Molecular phylogeny of centrohelid heliozoa,a novel lineage of bikont eukaryotes that arose by ciliary loss

Abstract Recent molecular and cellular evidence indicates that eukaryotes comprise three major lineages: the probably ancestrally uniciliate protozoan phylum Amoebozoa; the ancestrally posteriorly uniciliate opisthokont clade (animals, Choanozoa, and fungi); and a very diverse ancestrally biciliate clade, the bikonts—plants, chromalveolates, and excavate and rhizarian Protozoa. As Heliozoa are the only eukaryote phylum not yet placed on molecular sequence trees, we have sequenced the 18S rRNA genes of three centrohelid heliozoa, Raphidiophrys ambigua, Heterophrys marina, and Chlamydaster sterni, to investigate their phylogenetic position. Phylogenetic analysis by distance and maximum likelihood methods allowing for intersite rate variation and invariable sites confirms that centrohelid heliozoa are a robust clade that does not fall within any other phyla. In particular, they are decisively very distant from the heterokont pedinellid chromists, at one time thought to be related to heliozoa, and lack the unique heterokont signature sequence. They also appear not to be specifically related to either Amoebozoa or Radiolaria, with which they have sometimes been classified, so it is desirable to retain Heliozoa as a separate protozoan phylum. Even though centrohelids have no cilia or centrioles, the centrohelid clade branches among the bikont eukaryotes, but there is no strong bootstrap support for any particular position. Distance trees usually place centrohelids as sisters to haptophytes, whereas parsimony puts them as sisters to red algae, but there is no reason to think that either position is correct; both have very low bootstrap support. Quartet puzzling places them with fairly low support as sisters to the apusozoan zooflagellate Ancyromonas. As Ancyromonas is the only other eukaryote that shares the character combination of flat plate-like mitochondrial cristae and kinetocyst-type extrusomes with centrohelids, this position is biologically plausible, but because of weak support and conflict between trees it might not be correct. Irrespective of their precise position, our trees (together with previous evidence that Chlamydaster sterni has the derived dihydrofolate reductase/thymidylate synthetase gene fusion unique to bikonts) indicate that centrohelid heliozoa are ancestrally derived from a bikont flagellate by the loss of cilia. The centroplast that nucleates their axonemal microtubules is therefore almost certainly homologous with the centrosome of ciliated eukaryotes and should simply be called a centrosome.     

Molecular phylogeny in the Lardizabalaceae

Eleven species belonging to seven genera in the Lardizabalaceae were analyzed in terms of restriction fragment length polymorphism (RFLP) of chloroplast DNA and the sequence of the chloroplast gene,rbcL, of Lardizabalaceae and its related families. Phylogenetic trees inferred from parsimony, neighbor joining and maximum likelihood methods based on RFLP data showed that two South American genera,Boquila andLardizabala, and three East Asian genera,Akebia, Holboellia andStauntonia are closely related to each other, respectively. On the other hand, the parsimony, neighbor joining and maximum likelihood trees constructed using sequence data of therbcL gene showed thatAkebia, Stauntonia, Boquila andLardizabala clustered as(((Akebia, Stauntonia), Boquila), Lardizabala). This difference may be attributable to fewer informative sites inrbcL genes than in RFLP in this family.Decaisnea diverges at the very base of the Lardizabalaceae.     

Molecular phylogeny of the order Euryalida (Echinodermata: Ophiuroidea), based on mitochondrial and nuclear ribosomal genes

The existing taxonomy of Euryalida, one of the two orders of the Ophiuroidea (Echinodermata), is uncertain and characterized by controversial delimitation of taxonomic ranks from genus to family-level. Their phylogeny was not studied in detail until now. We investigated a dataset of sequence from a mitochondrial gene (16S rRNA) and two nucleic genes (18S rRNA and 28S rRNA) for 49 euryalid ophiuroids and four outgroup species from the order Ophiurida.The monophyly of the order Euryalida was supported as was the monophyly of Asteronychidae, Gorgonocephalidae and an Asteroschematidae + Euryalidae clade. However, the group currently known as the Asteroschematidae was paraphyletic with respect to the Euryalidae. The Asteroschematidae + Euryalidae clade, which we recognise as an enlarged Euryalidae, contains three natural groups: the Asteroschematinae (Asteroschema and Ophiocreas), a new subfamily Astrocharinae (Astrocharis) and the Euryalinae with remaining genera. These subfamilies can be distinguished by internal ossicle morphology.     

