Long-branch Attraction Artifact Research Articles

Reducing long-branch effects in multi-protein data uncovers a close relationship between Alveolata and Rhizaria

Rhizaria is a major eukaryotic group of tremendous diversity, including amoebae with spectacular skeletons or tests (Radiolaria and Foraminifera), plasmodial parasites (Plasmodiophorida) and secondary endosymbionts (Chlorarachniophyta). Current phylogeny places Rhizaria in an unresolved trichotomy with Stramenopila and Alveolata (supergroup “SAR”). We assembled a 147-protein data set with extensive rhizarian coverage (M147), including the first transcriptomic data for a euglyphid amoeba. Phylogenetic pre-screening of individual proteins indicated potential problems with radically misplaced sequences due either to contamination of rhizarian sequences amplified from wild collected material and/or extremely long branches (xLBs). Therefore, two data subsets were extracted containing either all proteins consistently recovering rhizarian monophyly (M34) or excluding all proteins with ⩾3 xLBs (defined as ⩾2× the average terminal branch length for the tree). Phylogenetic analyses of M147 give conflicting results depending on the outgroup and method of analysis but strongly support an exclusive Rhizaria+Alveolata (R+A) clade with both data subsets (M34 and M37) regardless of phylogenetic method used. Support for an R+A clade is most consistent when a close outgroup is used and decreases with more distant outgroups, suggesting that support for alternative SAR topologies may reflect a long-branch attraction artifact. A survey of xLB distribution among taxa and protein functional category indicates that small “informational” proteins in particular have highly variable evolutionary rates with no consistent pattern among taxa.

LS³: A Method for Improving Phylogenomic Inferences When Evolutionary Rates Are Heterogeneous among Taxa.

Phylogenetic inference artifacts can occur when sequence evolution deviates from assumptions made by the models used to analyze them. The combination of strong model assumption violations and highly heterogeneous lineage evolutionary rates can become problematic in phylogenetic inference, and lead to the well-described long-branch attraction (LBA) artifact. Here, we define an objective criterion for assessing lineage evolutionary rate heterogeneity among predefined lineages: the result of a likelihood ratio test between a model in which the lineages evolve at the same rate (homogeneous model) and a model in which different lineage rates are allowed (heterogeneous model). We implement this criterion in the algorithm Locus Specific Sequence Subsampling (LS³), aimed at reducing the effects of LBA in multi-gene datasets. For each gene, LS³ sequentially removes the fastest-evolving taxon of the ingroup and tests for lineage rate homogeneity until all lineages have uniform evolutionary rates. The sequences excluded from the homogeneously evolving taxon subset are flagged as potentially problematic. The software implementation provides the user with the possibility to remove the flagged sequences for generating a new concatenated alignment. We tested LS³ with simulations and two real datasets containing LBA artifacts: a nucleotide dataset regarding the position of Glires within mammals and an amino-acid dataset concerning the position of nematodes within bilaterians. The initially incorrect phylogenies were corrected in all cases upon removing data flagged by LS³.

On 20 years of Lophotrochozoa

Lophotrochozoa is a protostome clade that includes disparate animals such as molluscs, annelids, bryozoans, and flatworms, giving it the distinction of including the most body plans of any of the three major clades of Bilateria. This extreme morphological disparity has prompted numerous conflicting phylogenetic hypotheses about relationships among lophotrochozoan phyla. Here, I review the current understanding of lophotrochozoan phylogeny with emphasis on recent insights gained through approaches taking advantage of high-throughput DNA sequencing (phylogenomics). Of significance, Platyzoa, a hypothesized clade of mostly small-bodied animals, appears to be an artifact of long-branch attraction. Recent studies recovered Gnathifera (Syndermata, Gnathostomulida, and Micrognathozoa) sister to all other lophotrochozoans and a clade called Rouphozoa (Platyhelminthes and Gastrotricha) sister to the remaining non-gnathiferan lophotrochozoans. Although Bryozoa was traditionally grouped with Brachiopoda and Phoronida (Lophophorata), most molecular studies have supported a clade including Entoprocta, Cycliophora, and Bryozoa (Polyzoa). However, recent phylogenomic work has shown that entoprocts and bryozoans have compositionally heterogeneous genomes that may cause systematic artifacts affecting their phylogenetic placement. Lastly, relationships within Trochozoa (Mollusca, Annelida, and relatives) largely remain ambiguous. Recent work has shown that phylogenomic studies must identify and reduce sources of systematic error, such as amino acid compositional heterogeneity and long-branch attraction. Still, other approaches such as the analysis of rare genomic changes may be needed to overcome challenges to standard phylogenomic approaches. Resolving lophotrochozoan phylogeny will provide important insight into how these complex and diverse body plans evolved and provide a much-needed framework for comparative studies.

