Long-branch Attraction Artifact Research Articles

Multigene Analyses of Bilaterian Animals Corroborate the Monophyly of Ecdysozoa, Lophotrochozoa, and Protostomia

Almost a decade ago, a new phylogeny of bilaterian animals was inferred from small-subunit ribosomal RNA (rRNA) that claimed the monophyly of two major groups of protostome animals: Ecdysozoa (e.g., arthropods, nematodes, onychophorans, and tardigrades) and Lophotrochozoa (e.g., annelids, molluscs, platyhelminths, brachiopods, and rotifers). However, it received little additional support. In fact, several multigene analyses strongly argued against this new phylogeny. These latter studies were based on a large amount of sequence data and therefore showed an apparently strong statistical support. Yet, they covered only a few taxa (those for which complete genomes were available), making systematic artifacts of tree reconstruction more probable. Here we expand this sparse taxonomic sampling and analyze a large data set (146 genes, 35,371 positions) from a diverse sample of animals (35 species). Our study demonstrates that the incongruences observed between rRNA and multigene analyses were indeed due to long-branch attraction artifacts, illustrating the enormous impact of systematic biases on phylogenomic studies. A refined analysis of our data set excluding the most biased genes provides strong support in favor of the new animal phylogeny and in addition suggests that urochordates are more closely related to vertebrates than are cephalochordates. These findings have important implications for the interpretation of morphological and genomic data.

Phylogenetic Analysis of the Complete Genome Sequence of Encephalitozoon cuniculi Supports the Fungal Origin of Microsporidia and Reveals a High Frequency of Fast-Evolving Genes

Microsporidia are unicellular eukaryotes living as obligate intracellular parasites. Lacking mitochondria, they were initially considered as having diverged before the endosymbiosis at the origin of mitochondria. That microsporidia were primitively amitochondriate was first questioned by the discovery of microsporidial sequences homologous to genes encoding mitochondrial proteins and then refuted by the identification of remnants of mitochondria in their cytoplasm. Various molecular phylogenies also cast doubt on the early divergence of microsporidia, these organisms forming a monophyletic group with or within the fungi. The 2001 proteins putatively encoded by the complete genome of Encephalitozoon cuniculi provided powerful data to test this hypothesis. Phylogenetic analysis of 99 proteins selected as adequate phylogenetic markers indicated that the E. cuniculi sequences having the lowest evolutionary rates preferentially clustered with fungal sequences or, more rarely, with both animal and fungal sequences. Because sequences with low evolutionary rates are less sensitive to the long-branch attraction artifact, we concluded that microsporidia are evolutionarily related to fungi. This analysis also allowed comparing the accuracy of several phylogenetic algorithms for a fast-evolving lineage with real rather than simulated sequences.

Rabbits, if anything, are likely Glires

Rodentia (e.g., mice, rats, dormice, squirrels, and guinea pigs) and Lagomorpha (e.g., rabbits, hares, and pikas) are usually grouped into the Glires. Status of this controversial superorder has been evaluated using morphology, paleontology, and mitochondrial plus nuclear DNA sequences. This growing corpus of data has been favoring the monophyly of Glires. Recently, Misawa and Janke [Mol. Phylogenet. Evol. 28 (2003) 320] analyzed the 6441 amino acids of 20 nuclear proteins for six placental mammals (rat, mouse, rabbit, human, cattle, and dog) and two outgroups (chicken and xenopus), and observed a basal position of the two murine rodents among the former. They concluded that “the Glires hypothesis was rejected.” We here reanalyzed [loc. cit.] data set under maximum likelihood and Bayesian tree-building approaches, using phylogenetic models that take into account among-site variation in evolutionary rates and branch-length variation among proteins. Our observations support both the association of rodents and lagomorphs and the monophyly of Euarchontoglires (= Supraprimates) as the most likely explanation of the protein alignments. We conducted simulation studies to evaluate the appropriateness of lissamphibian and avian outgroups to root the placental tree. When the outgroup-to-ingroup evolutionary distance increases, maximum parsimony roots the topology along the long Mus- Rattus branch. Maximum likelihood, in contrast, roots the topology along different branches as a function of their length. Maximum likelihood appears less sensitive to the “long-branch attraction artifact” than is parsimony. Our phylogenetic conclusions were confirmed by the analysis of a different protein data set using a similar sample of species but different outgroups. We also tested the effect of the addition of afrotherian and xenarthran taxa. Using the linearized tree method, [loc. cit.] estimated that mice and rats diverged about 35 million years ago. Molecular dating based on the Bayesian relaxed molecular clock method suggests that the 95% credibility interval for the split between mice and rats is 7–17 Mya. We here emphasize the need for appropriate models of sequence evolution (matrices of amino acid replacement, taking into account among-site rate variation, and independent parameters across independent protein partitions) and for a taxonomically broad sample, and conclude on the likelihood that rodents and lagomorphs together constitute a monophyletic group (Glires).

