this topology is contradicted by mitochondrial phylogenies. To resolve this inconsistency, we have assembled phylogenomic mitochondrial and nuclear data sets from four distantly related taphrinomycotina taxa: Schizosaccharomyces pombe, Pneumocystis carinii, Saitoella complicata, and Taphrina deformans. Our phylogenomic analyses based on nuclear data (113 proteins) conclusively support the monophyly of Taphrinomycotina, diverging as a sister group to Saccharomycotina + Pezizomycotina. However, despite the improved taxon sampling, Taphrinomycotina continue to be paraphyletic with the mitochondrial data set (13 proteins): Schizosaccharomyces species associate with budding yeasts (Saccharomycotina) and the other Taphrinomycotina group as a sister group to Saccharomycotina + Pezizomycotina. Yet, as Schizosaccharomyces and Saccharomycotina species are fast evolving, the mitochondrial phylogeny may be influenced by a long-branch attraction (LBA) artifact. After removal of fast-evolving sequence positions from the mitochondrial data set, we recover the monophyly of Taphrinomycotina. Our combined results suggest that Taphrinomycotina is a legitimate taxon, that this group of species diverges as a sister group to Saccharomycotina + Pezizomycotina, and that phylogenetic positioning of yeasts and fission yeasts with mitochondrial data is plagued by a strong LBA artifact.

Parasitism and Mutualism in Wolbachia: What the Phylogenomic Trees Can and Cannot Say

Ecological and evolutionary theories predict that parasitism and mutualism are not fixed endpoints of the symbiotic spectrum. Rather, parasitism and mutualism may be host or environment dependent, induced by the same genetic machinery, and shifted due to selection. These models presume the existence of genetic or environmental variation that can spur incipient changes in symbiotic lifestyle. However, for obligate intracellular bacteria whose genomes are highly reduced, studies specify that discrete symbiotic associations can be evolutionarily stable for hundreds of millions of years. Wolbachia is an inherited obligate, intracellular infection of invertebrates containing taxa that act broadly as both parasites in arthropods and mutualists in certain roundworms. Here, we analyze the ancestry of mutualism and parasitism in Wolbachia and the evolutionary trajectory of this variation in symbiotic lifestyle with a comprehensive, phylogenomic analysis. Contrary to previous claims, we show unequivocally that the transition in lifestyle cannot be reconstructed with current methods due to long-branch attraction (LBA) artifacts of the distant Anaplasma and Ehrlichia outgroups. Despite the use of 1) site-heterogenous phylogenomic methods that can overcome systematic error, 2) a taxonomically rich set of taxa, and 3) statistical assessments of the genes, tree topologies, and models of evolution, we conclude that the LBA artifact is serious enough to afflict past and recent claims including the root lies in the middle of the Wolbachia mutualists and parasites. We show that different inference methods yield different results and high bootstrap support did not equal phylogenetic accuracy. Recombination was rare among this taxonomically diverse data set, indicating that elevated levels of recombination in Wolbachia are restricted to specific coinfecting groups. In conclusion, we attribute the inability to root the tree to rate heterogeneity between the ingroup and outgroup. Site-heterogenous models of evolution did improve the placement of aberrant taxa in the ingroup phylogeny. Finally, in the unrooted topology, the distribution of parasitism and mutualism across the tree suggests that at least two interphylum transfers shaped the origins of nematode mutualism and arthropod parasitism. We suggest that the ancestry of mutualism and parasitism is not resolvable without more suitable outgroups or complete genome sequences from all Wolbachia supergroups.

Newly discovered sister lineage sheds light on early ant evolution.

Ants are the world's most conspicuous and important eusocial insects and their diversity, abundance, and extreme behavioral specializations make them a model system for several disciplines within the biological sciences. Here, we report the discovery of a new ant that appears to represent the sister lineage to all extant ants (Hymenoptera: Formicidae). The phylogenetic position of this cryptic predator from the soils of the Amazon rainforest was inferred from several nuclear genes, sequenced from a single leg. Martialis heureka (gen. et sp. nov.) also constitutes the sole representative of a new, morphologically distinct subfamily of ants, the Martialinae (subfam. nov.). Our analyses have reduced the likelihood of long-branch attraction artifacts that have troubled previous phylogenetic studies of early-diverging ants and therefore solidify the emerging view that the most basal extant ant lineages are cryptic, hypogaeic foragers. On the basis of morphological and phylogenetic evidence we suggest that these specialized subterranean predators are the sole surviving representatives of a highly divergent lineage that arose near the dawn of ant diversification and have persisted in ecologically stable environments like tropical soils over great spans of time.

