Evolutionary relationships among heterokont algae (the autotrophic stramenopiles) based on combined analyses of small and large subunit ribosomal RNA

In order to study the phylogenetic relationships within the stramenopiles, and particularly among the heterokont algae, we have determined complete or nearly complete large-subunit ribosomal RNA sequences for different species of raphidophytes, phaeophytes, xanthophytes, chrysophytes, synurophytes and pinguiophytes. With the small- and large-subunit ribosomal RNA sequences of representatives for nearly all known groups of heterokont algae, phylogenetic trees were constructed from a concatenated alignment of both ribosomal RNAs, including more than 5,000 positions. By using different tree construction methods, inferred phylogenies showed phaeophytes and xanthophytes as sister taxa, as well as the pelagophytes and dictyochophytes, and the chrysophytes/synurophytes and eustigmatophytes. All these relationships are highly supported by bootstrap analysis. However, apart from these sister group relationships, very few other internodes are well resolved and most groups of heterokont algae seem to have diverged within a relatively short time frame.     

Early branchings in the evolution of eukaryotes: Ancient divergence of entamoeba that lacks mitochondria revealed by protein sequence data

Summary Phylogenetic analyses of ribosomal RNA sequences have played an important role in the study of early evolution of life. However, Loomis and Smith suggested that the ribosomal RNA tree is sometimes misleading—especially when G+C content differs widely among lineages—and that a protein tree from amino acid sequences may be more reliable. In this study, we analyzed amino acid sequence data of elongation factor-1 by a maximum likelihood method to clarify branching orders in the early evolution of eukaryotes. Contrary to Sogin et al.'s tree of small-subunit ribosomal RNA, a protozoan species, Entamoeba histolytica, that lacks mitochondria was shown to have diverged from the line leading to eukaryotes with mitochondria before the latter separated into several kingdoms. This indicates that Entamoeba is a living relic of the earliest phase of eukaryotic evolution before the symbiosis of protomitochondria occurred. Furthermore, this suggests that, among eukaryotic kingdoms with mitochondria, Fungi is the closest relative of Animalia, and that a cellular slime mold, Dictyostelium discoideum, had not diverged from the line leading to Plantae-Fungi-Animalia before these three kingdoms separated. Offprint requests to: M. Hasegawa     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.      

Characteristics and utility of nuclear-encoded large-subunit ribosomal gene sequences in phylogenetic studies of red algae

Primer sequences are described for amplifying and sequencing a large fragment (approximately 2500 b.p.) of the nuclear-encoded large-subunit ribosomal RNA gene (LSU) from red algae. In comparison to RuBisCo large-subunit gene (rbcL) and nuclear-encoded small-subunit ribosomal RNA gene (SSU) sequence data, LSU sequence data was intermediate in the number of phylogenetically informative positions and sequence divergence. Parsimony analysis of LSU sequences for 16 Gelidiales species resolved some nodes unresolved in rbcL and SSU parsimony trees. An analysis of LSU sequences from 13 species of red algae classified in 11 orders suggests that this gene may be useful in studies of higher-level relationships of red algae.     

Evaluating hypotheses of deuterostome phylogeny and chordate evolution with new LSU and SSU ribosomal DNA data

We investigated evolutionary relationships among deuterostome subgroups by obtaining nearly complete large-subunit ribosomal RNA (LSU rRNA)-gene sequences for 14 deuterostomes and 3 protostomes and complete small-subunit (SSU) rRNA-gene sequences for five of these animals. With the addition of previously published sequences, we compared 28 taxa using three different data sets (LSU only, SSU only, and combined LSU + SSU) under minimum evolution (with LogDet distances), maximum likelihood, and maximum parsimony optimality criteria. Additionally, we analyzed the combined LSU + SSU sequences with spectral analysis of LogDet distances, a technique that measures the amount of support and conflict within the data for every possible grouping of taxa. Overall, we found that (1) the LSU genes produced a tree very similar to the SSU gene tree, (2) adding LSU to SSU sequences strengthened the bootstrap support for many groups above the SSU-only values (e.g., hemichordates plus echinoderms as Ambulacraria; lancelets as the sister group to vertebrates), (3) LSU sequences did not support SSU-based hypotheses of pterobranchs evolving from enteropneusts and thaliaceans evolving from ascidians, and (4) the combined LSU + SSU data are ambiguous about the monophyly of chordates. No tree-building algorithm united urochordates conclusively with other chordates, although spectral analysis did so, providing our only evidence for chordate monophyly. With spectral analysis, we also evaluated several major hypotheses of deuterostome phylogeny that were constructed from morphological, embryological, and paleontological evidence. Our rRNA-gene analysis refutes most of these hypotheses and thus advocates a rethinking of chordate and vertebrate origins.     