Rooting the tree of life: the phylogenetic jury is still out.

This article aims to shed light on difficulties in rooting the tree of life (ToL) and to explore the (sociological) reasons underlying the limited interest in accurately addressing this fundamental issue. First, we briefly review the difficulties plaguing phylogenetic inference and the ways to improve the modelling of the substitution process, which is highly heterogeneous, both across sites and over time. We further observe that enriched taxon samplings, better gene samplings and clever data removal strategies have led to numerous revisions of the ToL, and that these improved shallow phylogenies nearly always relocate simple organisms higher in the ToL provided that long-branch attraction artefacts are kept at bay. Then, we note that, despite the flood of genomic data available since 2000, there has been a surprisingly low interest in inferring the root of the ToL. Furthermore, the rare studies dealing with this question were almost always based on methods dating from the 1990s that have been shown to be inaccurate for much more shallow issues! This leads us to argue that the current consensus about a bacterial root for the ToL can be traced back to the prejudice of Aristotle's Great Chain of Beings, in which simple organisms are ancestors of more complex life forms. Finally, we demonstrate that even the best models cannot yet handle the complexity of the evolutionary process encountered both at shallow depth, when the outgroup is too distant, and at the level of the inter-domain relationships. Altogether, we conclude that the commonly accepted bacterial root is still unproven and that the root of the ToL should be revisited using phylogenomic supermatrices to ensure that new evidence for eukaryogenesis, such as the recently described Lokiarcheota, is interpreted in a sound phylogenetic framework.

Caenogastropod mitogenomics

In order to further resolve the phylogenetic relationships within Caenogastropoda, the complete mitochondrial (mt) genomes of Cochlostoma hidalgoi (Cyclophoroidea), Naticarius hebraeus (Naticoidea), Galeodea echinophora (Tonnoidea), and Columbella adansoni (Buccinoidea), and the partial mt genome of Erosaria spurca (Cypraeoidea) were sequenced. All newly determined mt genomes conformed to the consensus gene order of caenogastropods, except that of C. hidalgoi, which differed in the relative positions of the trnD, trnQ, trnG, trnY, and trnT genes. Phylogenetic reconstruction of the caenogastropod tree was performed using probabilistic methods and based on the deduced amino acid sequences of concatenated mt protein coding genes. The reconstructed phylogeny recovered Architaenioglossa (superfamilies Cyclophoroidea, Ampullarioidea, and Viviparoidea) as a grade. The monophyly of Sorbeoconcha (all caenogastropods but Architaenioglossa) was supported by most but not all phylogenetic analyses (excluding Vermetoidea, which has a long branch). The relative phylogenetic position of Cerithioidea with respect to Hypsogastropoda remains unresolved. The monophyly of Hypsogastropoda (without Vermetoidea) is strongly supported. Within this clade, Littorinimorpha should be considered a grade. Several superfamilies (Abyssochrysoidea, Rissooidea, Truncatelloidea, and Naticoidea) branched off successively before a siphonate clade (Stromboidea, Cypraeoidea, Tonnoidea, Neogastropoda), which is strongly supported. The relative phylogenetic position of Vermetoidea could not be determined due to long-branch attraction artifacts. The superfamily Tonnoidea was recovered within Neogastropoda, which questions the monophyly of the latter as traditionally defined. The polyphyly of Muricoidea could be tentatively resolved excluding the families Volutidae and Babyloniidae, which would imply raising them to the rank of superfamilies.