Procaryote phylogeny

Summary The catalog of oligonucleotides produced by T₁ ribonuclease digestion ofAerobacter aerogenes 16 S ribosomal RNA has been determined and compared to that characterizingEscherichia coli. It is concluded that the two 16 SrRNAs are approximately 98% similar, making the organisms very closely related.     

Molecular phylogeny of Mugilidae fishes revised

Systematics derived from morphological characters often does not correspond with the evolutionary processes underlying the divergence within a group of organisms. In the family Mugilidae (Teleostei) morphological similarities have resulted in inconsistencies between taxonomy and phylogeny among its species, and particularly for the genera Mugil, Liza and Chelon where both intrageneric and intergeneric phylogenetic clarifications are needed. To address these issues, the direct sequencing of the mitochondrial region that encodes Phenylalanine (69 bp), 12S rRNA (842 bp), cytochrome c oxidase subunit I (651 bp) and cytochrome b (702 bp) was carried out. The data reveal that Mugil platanus and Mugil liza represent a continuum of a single species, closely related to but distinct from Mugil cephalus which itself appears to comprise a grouping of multiple and closely related species. This species complex was genetically distinct from Mugil curema, which, based on three clearly diverged species identified in this study along the Atlantic coast of the Americas, requires extensive taxonomic revision throughout its world-wide distribution. Unlike the monophyly supported within Mugil, relationships within Liza are paraphyletic, and a taxonomic revision of the genera Liza, Chelon and Oedalechilus is needed.     

Molecular phylogeny and evolution of the AsianAconitum subgenusAconitum (Ranunculaceae)

Classification of the genusAconitum (Ranunculaceae) has long been considered quite difficult because its species show high levels of morphological and ecological variability. The molecular phylogeny of Asian aconites,Aconitum subgenusAconitum was, therefore, studied based on RFLP and sequences of the intergenic spacer between thetrnL (UAA) 3′ exon andtrnF (GAA), and of thetrnL intron, of the chloroplast DNA. UsingAconitum subgenusLycoctonum as an outgroup, we obtained a statistically reliable molecular tree composed of six clades branched radiatively at the very base. There are three clades of Japanese aconites, a single clade of the species of Yunnan and Himalayas, and two clades of Siberian plants. All the tetraploid taxa of Japan we studied did not show any difference based on the molecular characters analyzed, though they have been classified into many taxa. Evolution and phytogeography of the Asian aconites as well as the phylogeny are discussed.     

Molecular phylogeny of Armillaria from the Patagonian Andes

A number of species in the plant pathogen genus Armillaria are known from South America where they cause root rot disease on a wide variety of hosts. Knowledge pertaining to phylogenetic relationships of these species with those of other Armillaria species is almost non-existent. In addition, very few cultures representing these species are available, making DNA-based phylogenetic analyses impossible. The aim of this study was to characterise a collection of Armillaria isolates from the Patagonian Andes using DNA sequences and to determine their phylogenetic relationships with other Armillaria species. DNA sequences were obtained from the internal transcribed regions (ITS1, 5.8S and ITS4) and ribosomal large subunit (LSU) gene and used in phylogenetic analyses. Phylogenetic trees generated from the sequences separated the Armillaria isolates into four lineages. Lineages I and II represented A. novae-zelandiae and A. luteobubalina, respectively. Isolates belonging to A. novae-zelandiae from Malaysia, New Zealand, Australia and South America showed considerable intra-clade sub-structure. Lineages III and IV are probably distinct species and are most closely related to A. hinnulea and an unnamed species isolated from New Zealand and Kenya. This is the first comprehensive study of the phylogenetic relationships of Armillaria species from Patagonia and it provides a foundation for future research in this region.     

