Phylogenomics and the number of characters required for obtaining an accurate phylogeny of eukaryote model species

MOTIVATION: Through the most extensive phylogenomic analysis carried out to date, complete genomes of 11 eukaryotic species have been examined in order to find the homologous of more than 25,000 amino acid sequences. These sequences correspond to the exons of more than 3000 genes and were used as presence/absence characters to test one of the most controversial hypotheses concerning animal evolution, namely the Ecdysozoa hypothesis. Distance, maximum parsimony and Bayesian methods of phylogenetic reconstruction were used to test the hypothesis. RESULTS: The reliability of the ecdysozoa, grouping arthropods and nematodes in a single clade was unequivocally rejected in all the consensus trees. The Coelomata clade, grouping arthropods and chordates, was supported by the highest statistical confidence in all the reconstructions. The study of the dependence of the genomes' tree accuracy on the number of exons used, demonstrated that an unexpectedly larger number of characters are necessary to obtain robust phylogenies. Previous studies supporting ecdysozoa, could not guarantee an accurate phylogeny because the number of characters used was clearly below the minimum required.     

Systematic searches for molecular synapomorphies in model metazoan genomes give some support for Ecdysozoa after accounting for the idiosyncrasies of Caenorhabditis elegans

There has been broad acceptance among evolutionary biologists of the Ecdysozoa hypothesis that, based principally on molecular phylogenetic studies of small and large subunit ribosomal RNA sequences, postulates a close relationship between molting taxa such as arthropods and nematodes. On the other hand, recent studies of as many as 100 additional genes do not support the Ecdysozoa hypothesis and instead favor the older Coelomata hypothesis that groups the coelomate arthropods with the coelomate vertebrates to the exclusion of the nematodes. Here, exploiting completely sequenced genomes, we examined this question using cladistic analyses of the phylogenetic distribution of 1712 orthologous genes and 2906 protein domain combinations; we found stronger support for the Coelomata hypothesis than for the Ecdysozoa hypothesis. However, although arrived at by considering very large data sets, we show that this conclusion is unreliable, biased toward grouping arthropods with chordates by systematic high rate of character loss in the nematode. When we addressed this problem, we found slightly more support for Ecdysozoa than for Coelomata. Our identification of this systematic bias even when using entire genomes has important implications for future phylogenetic studies. We conclude that the results from the intensively sampled ribosomal RNA genes supporting the Ecdysozoa hypothesis provide the most credible current estimates of metazoan phylogeny.     

Genome-scale evidence of the nematode-arthropod clade

Background  

The issue of whether coelomates form a single clade, the Coelomata, or whether all animals that moult an exoskeleton (such as the coelomate arthropods and the pseudocoelomate nematodes) form a distinct clade, the Ecdysozoa, is the most puzzling issue in animal systematics and a major open-ended subject in evolutionary biology. Previous single-gene and genome-scale analyses designed to resolve the issue have produced contradictory results. Here we present the first genome-scale phylogenetic evidence that strongly supports the Ecdysozoa hypothesis.     

The complete mitochondrial genome of the onychophoran Epiperipatus biolleyi reveals a unique transfer RNA set and provides further support for the ecdysozoa hypothesis

Onychophora (velvet worms) play a crucial role in current discussions on position of arthropods. The ongoing Articulata/Ecdysozoa debate is in need of additional ground pattern characters for Panarthropoda (Arthropoda, Tardigrada, and Onychophora). Hence, Onychophora is an important outgroup taxon in resolving the relationships among arthropods, irrespective of whether morphological or molecular data are used. To date, there has been a noticeable lack of mitochondrial genome data from onychophorans. Here, we present the first complete mitochondrial genome sequence of an onychophoran, Epiperipatus biolleyi (Peripatidae), which shows several characteristic features. Specifically, the gene order is considerably different from that in other arthropods and other bilaterians. In addition, there is a lack of 9 tRNA genes usually present in bilaterian mitochondrial genomes. All these missing tRNAs have anticodon sequences corresponding to 4-fold degenerate codons, whereas the persisting 13 tRNAs all have anticodons pairing with 2-fold degenerate codons. Sequence-based phylogenetic analysis of the mitochondrial protein-coding genes provides a robust support for a clade consisting of Onychophora, Priapulida, and Arthropoda, which confirms the Ecdysozoa hypothesis. However, resolution of the internal ecdysozoan relationships suffers from a cluster of long-branching taxa (including Nematoda and Platyhelminthes) and a lack of data from Tardigrada and further nemathelminth taxa in addition to nematodes and priapulids.     

