rRNA Phylogeny Research Articles

Andalucia (n. gen.)—the Deepest Branch Within Jakobids (Jakobida; Excavata), Based on Morphological and Molecular Study of a New Flagellate from Soil

A new heterotrophic flagellate (Andalucia godoyi n. gen. n. sp.) is described from soil. Earlier preliminary 18S rRNA analyses had indicated a relationship with the phylogenetically difficult-to-place jakobid Jakoba incarcerata. Andalucia godoyi is a small (3-5 mum) biflagellated cell with a ventral feeding groove. It has tubular mitochondrial cristae. There are two major microtubular roots (R1, R2) and a singlet root associated with basal body 1 (posterior). The microtubular root R1 is associated with non-microtubular fibres "I,""B," and "A," and divides in two parts, while R2 is associated with a "C" fibre. These structures support the anterior portion of the groove. Several features of A. godoyi are characteristic of jakobids: (i) there is a single dorsal vane on flagellum 2; (ii) the C fibre has the jakobid multilaminate substructure; (iii) the dorsal fan of microtubules originates in very close association with basal body 2; and (iv) there is no "R4" microtubular root associated with basal body 2. Morphological analyses incorporating the A. godoyi data strongly support the monophyly of all jakobids. Our 18S rRNA phylogenies place A. godoyi and J. incarcerata as a strong clade, which falls separately from other jakobids. Statistical tests do not reject jakobid monophyly, but a specific relationship between Jakoba libera and J. incarcerata and/or A. godoyi is rejected. Therefore, we have established a new genus Andalucia n. gen. with the type species Andalucia godoyi n. sp., and transfer Jakoba incarcerata to Andalucia as Andalucia incarcerata n. comb.

Molecular evolution of the nif gene cluster carrying nifI1 and nifI2 genes in the Gram-positive phototrophic bacterium Heliobacterium chlorum

A major nif cluster was detected in the strictly anaerobic, Gram-positive phototrophic bacterium Heliobacterium chlorum. The cluster consisted of 11 genes arranged within a 10 kb region in the order nifI1, nifI2, nifH, nifD, nifK, nifE, nifN, nifX, fdx, nifB and nifV. The phylogenetic position of Hbt. chlorum was the same in the NifH, NifD, NifK, NifE and NifN trees; Hbt. chlorum formed a cluster with Desulfitobacterium hafniense, the closest neighbour of heliobacteria based on the 16S rRNA phylogeny, and two species of the genus Geobacter belonging to the Deltaproteobacteria. Two nifI genes, known to occur in the nif clusters of methanogenic archaea between nifH and nifD, were found upstream of the nifH gene of Hbt. chlorum. The organization of the nif operon and the phylogeny of individual and concatenated gene products showed that the Hbt. chlorum nif operon carrying nifI genes upstream of the nifH gene was an intermediate between the nif operon with nifI downstream of nifH (group II and III of the nitrogenase classification) and the nif operon lacking nifI (group I). Thus, the phylogenetic position of Hbt. chlorum nitrogenase may reflect an evolutionary stage of a divergence of the two nitrogenase groups, with group I consisting of the aerobic diazotrophs and group II consisting of strictly anaerobic prokaryotes.

First genome data from uncultured upland soil cluster alpha methanotrophs provide further evidence for a close phylogenetic relationship to Methylocapsa acidiphila B2 and for high-affinity methanotrophy involving particulate methane monooxygenase.

Members of upland soil cluster alpha (USC alpha) are assumed to be methanotrophic bacteria (MB) adapted to the trace level of atmospheric methane. So far, these MB have eluded all cultivation attempts. While the 16S rRNA phylogeny of USC alpha members is still not known, phylogenies constructed for the active-site polypeptide (encoded by pmoA) of particulate methane monooxygenase (pMMO) placed USC alpha next to the alphaproteobacterial Methylocapsa acidiphila B2. To assess whether the pmoA tree reflects the evolutionary identity of USC alpha, a 42-kb genomic contig of a USC alpha representative was obtained from acidic forest soil by screening a metagenomic fosmid library of 250,000 clones using pmoA-targeted PCR. For comparison, a 101-kb genomic contig from M. acidiphila was analyzed, including the pmo operon. The following three lines of evidence confirmed a close phylogenetic relationship between USC alpha and M. acidiphila: (i) tetranucleotide frequency patterns of 5-kb genomic subfragments, (ii) annotation and comparative analysis of the genomic fragments against all completely sequenced genomes available in public domain databases, and (iii) three single gene phylogenies constructed using the deduced amino acid sequences of a putative prephenate dehydratase, a staphylococcal-like nuclease, and a putative zinc metalloprotease. A comparative analysis of the pmo operons of USC alpha and M. acidiphila corroborated previous reports that both the pmo operon structure and the predicted secondary structure of deduced pMMO are highly conserved among all MB.