Evidence of Statistical Inconsistency of Phylogenetic Methods in the Presence of Multiple Sequence Alignment Uncertainty.

Evolutionary studies usually use a two-step process to investigate sequence data. Step one estimates a multiple sequence alignment (MSA) and step two applies phylogenetic methods to ask evolutionary questions of that MSA. Modern phylogenetic methods infer evolutionary parameters using maximum likelihood or Bayesian inference, mediated by a probabilistic substitution model that describes sequence change over a tree. The statistical properties of these methods mean that more data directly translates to an increased confidence in downstream results, providing the substitution model is adequate and the MSA is correct. Many studies have investigated the robustness of phylogenetic methods in the presence of substitution model misspecification, but few have examined the statistical properties of those methods when the MSA is unknown. This simulation study examines the statistical properties of the complete two-step process when inferring sequence divergence and the phylogenetic tree topology. Both nucleotide and amino acid analyses are negatively affected by the alignment step, both through inaccurate guide tree estimates and through overfitting to that guide tree. For many alignment tools these effects become more pronounced when additional sequences are added to the analysis. Nucleotide sequences are particularly susceptible, with MSA errors leading to statistical support for long-branch attraction artifacts, which are usually associated with gross substitution model misspecification. Amino acid MSAs are more robust, but do tend to arbitrarily resolve multifurcations in favor of the guide tree. No inference strategies produce consistently accurate estimates of divergence between sequences, although amino acid MSAs are again more accurate than their nucleotide counterparts. We conclude with some practical suggestions about how to limit the effect of MSA uncertainty on evolutionary inference.

The Root of Flowering Plants and Total Evidence.

Support for Amborella as the sole survivor of an evolutionary lineage that is sister to all other angiosperms comes from positions in DNA multiple-sequence alignments that have a poor fit to time-reversible substitution models. These sites exhibit significant levels of homoplasy, compositional heterogeneity, and strong heterotachy. We report phylogenetic analyses with observed, randomized, and simulated data which show there is little or no expectation that these sites provide useful information for understanding relationships among basal angiosperms. Their inclusion in phylogenetic analyses leads to a long-branch attraction artifact that favors Amborella as sister to other angiosperms in reconstructed phylogenies. Using parametric simulations, we show that sites in chloroplast sequences that exhibit less homoplasy between angiosperms and gymnosperms provide more reliable information for inferring basal angiosperm relationships. We confirm our earlier findings that the basal angiosperm Amborella is most closely related to aquatic herbs. Our current and previously reported (Goremykin et al. 2013) analyses highlight an essential aspect of the total evidence approach to phylogenetic inference. They suggest that data partitioning aimed at identifying components of the data that better fit evolutionary models is a more reliable approach to phylogeny reconstruction at deep taxonomic levels.

Phylogenomic interrogation of arachnida reveals systemic conflicts in phylogenetic signal.

Chelicerata represents one of the oldest groups of arthropods, with a fossil record extending to the Cambrian, and is sister group to the remaining extant arthropods, the mandibulates. Attempts to resolve the internal phylogeny of chelicerates have achieved little consensus, due to marked discord in both morphological and molecular hypotheses of chelicerate phylogeny. The monophyly of Arachnida, the terrestrial chelicerates, is generally accepted, but has garnered little support from molecular data, which have been limited either in breadth of taxonomic sampling or in depth of sequencing. To address the internal phylogeny of this group, we employed a phylogenomic approach, generating transcriptomic data for 17 species in combination with existing data, including two complete genomes. We analyzed multiple data sets containing up to 1,235,912 sites across 3,644 loci, using alternative approaches to optimization of matrix composition. Here, we show that phylogenetic signal for the monophyly of Arachnida is restricted to the 500 slowest-evolving genes in the data set. Accelerated evolutionary rates in Acariformes, Pseudoscorpiones, and Parasitiformes potentially engender long-branch attraction artifacts, yielding nonmonophyly of Arachnida with increasing support upon incrementing the number of concatenated genes. Mutually exclusive hypotheses are supported by locus groups of variable evolutionary rate, revealing significant conflicts in phylogenetic signal. Analyses of gene-tree discordance indicate marked incongruence in relationships among chelicerate orders, whereas derived relationships are demonstrably robust. Consistently recovered and supported relationships include the monophyly of Chelicerata, Euchelicerata, Tetrapulmonata, and all orders represented by multiple terminals. Relationships supported by subsets of slow-evolving genes include Ricinulei + Solifugae; a clade comprised of Ricinulei, Opiliones, and Solifugae; and a clade comprised of Tetrapulmonata, Scorpiones, and Pseudoscorpiones. We demonstrate that outgroup selection without regard for branch length distribution exacerbates long-branch attraction artifacts and does not mitigate gene-tree discordance, regardless of high gene representation for outgroups that are model organisms. Arachnopulmonata (new name) is proposed for the clade comprising Scorpiones + Tetrapulmonata (previously named Pulmonata).