Effect of taxon sampling on recovering the phylogeny of squamate reptiles based on complete mitochondrial genome and nuclear gene sequence data

The complete nucleotide sequences of the mitochondrial (mt) genomes of three species of squamate lizards: Blanus cinereus (Amphisbaenidae), Anguis fragilis (Anguidae), and Tarentola mauritanica (Geckkonidae) were determined anew. The deduced amino acid sequences of all 13 mt protein-coding genes were combined into a single data set and phylogenetic relationships among main squamate lineages were analyzed under maximum likelihood (ML) and Bayesian Inference (BI). Within Squamata, the monophyly of Iguanidae, Anguimorpha, Amphisbaenia, Gekkota, Serpentes, and Acrodonta received high statistical support with both methods. It is particularly striking that this is the first molecular analysis that recovers the monophyly of Scincomorpha (including Scincidae, Xantusiidae, Cordylidae, and Lacertidae), although it is only supported in the Bayesian analysis, and it is sensitive to changes in the outgroup (see below). Phylogenetic relationships among the main squamate lineages could not be resolved with ML but received strong support with BI (above 95%). The newly reconstructed phylogeny of squamates does not support the Iguania–Scleroglossa split. Acrodonta and Serpentes form a clade, which is the sister group of the remaining squamate lineages. Within these, Gekkota were the first branching out, followed by Amphisbaenia, and a clade including Anguimorpha as sister group of Scincomorpha + Iguanidae. The recovered topology differed substantially from previously reported hypotheses on squamate relationships, and the relative effect of using different outgroups, genes, and taxon samplings were explored. The sister group relationship of Serpentes + Acrodonta, and their relative basal position within Squamata could be due to a long-branch attraction artifact. Phylogenetic relationships among Scincomorpha, Amphisbaenia, and Anguimorpha were found to be rather unresolved. Future improving of squamate phylogenetic relationships would rely on finding snake and acrodont species with slower mt evolutionary rates, ensuring thorough taxon coverage of squamate diversity, and incorporating more nuclear genes with appropriate evolutionary rates.

Phylogenetic placement of the enigmatic parasite, Polypodium hydriforme, within the Phylum Cnidaria.

BackgroundPolypodium hydriforme is a parasite with an unusual life cycle and peculiar morphology, both of which have made its systematic position uncertain. Polypodium has traditionally been considered a cnidarian because it possesses nematocysts, the stinging structures characteristic of this phylum. However, recent molecular phylogenetic studies using 18S rDNA sequence data have challenged this interpretation, and have shown that Polypodium is a close relative to myxozoans and together they share a closer affinity to bilaterians than cnidarians. Due to the variable rates of 18S rDNA sequences, these results have been suggested to be an artifact of long-branch attraction (LBA). A recent study, using multiple protein coding markers, shows that the myxozoan Buddenbrockia, is nested within cnidarians. Polypodium was not included in this study. To further investigate the phylogenetic placement of Polypodium, we have performed phylogenetic analyses of metazoans with 18S and partial 28S rDNA sequences in a large dataset that includes Polypodium and a comprehensive sampling of cnidarian taxa.ResultsAnalyses of a combined dataset of 18S and partial 28S sequences, and partial 28S alone, support the placement of Polypodium within Cnidaria. Removal of the long-branched myxozoans from the 18S dataset also results in Polypodium being nested within Cnidaria. These results suggest that previous reports showing that Polypodium and Myxozoa form a sister group to Bilateria were an artifact of long-branch attraction.ConclusionBy including 28S rDNA sequences and a comprehensive sampling of cnidarian taxa, we demonstrate that previously conflicting hypotheses concerning the phylogenetic placement of Polypodium can be reconciled. Specifically, the data presented provide evidence that Polypodium is indeed a cnidarian and is either the sister taxon to Hydrozoa, or part of the hydrozoan clade, Leptothecata. The former hypothesis is consistent with the traditional view that Polypodium should be placed in its own cnidarian class, Polypodiozoa.