小鲵科线粒体16S rRNA基因序列分析及其系统发育

To study the phylogeny of Hynobiidae, we amplified DNA fragments of 470 bp 16S ribosomal RNA (16S rRNA) gene on mitochondrial DNA from Ranodon sibiricus and Ranodon tsinpaensis. PCR products were cloned into PMD18 T vector after purification. These sequences were determined and deposited in the GenBank (accession numbers: AY373459 for Ranodon sibiricus, AY372534 for Ranodon tsinpaensis). By comparing the nucleotide differences of 16S ribosomal RNA sequences among Liua shihi, Pseudohynobius flavomaculatus and Batrachuperus genus from GenBank database, we analyzed the divergences and base substitution among these sequences with the MEGA software. The molecular results support that B. tibetanus, B. pinchonii and B. karlschmidti are classified into three valid species. Liua shihi has closer phylogenetic relationships to Ranodon tsinpaensis than to other species. More our results reveal that Pseudohynobius flavomaculatus is not a synonym of Ranodon tsinpaensis. [Acta Zoologica Sinica 50 (3) : 464 - 469,2004].     

PHYLOGENY, BIOGEOGRAPHY AND LIFE HISTORY EVOLUTION IN THE RED ALGAL FAMILY PHYLLOPHORACEAE (GIGARTINALES)

Red algae are exceptional for the great diversity in reproductive morphology and for their complex life histories. In particular, the family Phyllophoraceae, consisting of ∼100 species worldwide, stands out in exhibiting a wide spectrum of unique life history types that makes it unusually interesting for assessing the phylogenetic importance of reproductive traits relative to classification criteria. Type of life history and position of the reproductive structures on the plant body have traditionally formed the basis for separating eleven genera in the Phyllophoraceae; however, phylogenetic analyses inferred from three sets of DNA sequences [chloroplast-encoded rbcL , nuclear large-subunit ribosomal RNA gene (LSU), and internal transcribed spacer regions (ITS) of nuclear ribosomal DNA], instead indicate a lack of correlation between type of life history and phylogenetic relationships among the established taxa. This lack of correlation dramatically challenges all of the traditional taxonomy. The study will answer the question which morphological features and which aspects of life history evolution can be used as meaningful indicators of phylogenetic relationships in the Phyllophoraceae. The results are addressed in light of global biogeographic hypotheses for the family.     

MOLECULAR SYSTEMATICS OF THE FRESHWATER SNAIL FAMILY LYMNAEIDAE (PULMONATA: BASOMMATOPHORA) UTILISING MITOCHONDRIAL RIBOSOMAL DNA SEQUENCES

Nucleotide sequences were obtained from a region of the largesubunit mitochondrial ribosomal RNA gene from 18 lymnaeid taxaand a planorbid out-group. A molecular phylogeny was constructedutilising maximum-parsimony and distance analyses of the data.This agreed with certain aspects of kary-ology and immunology-basedlymnaeid taxonomy, but failed to support the only previouslypublished phylogenetk scheme or past inferences regarding basalrelationships in lymnaeids. The molecular phylogeny is alsoincongruent with previous progressive-evolution models basedon chromosome numbers because European genera having n = 18formed a group basal within lymnaeids whereas taxa belongingto the n = 16 group included the most derived lymnaeids. Relationshipsof selected problematic taxa inferred from the molecular phylogenywere compared to previous hypotheses based on other data sets.A partial, tentative bio-geographical hypothesis was derivedfrom this phylogeny. We propose a Laurasian origin for lymnaeidsin the late Jurassic. (Received 14 August 1996; accepted 3 October 1996)     