Rooting the Eukaryotic Tree with Mitochondrial and Bacterial Proteins

By exploiting the large body of genome data and the considerable progress in phylogenetic methodology, recent phylogenomic studies have provided new insights into the relationships among major eukaryotic groups. However, confident placement of the eukaryotic root remains a major challenge. This is due to the large evolutionary distance separating eukaryotes from their closest relatives, the Archaea, implying a weak phylogenetic signal and strong long-branch attraction artifacts. Here, we apply a new approach to the rooting of the eukaryotic tree by using a subset of genomic information with more recent evolutionary origin-mitochondrial sequences, whose closest relatives are α-Proteobacteria. For this, we identified and assembled a data set of 42 mitochondrial proteins (mainly encoded by the nuclear genome) and performed Bayesian and maximum likelihood analyses. Taxon sampling includes the recently sequenced Thecamonas trahens, a member of the phylogenetically elusive Apusozoa. This data set confirms the relationships of several eukaryotic supergroups seen before and places the eukaryotic root between the monophyletic "unikonts" and "bikonts." We further show that T. trahens branches sister to Opisthokonta with significant statistical support and question the bikont/excavate affiliation of Malawimonas species. The mitochondrial data set developed here (to be expanded in the future) constitutes a unique alternative means in resolving deep eukaryotic relationships.

MicroRNAs and phylogenomics resolve the relationships of Tardigrada and suggest that velvet worms are the sister group of Arthropoda.

Morphological data traditionally group Tardigrada (water bears), Onychophora (velvet worms), and Arthropoda (e.g., spiders, insects, and their allies) into a monophyletic group of invertebrates with walking appendages known as the Panarthropoda. However, molecular data generally do not support the inclusion of tardigrades within the Panarthropoda, but instead place them closer to Nematoda (roundworms). Here we present results from the analyses of two independent genomic datasets, expressed sequence tags (ESTs) and microRNAs (miRNAs), which congruently resolve the phylogenetic relationships of Tardigrada. Our EST analyses, based on 49,023 amino acid sites from 255 proteins, significantly support a monophyletic Panarthropoda including Tardigrada and suggest a sister group relationship between Arthropoda and Onychophora. Using careful experimental manipulations--comparisons of model fit, signal dissection, and taxonomic pruning--we show that support for a Tardigrada + Nematoda group derives from the phylogenetic artifact of long-branch attraction. Our small RNA libraries fully support our EST results; no miRNAs were found to link Tardigrada and Nematoda, whereas all panarthropods were found to share one unique miRNA (miR-276). In addition, Onychophora and Arthropoda were found to share a second miRNA (miR-305). Our study confirms the monophyly of the legged ecdysozoans, shows that past support for a Tardigrada + Nematoda group was due to long-branch attraction, and suggests that the velvet worms are the sister group to the arthropods.

As an evolutionary biologist. Debashish Bhattacharya.

As an evolutionary biologist. Debashish Bhattacharya.