Molecular phylogeny and systematics of the Lilium carniolicum group (Liliaceae) based on nuclear ITS sequences

The Lilium carniolicum group consists of several taxonomically dubious taxa endemic to the European flora. Internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA) were used to clarify both the delineation of, and relationships among, taxa in the group as well as to provide insight on the phylogenetic position of the group within the genus. Maximum parsimony, maximum likelihood and Bayesian analyses were in general agreement, with all taxa in the group being very closely related, and the entire group being monophyletic. L. pyrenaicum and L. pomponium are placed at the basal position in the group, while L. chalcedonicum is shown to be more closely related to L. carniolicum than previously thought. Our analyses suggested that L. albanicum and L. jankae are distinct from L. carniolicum, while no evidence was found to support the same separation for L. bosniacum.     

Molecular phylogeny of Drosophila based on ribosomal RNA sequences

Nucleotide sequences of 72 species of Drosophilidae were determined for divergent D1 and D2 domains (representing 200 and 341 nucleotides respectively in D. melanogaster) of large ribosomal RNA, using the rRNA direct sequencing method. Molecular phylogenetic trees were reconstructed using both distance and parsimony methods and the robustness of the nodes was evaluated by the bootstrap procedure. The trees obtained by these methods revealed four main lineages or clades which do not correspond to the taxonomical hierarchy. In our results, the genus Chymomyza is associated with the subgenus Scaptodrosophila of the genus Drosophila and their cluster constitutes the most ancient clade. The two other clades are constituted of groups belonging to the subgenus Sophophora of the genus Drosophila: the so-called Neotropical clade including the willistoni and saltans groups and the obscura-melanogaster clade itself split into three lineages: (1) obscura group + ananassae subgroup, (2) montium subgroup, and (3) melanogaster + Oriental subgroups. The fourth clade, the Drosophila one, contains three lineages. D. polychaeta, D. iri, and D. fraburu are branched together and constitute the most ancient lineage; the second lineage includes the annulimana, bromeliae, dreyfusi, melanica, mesophragmatica, repleta, robusta, and virilis groups. The third lineage is composed of the immigrans and the cardini, funebris, guaramunu, guarani, histrio, pallidipennis, quinaria, and tripunctata groups. The genera Samoaia, Scaptomyza, and Zaprionus are branched within the Drosophila clade. Although these four clades appear regularly in almost all tree calculations, additional sequencing will be necessary to determine their precise relationships.Correspondence to: M. Pelandakis     

Molecular phylogeny,taxonomy, and evolution of Geosiphon pyriformis and arbuscular mycorrhizal fungi

Geosiphon pyriformis(Kütz.) v. Wettstein is the only known example of a fungus living in endocytobiotic association with a cyanobacterium. The close phylogenetic relationship of Geosiphonwith some arbuscular mycorrhizal fungi (AMF) and the phylogenetic position of Geosiphonare shown in detail. Comprehensive small subunit (SSU) rRNA sequence analyses allow the erection of a new, molecular phylogeny-based taxonomic system for the AMF, including Geosiphon (Geosiphonaceae). Within the recently described phylum Glomeromycota (with one class, Glomeromycetes), a system including four orders was proposed. The erection of several new families will also be necessary. Evolutionary implications are discussed, referring to different possibilities of the influence of AMF on the colonization of the terrestrial habitat by plants.     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

Molecular phylogeny of Alnus (Betulaceae), inferred from nuclear ribosomal DNA ITS sequences