Evidence from the small and large ribosomal RNA structure suggests that Anoplostoma rectospiculum Gal'tsova, 1976 (Nematoda: Anoplostomatidae) is a member of the superfamily Enoploidea, not Oncholaimoidea

Abstract Analyses of the primary structure of the 18S rRNA gene and D3 segment of the 28S rRNA, as well as evidence from the secondary structure of the SSU rRNA V7 region, suggest that Anoplostoma rectospiculum (Anoplostomatidae) has closer relationships to the family Enoplidae than to the Oncholaimidae. In phylogenetic trees derived from full length SSU rRNA gene and partial LSU rRNA gene (D3 expansion segment) sequence analyses, A. rectospiculum exhibits long branches. The associated artefacts of long branch attraction (LBA) are circumvented because of the presence of an undoubted molecular synapomorphy – a low homoplastic 1. b. p. insertion in helix 43 of the SSU rRNA which is shared jointly by Anoplostomatidae and Enoplidae. Analysis of low homoplastic apomorphic characters is considered to be a tool for testing phylogenies against LBA artefacts.

Evolutionary history of "early-diverging" eukaryotes: the excavate taxon Carpediemonas is a close relative of Giardia.

Diplomonads, such as Giardia, and their close relatives retortamonads have been proposed as early-branching eukaryotes that diverged before the acquisition-retention of mitochondria, and they have become key organisms in attempts to understand the evolution of eukaryotic cells. In this phylogenetic study we focus on a series of eukaryotes suggested to be relatives of diplomonads on morphological grounds, the "excavate taxa". Phylogenies of small subunit ribosomal RNA (SSU rRNA) genes, alpha-tubulin, beta-tubulin, and combined alpha- + beta-tubulin all scatter the various excavate taxa across the diversity of eukaryotes. But all phylogenies place the excavate taxon Carpediemonas as the closest relative of diplomonads (and, where data are available, retortamonads). This novel relationship is recovered across phylogenetic methods and across various taxon-deletion experiments. Statistical support is strongest under maximum-likelihood (ML) (when among-site rate variation is modeled) and when the most divergent diplomonad sequences are excluded, suggesting a true relationship rather than an artifact of long-branch attraction. When all diplomonads are excluded, our ML SSU rRNA tree actually places retortamonads and Carpediemonas away from the base of the eukaryotes. The branches separating excavate taxa are mostly not well supported (especially in analyses of SSU rRNA data). Statistical tests of the SSU rRNA data, including an "expected likelihood weights" approach, do not reject trees where excavate taxa are constrained to be a clade (with or without parabasalids and Euglenozoa). Although diplomonads and retortamonads lack any mitochondria-like organelle, Carpediemonas contains double membrane-bounded structures physically resembling hydrogenosomes. The phylogenetic position of Carpediemonas suggests that it will be valuable in interpreting the evolutionary significance of many molecular and cellular peculiarities of diplomonads.

Nuclear and nucleomorph SSU rDNA phylogeny in the Cryptophyta and the evolution of cryptophyte diversity.

The plastid-bearing members of the Cryptophyta contain two functional eukaryotic genomes of different phylogenetic origin, residing in the nucleus and in the nucleomorph, respectively. These widespread and diverse protists thus offer a unique opportunity to study the coevolution of two different eukaryotic genomes within one group of organisms. In this study, the SSU rRNA genes of both genomes were PCR-amplified with specific primers and phylogenetic analyses were performed on different data sets using different evolutionary models. The results show that the composition of the principal clades obtained from the phylogenetic analyses of both genes was largely congruent, but striking differences in evolutionary rates were observed. These affected the topologies of the nuclear and nucleomorph phylogenies differently, resulting in long-branch attraction artifacts when simple evolutionary models were applied. Deletion of long-branch taxa stabilized the internal branching order in both phylogenies and resulted in a completely resolved topology in the nucleomorph phylogeny. A comparison of the tree topologies derived from SSU rDNA sequences with characters previously used in cryptophyte systematics revealed that the biliprotein type was congruent, but the type of inner periplast component incongruent, with the molecular trees. The latter is indicative of a hidden cellular dimorphism (cells with two periplast types present in a single clonal strain) of presumably widespread occurrence throughout cryptophyte diversity, which, in consequence, has far-reaching implications for cryptophyte systematics as it is practiced today.