Animal phylogeny and large-scale sequencing: progress and pitfalls

Phylogenomics, the inference of phylogenetic trees using genome-scale data, is becoming the rule for resolving difficult parts of the tree of life. Its promise resides in the large amount of information available, which should eliminate stochastic error. However, systematic error, which is due to limitations of reconstruction meth- ods, is becoming more apparent. We will illustrate, using animal phylogeny as a case study, the three most effi- cient approaches to avoid the pitfalls of phylogenomics: (1) using a dense taxon sampling, (2) using probabilistic methods with complex models of sequence evolution that more accurately detect multiple substitutions, and (3) removing the fastest evolving part of the data (e.g., species and positions). The analysis of a dataset of 55 animal species and 102 proteins (25712 amino acid positions) shows that standard site-homogeneous model inference is sensitive to long-branch attraction artifact, whereas the site-heterogeneous CAT model is less so. The latter model correctly locates three very fast evolving species, the appendicularian tunicate Oikopleura, the acoel Convoluta and the myxozoan Buddenbrockia. Overall, the resulting tree is in excellent agreement with the new animal phylogeny, confirming that simple organisms like platyhelminths and nematodes are not necessarily of basal emergence. This further emphasizes the importance of secondary simplification in animals, and for organismal evolution in general.

The complete mitochondrial genome of the sea spider Achelia bituberculata (Pycnogonida, Ammotheidae): arthropod ground pattern of gene arrangement.

BackgroundThe phylogenetic position of pycnogonids is a long-standing and controversial issue in arthropod phylogeny. This controversy has recently been rekindled by differences in the conclusions based on neuroanatomical data concerning the chelifore and the patterns of Hox expression. The mitochondrial genome of a sea spider, Nymphon gracile (Pycnogonida, Nymphonidae), was recently reported in an attempt to address this issue. However, N. gracile appears to be a long-branch taxon on the phylogenetic tree and exhibits a number of peculiar features, such as 10 tRNA translocations and even an inversion of several protein-coding genes. Sequences of other pycnogonid mitochondrial genomes are needed if the position of pycnogonids is to be elucidated on this basis.ResultsThe complete mitochondrial genome (15,474 bp) of a sea spider (Achelia bituberculata) belonging to the family Ammotheidae, which combines a number of anatomical features considered plesiomorphic with respect to other pycnogonids, was sequenced and characterized. The genome organization shows the features typical of most metazoan animal genomes (37 tightly-packed genes). The overall gene arrangement is completely identical to the arthropod ground pattern, with one exception: the position of the trnQ gene between the rrnS gene and the control region. Maximum likelihood and Bayesian inference trees inferred from the amino acid sequences of mitochondrial protein-coding genes consistently indicate that the pycnogonids (A. bituberculata and N. gracile) may be closely related to the clade of Acari and Araneae.ConclusionThe complete mitochondrial genome sequence of A. bituberculata (Family Ammotheidae) and the previously-reported partial sequence of Endeis spinosa show the gene arrangement patterns typical of arthropods (Limulus-like), but they differ markedly from that of N. gracile. Phylogenetic analyses based on mitochondrial protein-coding genes showed that Pycnogonida may be authentic arachnids (= aquatic arachnids) within Chelicerata sensu lato, as indicated by the name 'sea spider,' and suggest that the Cormogonida theory – that the pycnogonids are a sister group of all other arthropods – should be rejected. However, in view of the relatively weak node confidence, strand-biased nucleotide composition and long-branch attraction artifact, further more intensive studies seem necessary to resolve the exact position of the pycnogonids.

Acoel Flatworms Are Not Platyhelminthes: Evidence from Phylogenomics

Acoel flatworms are small marine worms traditionally considered to belong to the phylum Platyhelminthes. However, molecular phylogenetic analyses suggest that acoels are not members of Platyhelminthes, but are rather extant members of the earliest diverging Bilateria. This result has been called into question, under suspicions of a long branch attraction (LBA) artefact. Here we re-examine this problem through a phylogenomic approach using 68 different protein-coding genes from the acoel Convoluta pulchra and 51 metazoan species belonging to 15 different phyla. We employ a mixture model, named CAT, previously found to overcome LBA artefacts where classical models fail. Our results unequivocally show that acoels are not part of the classically defined Platyhelminthes, making the latter polyphyletic. Moreover, they indicate a deuterostome affinity for acoels, potentially as a sister group to all deuterostomes, to Xenoturbellida, to Ambulacraria, or even to chordates. However, the weak support found for most deuterostome nodes, together with the very fast evolutionary rate of the acoel Convoluta pulchra, call for more data from slowly evolving acoels (or from its sister-group, the Nemertodermatida) to solve this challenging phylogenetic problem.