Ecdysozoa: The Relationship between Cycloneuralia and Panarthropoda

The hypothesis that molting protostomes such as nematodes and arthropods form a monophyletic group known as Ecdysozoa is directly opposed to Articulata, in which some segmented protostomes such as annelids and arthropods form a monophyletic taxon. Ultrastructural and cladistic studies have led to the widely accepted hypothesis that nematodes belong among the protostomes. While early molecular studies suggested that nematodes were basal triploblasts, more recent molecular evidence suggests that this was an artifact of ‘long branch attraction’ and 18S rRNA gene, total evidence and hox gene studies all support the placement of nematodes within Ecdysozoa. The branching pattern within Ecdysozoa has been difficult to elucidate, but it now appears that priapulids and kinorhynchs form the earliest branching clade, followed by nematodes + nematomorphs, and finally the panarthropods. This suggests that Cycloneuralia is paraphyletic and that arthropods are the most derived of the ecdysozoans.     

双翅目昆虫（黑腹果蝇和冈比亚按蚊）内含子丢失的比较分析

内含子插入和丢失的进化动力及机制尚存在许多疑问。通过对真核生物的105个同源基因的蛋白质高度保守区域内含子-外显子结构的研究,对人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae和秀丽隐杆线虫Caenorhabditis elegans的3 574个内含子、1 001个的内含子保守位点进行分析,推断出不同系统中内含子的变化途径。发现在进化早期,脊椎动物、双翅目昆虫和线虫的共同祖先中含有大量内含子,在进化过程中,双翅目昆虫和线虫发生了大量的内含子丢失,甚至在双翅目昆虫中内含子丢失较线虫更严重。线虫获得的内含子略多于丢失的内含子, 而在双翅目昆虫中则显示出内含子的丢失明显多于内含子的获得。该结果合理地解释了内含子在脊椎动物、线虫及昆虫中数量的分布呈下降趋势。     

Near intron pairs and the metazoan tree

Gene structure data can substantially advance our understanding of metazoan evolution and deliver an independent approach to resolve conflicts among existing hypotheses. Here, we used changes of spliceosomal intron positions as novel phylogenetic marker to reconstruct the animal tree. This kind of data is inferred from orthologous genes containing mutually exclusive introns at pairs of sequence positions in close proximity, so-called near intron pairs (NIPs). NIP data were collected for 48 species and utilized as binary genome-level characters in maximum parsimony (MP) analyses to reconstruct deep metazoan phylogeny. All groupings that were obtained with more than 80% bootstrap support are consistent with currently supported phylogenetic hypotheses. This includes monophyletic Chordata, Vertebrata, Nematoda, Platyhelminthes and Trochozoa. Several other clades such as Deuterostomia, Protostomia, Arthropoda, Ecdysozoa, Spiralia, and Eumetazoa, however, failed to be recovered due to a few problematic taxa such as the mite Ixodes and the warty comb jelly Mnemiopsis. The corresponding unexpected branchings can be explained by the paucity of synapomorphic changes of intron positions shared between some genomes, by the sensitivity of MP analyses to long-branch attraction (LBA), and by the very unequal evolutionary rates of intron loss and intron gain during evolution of the different subclades of metazoans. In addition, we obtained an assemblage of Cnidaria, Porifera, and Placozoa as sister group of Bilateria + Ctenophora with medium support, a disputable, but remarkable result. We conclude that NIPs can be used as phylogenetic characters also within a broader phylogenetic context, given that they have emerged regularly during evolution irrespective of the large variation of intron density across metazoan genomes.     

Molecular cloning and sequencing of the Na+/K+-ATPase α-subunit of the medical leech Hirudo medicinalis (Annelida) – implications for modelling protostomian evolution

Within the past few years, the phylogenetic tree is discussed controversially regarding the position of the different bilaterian groups. There are two varying views of evolution: the classical one based on morphological structures where the annelids and arthropods are combined in the group of Articulata, and a new hypothesis based on molecular data sets, which divides the protostomian groups in Ecdysozoa (e.g. arthropods, and nematodes) and Lophotrochozoa (e.g. annelids, molluscs, and platyhelminthes). The Na⁺/K⁺-ATPase is a highly conserved protein and fulfils a very important role in physiology and maintaining the homeostasis of cells and can be found in almost all eukaryotic animals. Due to its similar molecular structure throughout the animal kingdom the Na⁺/K⁺-ATPase is an excellent marker for phylogenetic studies. Here we report the molecular cloning, sequencing and phylogenetic analysis of Na⁺/K⁺-ATPase complementary deoxyribonucleic acid (cDNA) of the medical leech Hirudo medicinalis . The cloned cDNA codes for a polypeptide of 1022 amino acids and possesses a predicted molecular mass of 113.33 kDa. Phylogenetic analysis of the complete Na⁺/K⁺-ATPase α -subunit of H. medicinalis and sequence data from other Na⁺/K⁺-ATPases supports the previously developed 'Ecdysozoa concept' with high posterior probabilities. A common clade comprising annelids and platyhelminthes can be defined, whereas nematodes are in a basal position at the arthropod stem line.     