Molecular phylogeny of glass sponges (Porifera, Hexactinellida): increased taxon sampling and inclusion of the mitochondrial protein-coding gene, cytochrome oxidase subunit I

Marine sponges of the class Hexactinellida (glass sponges) are among the most understudied groups of Porifera, and molecular approaches to investigating their evolution have only recently emerged. Although these first results appeared reliable as they largely corroborated morphology-based hypotheses, they were almost exclusively based on ribosomal RNA genes (rDNA) and should, therefore, be further tested with independent types of genetic data, such as protein-coding genes. To this end, we established the mitochondrial-encoded cytochrome oxidase subunit I gene (COI) as an additional marker, and conducted phylogenetic analyses on DNA- and amino-acid level, as well as a supermatrix analysis based on combined COI DNA and rDNA alignments. Furthermore, we increased taxon sampling compared to previous studies by adding seven additional species. The COI-based phylogenies were largely congruent with the rDNA-based phylogeny but suffered from poor bootstrap support for many nodes. However, addition of the COI sequences to the rDNA data set increased resolution of the overall molecular phylogeny. Thus, although obtaining COI sequences from glass sponges turned out to be quite challenging, this gene appears to be a valuable supplement to rDNA data for molecular evolutionary studies of this group. Some implications of our extended phylogeny for the evolution and systematics of Hexactinellida are discussed.     

The phylogeny of the Hyphochytriomycota as deduced from ribosomal RNA sequences of Hyphochytrium catenoides

Based on biochemical and ultrastructural data, hyphochytriomycetes are believed to share an ancestor with oomycetes and heterokont algae. In order to study the phylogeny of the hyphochytriomycetes, we determined both the small- and large-subunit ribosomal RNA sequence of Hyphochytrium catenoides. Phylogenetic trees were constructed using the neighbor-joining and maximum-parsimony method and include representatives of Chlorobionta, Fungi, Metazoa, Alveolata, and all known Heterokonta. Our main conclusion is that the hyphochytriomycetes form a monophyletic group with the oomycetes and heterokont algae and that they are probably the closest relatives of the oomycetes. However, the order of divergence between the various heterokont algal phyla and the oomycete-hyphochytriomycete cluster remains uncertain.      

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.     

Archaeal phylogeny based on ribosomal proteins

Until recently, phylogenetic analyses of Archaea have mainly been based on ribosomal RNA (rRNA) sequence comparisons, leading to the distinction of the two major archaeal phyla: the Euryarchaeota and the Crenarchaeota. Here, thanks to the recent sequencing of several archaeal genomes, we have constructed a phylogeny based on the fusion of the sequences of the 53 ribosomal proteins present in most of the archaeal species. This phylogeny was remarkably congruent with the rRNA phylogeny, suggesting that both reflected the actual phylogeny of the domain Archaea even if some nodes remained unresolved. In both cases, the branches leading to hyperthermophilic species were short, suggesting that the evolutionary rate of their genes has been slowed down by structural constraints related to environmental adaptation. In addition, to estimate the impact of lateral gene transfer (LGT) on our tree reconstruction, we used a new method that revealed that 8 genes out of the 53 ribosomal proteins used in our study were likely affected by LGT. This strongly suggested that a core of 45 nontransferred ribosomal protein genes existed in Archaea that can be tentatively used to infer the phylogeny of this domain. Interestingly, the tree obtained using only the eight ribosomal proteins likely affected by LGT was not very different from the consensus tree, indicating that LGT mainly brought random phylogenetic noise. The major difference involves organisms living in similar environments, suggesting that LGTs are mainly directed by the physical proximity of the organisms rather than by their phylogenetic proximity.     