The phylogenetic relationship between Astigmata and Oribatida (Acari) as indicated by molecular markers

Astigmata comprise a diverse group of acariform mite species with a remarkable range of life histories, most of which involve parasitic or commensal relationships with other organisms. Several authors have suggested that Astigmata evolved as a paedomorphic clade from within Oribatida, and both morphology and gland-chemistry strongly suggest that their sister-clade is within the oribatid subgroup Desmonomata. The biologies of these groups contrast greatly, since oribatid mites are mostly soil-living detritivores and fungivores, and have life cycles that are much longer than those in Astigmata. We tested the hypothesis that Astigmata evolved from within Desmonomata using two molecular markers, the ribosomal 18S region (18S) and the nuclear elongation factor 1 alpha (ef1alpha) gene. Representative acariform mites included 28 species of Oribatida, eight of Astigmata, two of Prostigmata and two of Endeostigmata; outgroups included members of Opilioacariformes, Parasitiformes and Ricinulei. To minimize the possibility of long-branch attraction artifacts, we limited highly variable sites by removing gaps (18S) and third codon positions (ef1alpha) from the sequences. Maximum parsimony, neighbor-joining and Bayesian algorithms formed trees that consistently placed Astigmata outside monophyletic Oribatida, usually as sister-group of the endeostigmatid mite Alicorhagia sp. Analyses with and without outgroups resulted in similar topologies, showing no evidence for long-branch artifacts and leaving the conflict with morphological and biochemical data unexplained.

A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis

BackgroundTo date, most fungal phylogenies have been derived from single gene comparisons, or from concatenated alignments of a small number of genes. The increase in fungal genome sequencing presents an opportunity to reconstruct evolutionary events using entire genomes. As a tool for future comparative, phylogenomic and phylogenetic studies, we used both supertrees and concatenated alignments to infer relationships between 42 species of fungi for which complete genome sequences are available.ResultsA dataset of 345,829 genes was extracted from 42 publicly available fungal genomes. Supertree methods were employed to derive phylogenies from 4,805 single gene families. We found that the average consensus supertree method may suffer from long-branch attraction artifacts, while matrix representation with parsimony (MRP) appears to be immune from these. A genome phylogeny was also reconstructed from a concatenated alignment of 153 universally distributed orthologs. Our MRP supertree and concatenated phylogeny are highly congruent. Within the Ascomycota, the sub-phyla Pezizomycotina and Saccharomycotina were resolved. Both phylogenies infer that the Leotiomycetes are the closest sister group to the Sordariomycetes. There is some ambiguity regarding the placement of Stagonospora nodurum, the sole member of the class Dothideomycetes present in the dataset.Within the Saccharomycotina, a monophyletic clade containing organisms that translate CTG as serine instead of leucine is evident. There is also strong support for two groups within the CTG clade, one containing the fully sexual species Candida lusitaniae, Candida guilliermondii and Debaryomyces hansenii, and the second group containing Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis and Lodderomyces elongisporus. The second major clade within the Saccharomycotina contains species whose genomes have undergone a whole genome duplication (WGD), and their close relatives. We could not confidently resolve whether Candida glabrata or Saccharomyces castellii lies at the base of the WGD clade.ConclusionWe have constructed robust phylogenies for fungi based on whole genome analysis. Overall, our phylogenies provide strong support for the classification of phyla, sub-phyla, classes and orders. We have resolved the relationship of the classes Leotiomyctes and Sordariomycetes, and have identified two classes within the CTG clade of the Saccharomycotina that may correlate with sexual status.

A close relationship between Cercozoa and Foraminifera supported by phylogenetic analyses based on combined amino acid sequences of three cytoskeletal proteins (actin, α-tubulin, and β-tubulin)