A Long-Branch Attraction Artifact Reveals an Adaptive Radiation in Pseudomonas

A significant proportion of protein-encoding gene phylogenies in bacteria is inconsistent with the species phylogeny. It was usually argued that such inconsistencies resulted from lateral transfers. Here, by further studying the phylogeny of the oprF gene encoding the major surface protein in the bacterial Pseudomonas genus, we found that the incongruent tree topology observed results from a long-branch attraction (LBA) artifact and not from lateral transfers. LBA in the oprF phylogeny could be explained by the faster evolution in a lineage adapted to the rhizosphere, highlighting an unexpected adaptive radiation. We argue that analysis of such artifacts in other inconsistent bacterial phylogenies could be a valuable tool in molecular ecology to highlight cryptic adaptive radiations in microorganisms.

Acoelomorph flatworms are deuterostomes related to Xenoturbella

Xenoturbellida and Acoelomorpha are marine worms with contentious ancestry. Both were originally associated with the flatworms (Platyhelminthes), but molecular data have revised their phylogenetic positions, generally linking Xenoturbellida to the deuterostomes and positioning the Acoelomorpha as the most basally branching bilaterian group(s). Recent phylogenomic data suggested that Xenoturbellida and Acoelomorpha are sister taxa and together constitute an early branch of Bilateria. Here we assemble three independent data sets-mitochondrial genes, a phylogenomic data set of 38,330 amino-acid positions and new microRNA (miRNA) complements-and show that the position of Acoelomorpha is strongly affected by a long-branch attraction (LBA) artefact. When we minimize LBA we find consistent support for a position of both acoelomorphs and Xenoturbella within the deuterostomes. The most likely phylogeny links Xenoturbella and Acoelomorpha in a clade we call Xenacoelomorpha. The Xenacoelomorpha is the sister group of the Ambulacraria (hemichordates and echinoderms). We show that analyses of miRNA complements have been affected by character loss in the acoels and that both groups possess one miRNA and the gene Rsb66 otherwise specific to deuterostomes. In addition, Xenoturbella shares one miRNA with the ambulacrarians, and two with the acoels. This phylogeny makes sense of the shared characteristics of Xenoturbellida and Acoelomorpha, such as ciliary ultrastructure and diffuse nervous system, and implies the loss of various deuterostome characters in the Xenacoelomorpha including coelomic cavities, through gut and gill slits.

Loss of different inverted repeat copies from the chloroplast genomes of Pinaceae and cupressophytes and influence of heterotachy on the evaluation of gymnosperm phylogeny.

The relationships among the extant five gymnosperm groups—gnetophytes, Pinaceae, non-Pinaceae conifers (cupressophytes), Ginkgo, and cycads—remain equivocal. To clarify this issue, we sequenced the chloroplast genomes (cpDNAs) from two cupressophytes, Cephalotaxus wilsoniana and Taiwania cryptomerioides, and 53 common chloroplast protein-coding genes from another three cupressophytes, Agathis dammara, Nageia nagi, and Sciadopitys verticillata, and a non-Cycadaceae cycad, Bowenia serrulata. Comparative analyses of 11 conifer cpDNAs revealed that Pinaceae and cupressophytes each lost a different copy of inverted repeats (IRs), which contrasts with the view that the same IR has been lost in all conifers. Based on our structural finding, the character of an IR loss no longer conflicts with the “gnepines” hypothesis (gnetophytes sister to Pinaceae). Chloroplast phylogenomic analyses of amino acid sequences recovered incongruent topologies using different tree-building methods; however, we demonstrated that high heterotachous genes (genes that have highly different rates in different lineages) contributed to the long-branch attraction (LBA) artifact, resulting in incongruence of phylogenomic estimates. Additionally, amino acid compositions appear more heterogeneous in high than low heterotachous genes among the five gymnosperm groups. Removal of high heterotachous genes alleviated the LBA artifact and yielded congruent and robust tree topologies in which gnetophytes and Pinaceae formed a sister clade to cupressophytes (the gnepines hypothesis) and Ginkgo clustered with cycads. Adding more cupressophyte taxa could not improve the accuracy of chloroplast phylogenomics for the five gymnosperm groups. In contrast, removal of high heterotachous genes from data sets is simple and can increase confidence in evaluating the phylogeny of gymnosperms.