A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models.

Over the past decade, nearly 1,000 variants have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in classic and atypical cystic fibrosis (CF) patients worldwide, and an enormous wealth of information concerning the structure and function of the protein has also been accumulated. These data, if evaluated together in a sequence comparison of all currently available CFTR homologs, are likely to refine the global structure-function relationship of the protein, which will, in turn, facilitate interpretation of the identified mutations in the gene. Based on such a combined analysis, we had recently defined a "functional R domain" of the CFTR protein. First, presenting two full-length cDNA sequences (termed sCFTR-I and sCFTR-II) from the Atlantic salmon (Salmo salar) and an additional partial coding sequence from the eastern gray kangaroo (Macropus giganteus), this study went further to refine the boundaries of the two nucleotide-binding domains (NBDs) and the COOH-terminal tail (C-tail), wherein NBD1 was defined as going from P439 to G646, NBD2 as going from A1225 to E1417, and the C-tail as going from E1418 to L1480. This approach also provided further insights into the differential roles of the two halves of CFTR and highlighted several well-conserved motifs that may be involved in inter- or intramolecular interactions. Moreover, a serious concern that a certain fraction of missense mutations identified in the CFTR gene may not have functional consequences was raised. Finally, phylogenetic analysis of all the full-length CFTR amino acid sequences and an extended set of exon 13--coding nucleotide sequences reinforced the idea that the rabbit may represent a better CF model than the mouse and strengthened the assertion that a long-branch attraction artifact separates the murine rodents from the rabbit and the guinea pig, the other Glires.

Evolution of cyclin-dependent kinases (CDKs) and CDK-activating kinases (CAKs): differential conservation of CAKs in yeast and metazoa.

Cyclin-dependent kinases (CDKs) function as central regulators of both the cell cycle and transcription. CDK activation depends on phosphorylation by a CDK-activating kinase (CAK). Different CAKs have been identified in budding yeast, fission yeast, and metazoans. All known CAKs belong to the extended CDK family. The sole budding yeast CAK, CAK1, and one of the two CAKs in fission yeast, csk1, have diverged considerably from other CDKs. Cell cycle regulatory components have been largely conserved in eukaryotes; however, orthologs of neither CAK1 nor csk1 have been identified in other species to date. To determine the evolutionary relationships of yeast and metazoan CAKs, we performed a phylogenetic analysis of the extended CDK family in budding yeast, fission yeast, humans, the fruit fly Drosophila melanogaster, and the nematode Caenorhabditis elegans. We observed that there were 10 clades for CDK-related genes, of which seven appeared ancestral, containing both yeast and metazoan genes. The four clades that contain CDKs that regulate transcription by phosphorylating the carboxyl-terminal domain (CTD) of RNA Polymerase II generally have only a single orthologous gene in each species of yeast and metazoans. In contrast, the ancestral cell cycle CDK (analogous to budding yeast CDC28) gave rise to a number of genes in metazoans, as did the ancestor of budding yeast PHO85. One ancestral clade is unique in that there are fission yeast and metazoan members, but there is no budding yeast ortholog, suggesting that it was lost subsequent to evolutionary divergence. Interestingly, CAK1 and csk1 branch together with high bootstrap support values. We used both the relative apparent synapomorphy analysis (RASA) method in combination with the S-F method of sampling reduced character sets and gamma-corrected distance methods to confirm that the CAK1/csk1 association was not an artifact of long-branch attraction. This result suggests that CAK1 and csk1 are orthologs and that a central aspect of CAK regulation has been conserved in budding and fission yeast. Although there are metazoan CDK-family members for which we could not define ancestral lineage, our analysis failed to identify metazoan CAK1/csk1 orthologs, suggesting that if the CAK1/csk1 gene existed in the metazoan ancestor, it has not been conserved.

What is the phylogenetic position of Cryptosporidium?