Recently, there has been increasing molecular evidence of phylogenetic affinity between Cercozoa and Foraminifera in the eukaryotic lineage. We performed phylogenetic analyses based on the combined (concatenated) amino acid sequence data of actin, α-tubulin, and β-tubulin from a wide variety of eukaryotes, including the foraminifers Planoglabratella opercularis and Reticulomyxa filosa, as well as cercomonad and chlorarachniophyte members of Cercozoa. A monophyletic lineage composed of two foraminiferan species branched with the centroheliozoan species Raphidiophrys contractilis was reconstructed in both Bayesian and maximum-likelihood (ML) analyses under ‘linked’ models, enforcing a single set of the parameters (the parameter for among-site rate variation and branch lengths) on the entire combined alignment. Considering the extremely divergent nature of Foraminifera and Raphidiophyrs tubulins, the union of these lineages recovered is most probably a long-branch attraction artifact due to ignoring gene-specific evolutionary processes. On the other hand, the foraminiferan lineage was within the radiation of Cercozoa in Bayesian analyses under ‘unlinked’ model conditions, accommodating differences in evolutionary processes across the three genes in the combined alignment. The Foraminifera + Cercozoa affinity recovered in the latter multi-gene analyses is most likely genuine, and thus our data presented here provide further support for the close relationship between these two protist lineages.

Rate Asymmetry After Genome Duplication Causes Substantial Long-Branch Attraction Artifacts in the Phylogeny of Saccharomyces Species

Whole-genome duplication (WGD) produces sets of gene pairs that are all of the same age. We therefore expect that phylogenetic trees that relate these pairs to their orthologs in other species should show a single consistent topology. However, a previous study of gene pairs formed by WGD in the yeast Saccharomyces cerevisiae found conflicting topologies among neighbor-joining (NJ) trees drawn from different loci and suggested that this conflict was the result of "asynchronous functional divergence" of duplicated genes (Langkjaer, R. B., P. F. Cliften, M. Johnston, and J. Piskur. 2003. Yeast genome duplication was followed by asynchronous differentiation of duplicated genes. Nature 421:848-852). Here, we test whether the conflicting topologies might instead be due to asymmetrical rates of evolution leading to long-branch attraction (LBA) artifacts in phylogenetic trees. We constructed trees for 433 pairs of WGD paralogs in S. cerevisiae with their single orthologs in Saccharomyces kluyveri and Candida albicans. We find a strong correlation between the asymmetry of evolutionary rates of a pair of S. cerevisiae paralogs and the topology of the tree inferred for that pair. Saccharomyces cerevisiae gene pairs with approximately equal rates of evolution tend to give phylogenies in which the WGD postdates the speciation between S. cerevisiae and S. kluyveri (B-trees), whereas trees drawn from gene pairs with asymmetrical rates tend to show WGD pre-dating this speciation (A-trees). Gene order data from throughout the genome indicate that the "A-trees" are artifacts, even though more than 50% of gene pairs are inferred to have this topology when the NJ method as implemented in ClustalW (i.e., with Poisson correction of distances) is used to construct the trees. This LBA artifact can be ameliorated, but not eliminated, by using gamma-corrected distances or by using maximum likelihood trees with robustness estimated by the Shimodaira-Hasegawa test. Tests for adaptive evolution indicated that positive selection might be the cause of rate asymmetry in a substantial fraction (19%) of the paralog pairs.

An Empirical Assessment of Long-Branch Attraction Artefacts in Deep Eukaryotic Phylogenomics

In the context of exponential growing molecular databases, it becomes increasingly easy to assemble large multigene data sets for phylogenomic studies. The expected increase of resolution due to the reduction of the sampling (stochastic) error is becoming a reality. However, the impact of systematic biases will also become more apparent or even dominant. We have chosen to study the case of the long-branch attraction artefact (LBA) using real instead of simulated sequences. Two fast-evolving eukaryotic lineages, whose evolutionary positions are well established, microsporidia and the nucleomorph of cryptophytes, were chosen as model species. A large data set was assembled (44 species, 133 genes, and 24,294 amino acid positions) and the resulting rooted eukaryotic phylogeny (using a distant archaeal outgroup) is positively misled by an LBA artefact despite the use of a maximum likelihood-based tree reconstruction method with a complex model of sequence evolution. When the fastest evolving proteins from the fast lineages are progressively removed (up to 90%), the bootstrap support for the apparently artefactual basal placement decreases to virtually 0%, and conversely only the expected placement, among all the possible locations of the fast-evolving species, receives increasing support that eventually converges to 100%. The percentage of removal of the fastest evolving proteins constitutes a reliable estimate of the sensitivity of phylogenetic inference to LBA. This protocol confirms that both a rich species sampling (especially the presence of a species that is closely related to the fast-evolving lineage) and a probabilistic method with a complex model are important to overcome the LBA artefact. Finally, we observed that phylogenetic inference methods perform strikingly better with simulated as opposed to real data, and suggest that testing the reliability of phylogenetic inference methods with simulated data leads to overconfidence in their performance. Although phylogenomic studies can be affected by systematic biases, the possibility of discarding a large amount of data containing most of the nonphylogenetic signal allows recovering a phylogeny that is less affected by systematic biases, while maintaining a high statistical support.