Efficient selection of branch-specific models of sequence evolution

The analysis of extant sequences shows that molecular evolution has been heterogeneous through time and among lineages. However, for a given sequence alignment, it is often difficult to uncover what factors caused this heterogeneity. In fact, identifying and characterizing heterogeneous patterns of molecular evolution along a phylogenetic tree is very challenging, for lack of appropriate methods. Users either have to a priori define groups of branches along which they believe molecular evolution has been similar or have to allow each branch to have its own pattern of molecular evolution. The first approach assumes prior knowledge that is seldom available, and the second requires estimating an unreasonably large number of parameters. Here we propose a convenient and reliable approach where branches get clustered by their pattern of molecular evolution alone, with no need for prior knowledge about the data set under study. Model selection is achieved in a statistical framework and therefore avoids overparameterization. We rely on substitution mapping for efficiency and present two clustering approaches, depending on whether or not we expect neighbouring branches to share more similar patterns of sequence evolution than distant branches. We validate our method on simulations and test it on four previously published data sets. We find that our method correctly groups branches sharing similar equilibrium GC contents in a data set of ribosomal RNAs and recovers expected footprints of selection through dN/dS. Importantly, it also uncovers a new pattern of relaxed selection in a phylogeny of Mantellid frogs, which we are able to correlate to life-history traits. This shows that our programs should be very useful to study patterns of molecular evolution and reveal new correlations between sequence and species evolution. Our programs can run on DNA, RNA, codon, or amino acid sequences with a large set of possible models of substitutions and are available at http://biopp.univ-montp2.fr/forge/testnh.     

The Most Conserved Genome Segments for Life Detection on Earth and Other Planets

On Earth, very simple but powerful methods to detect and classify broad taxa of life by the polymerase chain reaction (PCR) are now standard practice. Using DNA primers corresponding to the 16S ribosomal RNA gene, one can survey a sample from any environment for its microbial inhabitants. Due to massive meteoritic exchange between Earth and Mars (as well as other planets), a reasonable case can be made for life on Mars or other planets to be related to life on Earth. In this case, the supremely sensitive technologies used to study life on Earth, including in extreme environments, can be applied to the search for life on other planets. Though the 16S gene has become the standard for life detection on Earth, no genome comparisons have established that the ribosomal genes are, in fact, the most conserved DNA segments across the kingdoms of life. We present here a computational comparison of full genomes from 13 diverse organisms from the Archaea, Bacteria, and Eucarya to identify genetic sequences conserved across the widest divisions of life. Our results identify the 16S and 23S ribosomal RNA genes as well as other universally conserved nucleotide sequences in genes encoding particular classes of transfer RNAs and within the nucleotide binding domains of ABC transporters as the most conserved DNA sequence segments across phylogeny. This set of sequences defines a core set of DNA regions that have changed the least over billions of years of evolution and provides a means to identify and classify divergent life, including ancestrally related life on other planets.     

18S rDNA phylogeny of lamproderma and allied genera (Stemonitales, Myxomycetes, Amoebozoa)

The phylogenetic position of the slime-mould genus Lamproderma (Myxomycetes, Amoebozoa) challenges traditional taxonomy: although it displays the typical characters of the order Stemonitales, it appears to be sister to Physarales. This study provides a small subunit (18S or SSU) ribosomal RNA gene-based phylogeny of Lamproderma and its allies, with new sequences from 49 specimens in 12 genera. We found that the order Stemonitales and Lamproderma were both ancestral to Physarales and that Lamproderma constitutes several clades intermingled with species of Diacheopsis, Colloderma and Elaeomyxa. We suggest that these genera may have evolved from Lamproderma by multiple losses of fruiting body stalks and that many taxonomic revisions are needed. We found such high genetic diversity within three Lamproderma species that they probably consist of clusters of sibling species. We discuss the contrasts between genetic and morphological divergence and implications for the morphospecies concept, highlighting the phylogenetically most reliable morphological characters and pointing to others that have been overestimated. In addition, we showed that the first part (~600 bases) of the SSU rDNA gene is a valuable tool for phylogeny in Myxomycetes, since it displayed sufficient variability to distinguish closely related taxa and never failed to cluster together specimens considered of the same species.     