The nuclear ITS region of 19 species of Alnus was amplified and sequenced. The inferred molecular phylogeny shows that all species of the genus Alnus form a monophyletic group close to Betula and that the fundamental dichotomy within the genus lies between the subgenera Alnaster and Gymnothyrsus, sensu Murai (1964). The subgenus Alnaster appears to be basal in the genus, based on archaism of morphological features, and branching close to the root of the trees due to low ITS divergence from genus Betula. The monophyly of the section Clethropsis is not supported by the present data: Alnus nepalensis is positioned in the subgenus Gymnothyrsus, away from A. nitida and A. maritima. Surprisingly, A. formosana sect. Japonicae is closely tied to A. maritima sect. Clethropsis, with which it shares few morphological traits, and is separate from A. japonica sect. Japonicae with which it shares many traits. An increase in substitution rate is noted in the group comprising A. formosana, A. maritima and A. nitida relative to the rest of the genus, which appears to have had, on the average, a very slow mutation rate. Alnus glutinosa, the designated type for the genus, appears to be representative of the genus both for morphological characters and evolutionary rate. North-East Asia is comforted in its position of origin of the genus since not only does it have the highest number of species and representatives in all deep branching lineages, there are also fewer transcontinental migrations when a North-East Asian ancestor is postulated than when a North American ancestor is postulated.     

Molecular phylogeny of geminivirus infecting wild plants in Japan

Few studies have been made on the molecular divergence of plant viruses. To remedy this deficiency, we examined the molecular divergence of the tobacco leaf curl geminivirus (TLCV). TLCV infects not only tobacco but alsoEupatorium andLonicera in the field and causes yellow vein disease. A total of 29 nucleotide sequences of the replication protein gene (ORF C1) of geminiviruses infecting wild plants ofE. makinoi, E. glehni andL. japonica collected from ten localities was determined. Highly divergent sequences were obtained not only among host plant populations but also within a host population. Phylogenetic analyses showed that the TLCVs infectingEupatorium andLonicera were clustered into three different clades, and were either paraphyletic or polyphyletic. This result is the first evidence demonstrating that wild populations of single plant species possess genetically diversified virus strains. Comparison with recently reported genetic variations of tobacco mild green mosaic tobamovirus (TMGMV) revealed three characteristics of TLCV evolution: (1) a higher nucleotide substitution rate, (2) more frequent migration among geographically isolated host populations, and (3) more frequent host changes to different plant families. While TMGMV is an RNA virus, TLCV has DNA genomes. In animal viruses, RNA viruses tend to evolve faster than DNA viruses. Our results indicated that this trend may not hold for plant viruses.     

Molecular phylogeny of Old WorldTrifolium (Fabaceae), based on plastid and nuclear markers

Phylogenies of Old WorldTrifolium species were constructed using nucleotide sequence data of the internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast DNA restriction site data from PCR-amplified genes and genic regions (rbcL,trnK, andrpoC1–C2). Biogeography, morphological evolution, and the existing classification forTrifolium were examined. The genusTrifolium is strongly supported as monophyletic, however, only one small section (Chronosemium) is monophyletic, although the data are in conflict regarding its placement. The two largest sections of the genus, Sects.Lotoidea andTrifolium, are not supported as monophyletic, as currently circumscribed. Many members of Sect.Lotoidea are basal within the genus, supporting previously-proposed hypotheses concerning plesiomorphic morphological characters and a Mediterranean-Mideast biogeographic origin of the genus.     

Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) based on the internal transcribed spacer of the nuclear ribosomal DNA

The internal transcribed spacer (ITS1, 5.8S rDNA, and ITS2) region of nuclear ribosomal DNA (nrDNA) was sequenced from 53 species, which represent most of the living species diversity in the genus Phalaenopsis (Orchidaceae). A phylogeny was developed for the genus based on the neighbor-joining and maximum parsimony analyses of molecular data. Results of these analyses provided support for the monophyly of the genus Phalaenopsis and concurred in that the genera Doritis and Kingidium should be treated as being parts of the genus Phalaenopsis as suggested by Christenson (2001). Within the genus Phalaenopsis, neither subgenera Aphyllae nor Parishianae were monophyletic, and they were highly clustered with subgenus Proboscidioides plus sections Esmeralda and Deliciosae of subgenus Phalaenopsis based on ITS data. Those species also have the same characters of morphology of four pollinia and similar biogeographies. Furthermore, neither subgenus Phalaenopsis nor Polychilos was monophyletic. Within the subgenus Phalaenopsis, only section Phalaenopsis was highly supported as being monophyletic. As for the subgenus Polychilos, only section Polychilos was moderately supported as being monophyletic. In conclusion, the present molecular data obtained from the ITS sequence of nrDNA of the genus Phalaenopsis provide valuable information for elucidating the phylogeny of this genus.     