Automated Removal of Noisy Data in Phylogenomic Analyses

Noisy data, especially in combination with misalignment and model misspecification can have an adverse effect on phylogeny reconstruction; however, effective methods to identify such data are few. One particularly important class of noisy data is saturated positions. To avoid potential errors related to saturation in phylogenomic analyses, we present an automated procedure involving the step-wise removal of the most variable positions in a given data set coupled with a stopping criterion derived from correlation analyses of pairwise ML distances calculated from the deleted (saturated) and the remaining (conserved) subsets of the alignment. Through a comparison with existing methods, we demonstrate both the effectiveness of our proposed procedure for identifying noisy data and the effect of the removal of such data using a well-publicized case study involving placental mammals. At the least, our procedure will identify data sets requiring greater data exploration, and we recommend its use to investigate the effect on phylogenetic analyses of removing subsets of variable positions exhibiting weak or no correlation to the rest of the alignment. However, we would argue that this procedure, by identifying and removing noisy data, facilitates the construction of more accurate phylogenies by, for example, ameliorating potential long-branch attraction artefacts.

A Gene in the Process of Endosymbiotic Transfer

BackgroundThe endosymbiotic birth of organelles is accompanied by massive transfer of endosymbiont genes to the eukaryotic host nucleus. In the centric diatom Thalassiosira pseudonana the Psb28 protein is encoded in the plastid genome while a second version is nuclear-encoded and possesses a bipartite N-terminal presequence necessary to target the protein into the diatom complex plastid. Thus it can represent a gene captured during endosymbiotic gene transfer.Methodology/Principal FindingsTo specify the origin of nuclear- and plastid-encoded Psb28 in T. pseudonana we have performed extensive phylogenetic analyses of both mentioned genes. We have also experimentally tested the intracellular location of the nuclear-encoded Psb28 protein (nuPsb28) through transformation of the diatom Phaeodactylum tricornutum with the gene in question fused to EYFP.Conclusions/SignificanceWe show here that both versions of the psb28 gene in T. pseudonana are transcribed. We also provide experimental evidence for successful targeting of the nuPsb28 fused with EYFP to the diatom complex plastid. Extensive phylogenetic analyses demonstrate that nucleotide composition of the analyzed genes deeply influences the tree topology and that appropriate methods designed to deal with a compositional bias of the sequences and the long branch attraction artefact (LBA) need to be used to overcome this obstacle. We propose that nuclear psb28 in T. pseudonana is a duplicate of a plastid localized version, and that it has been transferred from its endosymbiont.

The Position of Gnetales among Seed Plants: Overcoming Pitfalls of Chloroplast Phylogenomics

The phylogenetic position of Gnetales is one of the most contentious issues in the seed plant systematics. To elucidate the Gnetales position, an improved amino acid substitution matrix was estimated based on 64 chloroplast (cp) genomes and was applied to cp genome data including all three lineages of Gnetales in maximum likelihood analyses of proteins. Although the initial analysis strongly supported the sister relation of Gnetales with Cryptomeria (Cupressophyta or non-Pinaceae conifers) (the "Gnecup" hypothesis), the support seems to be caused by a long-branch attraction (LBA) artifact. Indeed, by removing fastest evolving proteins that are most likely associated with the LBA, the support drastically declined. Furthermore, another analysis of partial genome data with dense taxon sampling of conifers showed that, in psbC, rpl2, and rps7 proteins, there are many parallel amino acid substitutions between the lineages leading to Gnetales and to Cryptomeria, and by further excluding these three genes, the sister relation of Gnetales with Pinaceae (the "Gnepine" hypothesis) became supported. Overall, our analyses indicate that the LBA and parallel substitutions cause a seriously biased inference of phylogenetic position of Gnetales with the cp genome data.