The phylogenetic position of Cryptosporidium is elusive. Although previous studies based solely upon small-subunit (SSU) rRNA sequences suggested that the genus was an early emerging lineage among the Apicomplexa, bootstrap support for this placement was low. Here, the phylogenetic position of Cryptosporidium has been re-evaluated for SSU rRNA, fused SSU/large-subunit (LSU) rRNA and six protein sequences using traditional distance-based neighbour-joining, maximum-parsimony and maximum-likelihood methods of phylogenetic reconstruction as well as the new Slow-Fast analysis, which focuses upon the slowly evolving positions within sequences and is especially useful if a long-branch attraction (LBA) artefact is suspected. All the methods of reconstruction indicated a trend for the early emergence of Cryptosporidium at the base of the Apicomplexa and showed that an LBA artefact plays no role in this placement. Although the inclusion of additional numbers of neither species nor genes has significantly enhanced the bootstrap support for this phylogenetic position, recent biochemical, molecular and ultrastructural data are congruent with it. Therefore, we favour a working hypothesis that this genus constitutes an early emerging branch of the Apicomplexa.

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

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

New insights into the phylogeny of eukaryotes based on ciliate Hsp70 sequences.

The current framework of the eukaryotic phylogeny is based on the analysis of a comprehensive set of sequences of the small subunit ribosomal RNA. However, phylogenies based on protein-encoding genes are not completely congruent with this picture. Since congruence between different markers is the best tool to determine evolutionary history, we focused on Hsp70 (heat-shock protein of 70 kDa), a chaperone protein which is highly conserved and is a potentially reliable phylogenetic marker. We used a PCR-based approach to sequence Hsp70s in two distinct classes of Ciliates. Seven Hsp70s were identified from Paramecium tetraurelia (Oligohymenophora) and six Hsp70s from Euplotes aediculatus (Hypotricha), encompassing orthologous genes for all major Hsp70 classes of Eukaryotes, i.e., those localized in cytosol, in endoplasmic reticulum, and in mitochondria. Three independent phylogenies of eukaryotes, based on each set of orthologous genes, have been constructed using different tree reconstruction methods. A significant advantage of Hsp70s is the existence of outgroups close to Eukaryotes for these major classes, reducing the long-branch attraction artifact due to the outgroup. The monophyly of Ciliates is supported by good bootstrap proportions in the phylogenetic reconstructions, and this phylum is generally a sister-group of Sporozoa, forming the expected Alveolates clade. The Hsp70 seems to be a suitable phylogenetic marker since it recovers all the monophyletic groups, undoubtedly defined by morphological criteria. The Hsp70 trees are, however, notably different from the rRNA ones and do not show two aspects of the classical topology, i.e., the successive emergence of deeply branching groups and the vast assembly of the major eukaryotic groups, emerging at the tip of the tree, i.e., the "terminal crown". More precisely, the Hsp70 trees do not resolve the relationships between the major groups of Eukaryotes with confidence, in keeping with the hypothesis that all these groups emerged in a great radiation that occurred at the origin of all the extant Eukaryotes.

Phylogenetic analysis of cystic fibrosis transmembrane conductance regulator gene in mammalian species argues for the development of a rabbit model for cystic fibrosis.

The species-specific pattern of cystic fibrosis transmembrane conductance regulator (CFTR) expression was investigated in order to identify species closely related to man which can be used as potential cystic fibrosis (CF) animal models. To this purpose, the nucleotide sequences of the CFTR promoter region of eight mammalian species representing four different orders (Primates, Artiodactyla, Lagomorpha and Rodentia) were analyzed. Distance matrices and unrooted trees of the CFTR promoter region sequences yielded two deeply separated groups, one including man (Homo sapiens), nonhuman primates (Hylobates lar, Macaca fascicularis, Saimiri sciureus), cow (Bos taurus), and rabbit (Oryctolagus cuniculus) and the other including the rodents (Rattus norvegicus, Mus musculus). Divergences between rodent and nonrodent groups have been observed in putative cis transcriptional regulatory elements and can be involved in the differences of pattern of expression between these two groups. Comparison of the available CFTR cDNA sequences enabled us to root the tree with a noneutherian outgroup and to perform a phylogenetic analysis. This analysis did not detect any base composition bias and supported polyphyletic Glires. Although a long-branch attraction artifact cannot be completely excluded, these findings converge toward the recent statement (Graur, Duret, and Gouy 1996) that Lagomorpha is more closely related to Primates than to Rodentia. In addition, the phenylalanine residue in exon 10 involved in the most common CF mutation in man is conserved in rabbit. These phylogenetic analyses as well as anatomical and developmental data suggest that, once rabbit embryonic stem cells become available, the rabbit will provide a suitable tool for both gene transfer and pharmacological investigations and could lead to a better CF model than the current murine models.