The evolution of the Ecdysozoa

Ecdysozoa is a clade composed of eight phyla: the arthropods, tardigrades and onychophorans that share segmentation and appendages and the nematodes, nematomorphs, priapulids, kinorhynchs and loriciferans, which are worms with an anterior proboscis or introvert. Ecdysozoa contains the vast majority of animal species and there is a great diversity of body plans among both living and fossil members. The monophyly of the clade has been called into question by some workers based on analyses of whole genome datasets. We review the evidence that now conclusively supports the unique origin of these phyla. Relationships within Ecdysozoa are also controversial and we discuss the molecular and morphological evidence for a number of monophyletic groups within this superphylum.     

Rare genomic characters do not support Coelomata: intron loss/gain

Recently, a new phylogenetic method employing intron positionsharing across species was proposed and support for a Coelomateclade reported (Zheng et al. 2007. A rigorous analysis of thepattern of intron conservation supports the Coelomata cladeof animals. Mol Biol Evol. 24:2583–2592.). Here, we showthat the previous analysis depends on: 1) an idiosyncratic definitionof "conserved" introns, 2) exclusion of all phylogeneticallyinformative introns present in outgroups, 3) incorrect inferenceof change along the critical branch, and 4) lack of variationin rates across branches. The method thus seems unlikely togive accurate results. In addition, we address differences inrates of loss across intron sites, which Zheng et al. claimedinvalidates our previous analysis that supported Ecdysozoa (Royand Gilbert. 2005a. Resolution of a deep animal divergence bythe pattern of intron conservation. Proc Natl Acad Sci USA.102:4403–4408.). Instead, we show that our conclusionsare likely to be robust to such concerns.     

Conserved intron positions in FGFR genes reflect the modular structure of FGFR and reveal stepwise addition of domains to an already complex ancestral FGFR

We have analyzed the evolution of fibroblast growth factor receptor (FGFR) tyrosine kinase genes throughout a wide range of animal phyla. No evidence for an FGFR gene was found in Porifera, but we tentatively identified an FGFR gene in the placozoan Trichoplax adhaerens. The gene encodes a protein with three immunoglobulin-like domains, a single-pass transmembrane, and a split tyrosine kinase domain. By superimposing intron positions of 20 FGFR genes from Placozoa, Cnidaria, Protostomia, and Deuterostomia over the respective protein domain structure, we identified ten ancestral introns and three conserved intron groups. Our analysis shows (1) that the position of ancestral introns correlates to the modular structure of FGFRs, (2) that the acidic domain very likely evolved in the last common ancestor of triploblasts, (3) that splicing of IgIII was enabled by a triploblast-specific insertion, and (4) that IgI is subject to substantial loss or duplication particularly in quickly evolving genomes. Moreover, intron positions in the catalytic domain of FGFRs map to the borders of protein subdomains highly conserved in other serine/threonine kinases. Nevertheless, these introns were introduced in metazoan receptor tyrosine kinases exclusively. Our data support the view that protein evolution dating back to the Cambrian explosion took place in such a short time window that only subtle changes in the domain structure are detectable in extant representatives of animal phyla. We propose that the first multidomain FGFR originated in the last common ancestor of Placozoa, Cnidaria, and Bilateria. Additional domains were introduced mainly in the ancestor of triploblasts and in the Ecdysozoa.     