Rooting for the root of elongation factor-like protein phylogeny

Lateral gene transfer (LGT) may play a pivotal role in the evolution of elongation factor-like (EFL) genes in eukaryotes. To date, numbers of putative cases for lateral transfer of EFL genes have been postulated based on unrooted EFL phylogenies. Nevertheless, the root position in EFL phylogeny is important to validate lateral EFL gene transfer: for instance, a clade of two EFL homologs from distantly related organisms in an unrooted EFL tree does not necessarily confirm the LGT, since the possibility that the root may locate in this clade cannot be excluded. Cocquyt et al. (2009, p. 39) recently demonstrated that a putative case of lateral EFL gene transfer, which was originally proposed based on an unrooted phylogeny, could not be endorsed by the corresponding rooted analysis. Although rooting EFL phylogeny is indispensable to elucidate various aspects in EFL gene evolution, we suspected that the outgroup clade comprised of EF-1α and eukaryote-specific EF-1α paralogs erroneously attached to long EFL branches in Cocquyt et al. (2009) — a typical long branch attraction (LBA) artifact. Here, we systematically assessed the putative LBA artifact between the branch leading to the outgroup clade and long ingroup branches by analyzing the original dataset used in Cocquyt et al. (2009) with and without modifying ingroup-sequence sampling. A series of the rooted EFL analyses indicated that the root inference was highly susceptible to presence and absence of long-branched ingroup-sequences, suggesting that the rooted EFL phylogenies cannot be free from severe LBA artifact. We also discussed a new aspect in EFL gene evolution in stramenopiles identified in the course of the EFL analyses described above. Finally, the relative timing of the first emergence of EFL gene in eukaryotes was contemplated based on the current EF-1α/EFL distribution.

Molecular phylogeny of acariform mites (Acari, Arachnida): Strong conflict between phylogenetic signal and long-branch attraction artifacts

Acariformes (one of the two main lineages of Acari) represent an exceptionally diverse group of Arachnida. We performed first comprehensive phylogenetic analysis of Acariformes using sequence data from the nuclear small subunit rRNA gene (18S rDNA) and the mitochondrial cytochrome c oxidase subunit I (COI, amino acids). Our analyses confirm the monophyly of Acariformes and recognize two orders within Acariformes: Sarcoptiformes, consisting of Endeostigmata and Oribatida+Astigmata, and Trombidiformes. The data revealed the origin of Astigmata within Oribatida with the desmonomatan superfamily Crotonioidea as the source of astigmatan radiation and the sexual family Hermanniidae as the sister group, which generally supports previous morphological hypotheses. These results were found despite the strong conflict between long-branch attraction (LBA) artifacts and phylogenetic signal. It is likely that the conflict resulted from differences in the substitution rates among acariform lineages, especially comparing slowly evolving Oribatida with rapidly evolving Astigmata. The use of likelihood methods considered more resistant to LBA only slightly decreased the chance of falling into the LBA trap; the probability of recovering the origin of Astigmata within Desmonomata differs only by about 10% from that of having the long branched Astigmata and Trombidiformes either connected directly or shifted to deep parts of the tree due to outgroup attraction. Molecular dating using the rate-smoothing method PATHd8 shows that Acariformes originated c. 435 MYA and were probably among the earliest arthropods invading terrestrial habitats in late Silurian or the Lower Devonian, when the first vascular plants are thought to have arisen. Our analyses did not support the monophyly of Acari because we recovered clades Acariformes-Solifugae and Parasitiformes-Pseudoscorpionida. However, a formal revision of arachnid classification that would reflect these results must await future analyses.

Large-Scale Parsimony Analysis of Metazoan Indels in Protein-Coding Genes

Insertions and deletions (indels) are considered to be rare evolutionary events, the analysis of which may resolve controversial phylogenetic relationships. Indeed, indel characters are often assumed to be less homoplastic than amino acid and nucleotide substitutions and, consequently, more reliable markers for phylogenetic reconstruction. In this study, we analyzed indels from over 1,000 metazoan orthologous genes. We studied the impact of different species sampling, ortholog data sets, lengths of included indels, and indel-coding methods on the resulting metazoan tree. Our results show that, similar to sequence substitutions, indels are homoplastic characters, and their analysis is sensitive to the long-branch attraction artifact. Furthermore, improving the taxon sampling and choosing a closely related outgroup greatly impact the phylogenetic inference. Our indel-based inferences support the Ecdysozoa hypothesis over the Coelomata hypothesis and suggest that sponges are a sister clade to other animals.