Recurrent structural RNA motifs, Isostericity Matrices and sequence alignments

The occurrences of two recurrent motifs in ribosomal RNA sequences, the Kink-turn and the C-loop, are examined in crystal structures and systematically compared with sequence alignments of rRNAs from the three kingdoms of life in order to identify the range of the structural and sequence variations. Isostericity Matrices are used to analyze structurally the sequence variations of the characteristic non-Watson–Crick base pairs for each motif. We show that Isostericity Matrices for non-Watson–Crick base pairs provide important tools for deriving the sequence signatures of recurrent motifs, for scoring and refining sequence alignments, and for determining whether motifs are conserved throughout evolution. The systematic use of Isostericity Matrices identifies the positions of the insertion or deletion of one or more nucleotides relative to the structurally characterized examples of motifs and, most importantly, specifies whether these changes result in new motifs. Thus, comparative analysis coupled with Isostericity Matrices allows one to produce and refine structural sequence alignments. The analysis, based on both sequence and structure, permits therefore the evaluation of the conservation of motifs across phylogeny and the derivation of rules of equivalence between structural motifs. The conservations observed in Isostericity Matrices form a predictive basis for identifying motifs in sequences.     

Effects of sequence alignment and structural domains of ribosomal DNA on phylogeny reconstruction for the protozoan family sarcocystidae

Finding correct species relationships using phylogeny reconstruction based on molecular data is dependent on several empirical and technical factors. These include the choice of DNA sequence from which phylogeny is to be inferred, the establishment of character homology within a sequence alignment, and the phylogeny algorithm used. Nevertheless, sequencing and phylogeny tools provide a way of testing certain hypotheses regarding the relationship among the organisms for which phenotypic characters demonstrate conflicting evolutionary information. The protozoan family Sarcocystidae is one such group for which molecular data have been applied phylogenetically to resolve questionable relationships. However, analyses carried out to date, particularly based on small-subunit ribosomal DNA, have not resolved all of the relationships within this family. Analysis of more than one gene is necessary in order to obtain a robust species signal, and some DNA sequences may not be appropriate in terms of their phylogenetic information content. With this in mind, we tested the informativeness of our chosen molecule, the large-subunit ribosomal DNA (lsu rDNA), by using subdivisions of the sequence in phylogenetic analysis through PAUP, fastDNAml, and neighbor joining. The segments of sequence applied correspond to areas of higher nucleotide variation in a secondary-structure alignment involving 21 taxa. We found that subdivision of the entire lsu rDNA is inappropriate for phylogenetic analysis of the Sarcocystidae. There are limited informative nucleotide sites in the lsu rDNA for certain clades, such as the one encompassing the subfamily Toxoplasmatinae. Consequently, the removal of any segment of the alignment compromises the final tree topology. We also tested the effect of using two different alignment procedures (CLUSTAL W and the structure alignment using DCSE) and three different tree-building methods on the final tree topology. This work shows that congruence between different methods in the formation of clades may be a feature of robust topology; however, a sequence alignment based on primary structure may not be comparing homologous nucleotides even though the expected topology is obtained. Our results support previous findings showing the paraphyly of the current genera Sarcocystis and Hammondia and again bring to question the relationships of Sarcocystis muris, Isospora felis, and Neospora caninum. In addition, results based on phylogenetic analysis of the structure alignment suggest that Sarcocystis zamani and Sarcocystis singaporensis, which have reptilian definitive hosts, are monophyletic with Sarcocystis species using mammalian definitive hosts if the genus Frenkelia is synonymized with Sarcocystis.     

Molecular phylogenetic analysis among bryophytes and tracheophytes based on combined data of plastid coded genes and the 18S rRNA gene.

The basal relationship of bryophytes and tracheophytes is problematic in land plant phylogeny. In addition to cladistic analyses of morphological data, molecular phylogenetic analyses of the nuclear small-subunit ribosomal RNA gene and the plastic gene rbcL have been performed, but no confident conclusions have been reached. Using the maximum-likelihood (ML) method, we analyzed 4,563 bp of aligned sequences from plastid protein-coding genes and 1,680 bp from the nuclear 18S rRNA gene. In the ML tree of deduced amino acid sequences of the plastid genes, hornworts were basal among the land plants, while mosses and liverworts each formed a clade and were sister to each other. Total-evidence evaluation of rRNA data and plastid protein-coding genes by TOTALML had an almost identical result.