Analysis of evolution of exon-intron structure of eukaryotic genes

The availability of multiple, complete eukaryotic genome sequences allows one to address many fundamental evolutionary questions on genome scale. One such important, long-standing problem is evolution of exon-intron structure of eukaryotic genes. Analysis of orthologous genes from completely sequenced genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists. The data on shared and lineage-specific intron positions were used as the starting point for evolutionary reconstruction with parsimony and maximum-likelihood approaches. Parsimony methods produce reconstructions with intron-rich ancestors but also infer lineage-specific, in many cases, high levels of intron loss and gain. Different probabilistic models gave opposite results, apparently depending on model parameters and assumptions, from domination of intron loss, with extremely intron-rich ancestors, to dramatic excess of gains, to the point of denying any true conservation of intron positions among deep eukaryotic lineages. Development of models with adequate, realistic parameters and assumptions seems to be crucial for obtaining more definitive estimates of intron gain and loss in different eukaryotic lineages. Many shared intron positions were detected in ancestral eukaryotic paralogues which evolved by duplication prior to the divergence of extant eukaryotic lineages. These findings indicate that numerous introns were present in eukaryotic genes already at the earliest stages of evolution of eukaryotes and are compatible with the hypothesis that the original, catastrophic intron invasion accompanied the emergence of the eukaryotic cells. Comparison of various features of old and younger introns starts shedding light on probable mechanisms of intron insertion, indicating that propagation of old introns is unlikely to be a major mechanism for origin of new ones. The existence and structure of ancestral protosplice sites were addressed by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and, accordingly, are not subject to selection for splicing efficiency. It was shown that introns indeed predominantly insert into or are fixed in specific protosplice sites which have the consensus sequence (A/C)AG|Gt.     

Exclusive conservation of mitochondrial group II intron nad4i548 among liverworts and its use for phylogenetic studies in this ancient plant clade

Liverworts occupy a pivotal position in land plant (embryophyte) phylogeny as the presumed earliest-branching major clade, sister to all other land plants, including the mosses, hornworts, lycophytes, monilophytes and seed plants. Molecular support for this earliest dichotomy in land plant phylogeny comes from strikingly different occurrences of introns in mitochondrial genes distinguishing liverworts from all other embryophytes. Exceptionally, however, the nad5 gene--the mitochondrial locus hitherto used most widely to elucidate early land plant phylogeny--carries a group I type intron that is shared between liverworts and mosses. We here explored whether a group II intron, the other major type of organellar intron, would similarly be conserved in position across the entire diversity of extant liverworts and could be of use for phylogenetic analyses in this supposedly most ancient embryophyte clade. To this end, we investigated the nad4 gene as a candidate locus possibly featuring different introns in liverworts as opposed to the non-liverwort embryophyte (NLE) lineage. We indeed found group II intron nad4i548 universally conserved in a wide phylogenetic sampling of 55 liverwort taxa, confirming clade specificity and surprising evolutionary stability of plant mitochondrial introns. As expected, intron nad4i548g2 carries phylogenetic information in its variable sequences, which confirms and extends previous cladistic insights on liverwort evolution. We integrate the new nad4 data with those of the previously established mitochondrial nad5 and the chloroplast rbcL and rps4 genes and present a phylogeny based on the fused datasets. Notably, the phylogenetic analyses suggest a reconsideration of previous phylogenetic and taxonomic assignments for the genera Calycularia and Mylia and resolve a sister group relationship of Ptilidiales and Porellales.     

Molecular evolution of calmodulin-like domain protein kinases (CDPKs) in plants and protists

Many genes for calmodulin-like domain protein kinases (CDPKs) have been identified in plants and Alveolate protists. To study the molecular evolution of the CDPK gene family, we performed a phylogenetic analysis of CDPK genomic sequences. Analysis of introns supports the phylogenetic analysis; CDPK genes with similar intron/exon structure are grouped together on the phylogenetic tree. Conserved introns support a monophyletic origin for plant CDPKs, CDPK-related kinases, and phosphoenolpyruvate carboxylase kinases. Plant CDPKs divide into two major branches. Plant CDPK genes on one branch share common intron positions with protist CDPK genes. The introns shared between protist and plant CDPKs presumably originated before the divergence of plants from Alveolates. Additionally, the calmodulin-like domains of protist CDPKs have intron positions in common with animal and fungal calmodulin genes. These results, together with the presence of a highly conserved phase zero intron located precisely at the beginning of the calmodulin-like domain, suggest that the ancestral CDPK gene could have originated from the fusion of protein kinase and calmodulin genes facilitated by recombination of ancient introns. Received: 11 July 2000 / Accepted: 18 April 2001     

Phylogenetic and Exon–Intron Structure Analysis of Fungal Subtilisins: Support for a Mixed Model of Intron Evolution