Molecular detection,community structure and phylogeny of ericoid mycorrhizal fungi

Through traditional culturing and molecular characterization, we have determined that five putative species and 2 polyphyletic assemblages of fungi produce ericoid mycorrhizae in Gaultheria shallon, other Ericaceae and Epacridaceae. Using phylogenetic analysis of ITS2 sequences in GenBank, we have confirmed that most of these fungi occur in North America, Europe, and Australia. The low recovery rate of culturable ericoid mycorrhizal fungi from Gaultheria shallon may partly be explained by the fact that most mycorrhizal root segments contain an unculturable basidiomycete, revealed by direct amplification, cloning, and sequencing of LSU fungal DNA from root. Molecular characterization and phylogenetic analysis are powerful tools in revealing the geographic distribution and identity of ericoid mycorrhizal fungi.     

Molecular phylogeny of the genus Triticum L

The genus Triticum L. includes the major cereal crop, common or bread wheat (hexaploid Triticum aestivum L.), and other important cultivated species. Here, we conducted a phylogenetic analysis of all known wheat species and the closely related Aegilops species. This analysis was based on chloroplast matK gene comparison along with trnL intron sequences of some species. Polyploid wheat species are successfully divided only into two groups – Emmer (sections Dicoccoides and Triticum) and Timopheevii (section Timopheevii). Results reveal strictly maternal plastid inheritance of synthetic wheat amphiploids included in the study. A concordance of chloroplast origin with the definite nuclear genomes of polyploid species that were inherited at the last hybridization events was found. Our analysis suggests that there were two ancestral representatives of Aegilops speltoides Tausch that participated in the speciation of polyploid wheats with B and G genome in their genome composition. However, G genome species are younger in evolution than ones with B genome. B genome-specific PCR primers were developed for amplification of Acc-1 gene.     

Molecular phylogeny of Pottiaceae (Musci) based on chloroplast rps4 sequence data

Comparative sequencing of the chloroplast rps4 gene was used to reconstruct the phylogenetic relationships within the family Pottiaceae (Musci). The results confirm that Ephemerum spinulosum, Splachnobryum obtusum, Goniomitrium acuminatum and Cinclidotus fontinaloides are clearly positioned within the Pottiaceae and that Hypodontium dregei is not a member. At subfamily level, the data support the subfamily Pottioideae as being a monophyletic clade. The Trichostomoideae are probably paraphyletic. Neither the subfamily Chionolomideae, represented in this study by Pseudosymblepharis schimperiana, nor the subfamily Erythrophyllopsoideae, represented by both known species, Erythrophyllastrum andinum and Erythrophyllopsis fuscula, are supported by the sequence data. The Timmielloideae should be excluded from the Pottiaceae. The Merceyoideae, represented in this study by Scopelophila cataractae, might form a sister clade to all other Pottiaceae, but their position is not fully resolved. At the genus level, Barbula is clearly polyphyletic since Barbula bolleana and Barbula indica appear in a clade clearly separated from Barbula unguiculata. Pottiopsis caespitosa and Leptobarbula berica are placed within the Trichostomoideae. Likewise, the genera Gymnostomum and Anoectangium are excluded from the Pottioideae and placed within the Trichostomoideae. Leptophascum leptophyllum is closely related with Syntrichia; Aloina is not closely related to Tortula or Crossidium. Evidence of a clade within the Pottioideae, formed of Leptodontium and Triquetrella, is provided.     

Procaryote phylogeny IV: Concerning the phylogenetic status of a photosynthetic bacterium

Summary The 16S ribosomal RNA (30S subunit) ofRhodopseudomonas spheroides has been characterized in terms of T1 ribonuclease digestion products. This fingerprint ultimately permits the placement ofR. spheroides into a detailed procaryotic phylogenetic tree. Given the number of major procaryotic lines that have been characterized in these terms to date, one can tentatively place the Athiorhodaceae closer to the Vibrio-Enteric group than to the Bacillaceae or Cyanophyta.