Identifying the Basal Angiosperm Node in Chloroplast Genome Phylogenies: Sampling One's Way Out of the Felsenstein Zone

While there has been strong support for Amborella and Nymphaeales (water lilies) as branching from basal-most nodes in the angiosperm phylogeny, this hypothesis has recently been challenged by phylogenetic analyses of 61 protein-coding genes extracted from the chloroplast genome sequences of Amborella, Nymphaea, and 12 other available land plant chloroplast genomes. These character-rich analyses placed the monocots, represented by three grasses (Poaceae), as sister to all other extant angiosperm lineages. We have extracted protein-coding regions from draft sequences for six additional chloroplast genomes to test whether this surprising result could be an artifact of long-branch attraction due to limited taxon sampling. The added taxa include three monocots (Acorus, Yucca, and Typha), a water lily (Nuphar), a ranunculid (Ranunculus), and a gymnosperm (Ginkgo). Phylogenetic analyses of the expanded DNA and protein data sets together with microstructural characters (indels) provided unambiguous support for Amborella and the Nymphaeales as branching from the basal-most nodes in the angiosperm phylogeny. However, their relative positions proved to be dependent on the method of analysis, with parsimony favoring Amborella as sister to all other angiosperms and maximum likelihood (ML) and neighbor-joining methods favoring an Amborella + Nymphaeales clade as sister. The ML phylogeny supported the later hypothesis, but the likelihood for the former hypothesis was not significantly different. Parametric bootstrap analysis, single-gene phylogenies, estimated divergence dates, and conflicting indel characters all help to illuminate the nature of the conflict in resolution of the most basal nodes in the angiosperm phylogeny. Molecular dating analyses provided median age estimates of 161 MYA for the most recent common ancestor (MRCA) of all extant angiosperms and 145 MYA for the MRCA of monocots, magnoliids, and eudicots. Whereas long sequences reduce variance in branch lengths and molecular dating estimates, the impact of improved taxon sampling on the rooting of the angiosperm phylogeny together with the results of parametric bootstrap analyses demonstrate how long-branch attraction might mislead genome-scale phylogenetic analyses.

Evidence for Multiple Reversals of Asymmetric Mutational Constraints during the Evolution of the Mitochondrial Genome of Metazoa, and Consequences for Phylogenetic Inferences

Mitochondrial DNA (mtDNA) sequences are comonly used for inferring phylogenetic relationships. However, the strand-specific bias in the nucleotide composition of the mtDNA, which is thought to reflect assymetric mutational constraints, combined with the important compositional heterogeneity among taxa, are known to be highly problematic for phylogenetic analyses. Here, nucleotide composition was compared across 49 species of Metazoa (34 arthropods, 2 annelids, 2 molluscs, and 11 deuterosomes), and analyzed for a mtDNA fragment including six protein-coding genes, i.e., atp6, atp8, cox1, cox2, cox3, and nad2. The analyses show that most metazoan species present a clear strand assymetry, where one strand is biased in favor of A and C, whereas the other strand has reverse bias, i.e. in favor of T and G. the origin of this strand bias can be related to assymetric mutational constraints involving deaminations of A and C nucleotides during the replication and/or transcription processes. The analyses reveal that six unrelated genera are characterized by a reversal of the usual strand bias, i.e., Argiope (Araneae), Euscorpius (Scorpiones), Tigrioupus (Maxillopoda), Branchiostoma (Cephalochordata) Florometra (Echinodermata), and Katharina (Mollusca). It is proposed that assymetric mutational constraints have been independantly reversed in these six genera, through an inversion of the control region, i.e., the region that contains most regulatory elements for replication and transcription of the mtDNA. We show that reversals of assymetric mutational constraints have dramatic consequences on the phylogenetic analyses, as taxa characterized by reverse strand bias tend to group together due to long-branch attraction artifacts. We propose a new method for limiting this specific problem in tree reconstruction under the Bayesian approach. We apply our method to deal with the question of phylogenetic relationships of the major lineages of Arthropoda, This new approach provides a better congruence with nuclear analyses based on mtDNA sequences, our data suggest that Chelicerata, Crustacea, Myriapoda, Pancrustacea, and Paradoxopoda are monophyletic.