Phylogenetic and exon–intron structure analyses of intra- and interspecific fungal subtilisins in this study provided support for a mixed model of intron evolution: a synthetic theory of introns-early and introns-late speculations. Intraspecifically, there were three phase zero introns in Pr1A and its introns 1 and 2 located at the highly conserved positions were phylogentically congruent with coding region, which is in favor of the view of introns-early speculation, while intron 3 had two different sizes and was evolutionarily incongruent with coding region, the evidence for introns-late speculation. Noticeably, the subtilisin Pr1J gene from different strains of M. ansiopliae contained different number of introns, the strong evidence in support of introns-late theory. Interspecifically, phylogenetic analysis of 60 retrievable fungal subtilisins provided a clear relationship between amino acid sequence and gene exon–intron structure that the homogeneous sequences usually have a similar exon–infron structure. There were 10 intron positions inserted by highly biased phase zero introns across examined fungal subtilisin genes, half of these positions were highly conserved, while the others were species-specific, appearing to be of recent origins due to intron insertion, in favor of the introns-late theory. High conservations of positions 1 and 2 inserted by the high percentage of phase zero introns as well as the evidence of phylogenetic congruence between the evolutionary histories of intron sequences and coding region suggested that the introns at these two positions were primordial.Reviewing Editor:Dr. Manyuan Long     

Spliceosomal introns as tools for genomic and evolutionary analysis

Over the past 5 years, the availability of dozens of whole genomic sequences from a wide variety of eukaryotic lineages has revealed a very large amount of information about the dynamics of intron loss and gain through eukaryotic history, as well as the evolution of intron sequences. Implicit in these advances is a great deal of information about the structure and evolution of surrounding sequences. Here, we review the wealth of ways in which structures of spliceosomal introns as well as their conservation and change through evolution may be harnessed for evolutionary and genomic analysis. First, we discuss uses of intron length distributions and positions in sequence assembly and annotation, and for improving alignment of homologous regions. Second, we review uses of introns in evolutionary studies, including the utility of introns as indicators of rates of sequence evolution, for inferences about molecular evolution, as signatures of orthology and paralogy, and for estimating rates of nucleotide substitution. We conclude with a discussion of phylogenetic methods utilizing intron sequences and positions.     

The multimeric beta-thymosin found in nematodes and arthropods is not a synapomorphy of the Ecdysozoa

The Ecdysozoa hypothesis proposes a clade of animals including arthropods and nematodes that share the characteristic of periodic molting or ecdysis. The original evidence supporting this hypothesis came from molecular phylogenies based on ribosomal RNA gene sequences. Contrary evidence has come from studies of multiple protein coding genes. One of the most convincing bits of supporting evidence for this theory has been the observation of an unusual multimeric form of the beta-thymosin gene in the genomes of Drosophila melanogaster and Caenorhabditis elegans where, in other metazoans that had been studied, a monomeric form has been found. Here I show that recently deposited sequence data reveal that the multimeric form is in fact a characteristic of all major subdivisions of the Metazoa. The multimeric form is present in a deuterostome, Ciona intestinalis, a lophotrochozoan, Hermissenda crassicornis, and in the ecdysozoans and also exists outside the Metazoa in a fungus. The presence of the multimeric form in nematodes and arthropods, therefore, although not contradicting the Ecdysozoa hypothesis, gives it no support. The absence of the monomeric form in the completely sequenced flies and nematodes may suggest they are linked but, lacking the complete genomes of other ecdysozoans, proving its total absence from the Ecdysozoa is not possible. Furthermore, the absence of the monomeric form from the genome of the deuterostome Ciona suggests that the absence of this character is an unreliable indicator of relationships.     

Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns

As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5′ end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns’ features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts.     

Intron evolution in a phylogenetic perspective: Divergent trends in the two copies of the duplicated def gene in Impatiens L. (Balsaminaceae)

The history of MADS box genes is well-known in angiosperms. While duplication events and gene losses occur frequently, gene structure and intron positions are very conserved. We investigated all six introns in a duplicated MADS box gene (deficiens, def) in selected Impatiens taxa, thereby assessing intron features. For the first time, our study provides a comparison of molecular changes in all introns of a gene from a phylogenetic perspective. Interestingly, a uniform pattern of molecular evolution in the introns of each copy was not observed, but intron length increases, decreases, and size retention can be found in each copy. A tendency to accumulate long autapomorphic indels is also present, thus, a longer intron length does not reflect a higher number of parsimony-informative characters. Substitution rates vary between introns of each gene copy. While four of the six introns of def1 exhibit a change in their substitution rate, five of the six def2 introns maintain their rates throughout the genus albeit at different levels. In MADS box genes several regulatory sequences are found residing in introns. Thus, presence of putative regulatory motifs was investigated. Most of them are not conserved in position and usually present in only one of the gene copies. In addition, the potential for phylogenetic reconstruction of introns in both def copies is shortly discussed.