Unravelling the evolution of the head lice and body lice of humans

Recent studies of mitochondrial genes of the head and body lice of humans indicate that present-day lice comprise two lineages that diverged before the evolution of modern humans. To test if this was a locus-specific phenomenon, we studied two nuclear genes, elongation factor-1alpha (EF-1alpha) and small subunit ribosomal RNA (ssu rRNA). Our ssu rRNA phylogeny was concordant with the phylogenies from mitochondrial genes, but the EF-1alpha phylogeny was not concordant either with the mitochondrial phylogenies or with the ssu rRNA phylogeny. So both nuclear (ssu rRNA) and mitochondrial data indicate that there are two lineages of lice: one lineage with head lice only (H-only lineage) the other lineage with head and body lice (H+B lineage). Thus, body lice apparently evolved from just one of the two main lineages of lice. However, the date of divergence and geographical origins of the two lineages are controversial. Kittler et al. (Curr Biol 13:1414-1417, 2003; Curr Biol 14:2309, 2004) proposed that these two lineages diverged 0.77 mya, whereas Reed et al. (PLoS Biol 2:e340, 2004) proposed that they diverged 1.18 mya and suggested that one of the lineages, the H-only lineage, evolved in the New World on Homo erectus. We discuss this hypothesis in light of our results from ssu rRNA.

Phylogeny of the Centrohelida Inferred from SSU rRNA, Tubulins, and Actin Genes

Amoeboid protists are major targets of recent molecular phylogeny in connection with reconstruction of global phylogeny of eukaryotes as well as the search for the root of eukaryotes. The Centrohelida are one of the major groups of Heliozoa, classified in the Actinopodida, whose evolutionary position is not well understood. To clarify the relationships between the Centrohelida and other eukaryotes, we sequenced SSU rRNA, alpha-tubulin, and beta-tubulin genes from a centroheliozoan protist, Raphidiophrys contractilis. The SSU rRNA phylogeny showed that the Centrohelida are not closely related to other heliozoan groups, Actinophryida, Desmothoracida, or Taxopodida. Maximum likelihood analyses of the combined phylogeny using a concatenate model for an alpha- + beta-tubulin + actin data set, and a separate model for SSU rRNA, alpha- and beta-tubulin, and actin gene data sets revealed the best tree, in which the Centrohelida have a closer relationship to Rhodophyta than to other major eukaryotic groups. However, both weighted Shimodaira-Hasegawa and approximately unbiased tests for the concatenate protein phylogeny did not reject alternative trees in which Centrohelida were constrained to be sisters to the Amoebozoa. Moreover, alternative trees in which Centrohelida were placed at the node branching before and after Amoebozoa or Viridiplantae were not rejected by the WSH tests. These results narrowed the possibilities for the position of Centrohelida to a sister to the Rhodophyta, to the Amoebozoa, or to an independent branch between the branchings of Amoebozoa and Rhodophyta (or possibly Plantae) at the basal position within the bikonts clade in the eukaryotic tree.

Phylogeny of lobose amoebae based on actin and small‐subunit ribosomal RNA genes

Lobose amoebae are abundant free‐living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyse actin and small‐subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, as well as the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well‐defined clades of amoebae, the “Gymnamoebia sensu stricto” and the Archamoebae (pelobionts+entamoebids), and one weakly supported and ill‐resolved group comprising some naked, lobose amoebae and the Mycetozoa.

Critical analysis of the topology and rooting of the parabasalian 16S rRNA tree

The morphological classification of the protozoan phylum Parabasala is not in absolute agreement with the 16S rRNA phylogeny. However, there are strong indications that tree‐construction artifacts play a considerable role in the shaping of the 16S rRNA tree. We have performed rigorous analyses designed to minimize such artifacts using the slow–fast and taxa‐exclusion methods. The analyses, which included new sequences from the genera Monocercomonas and Hexamastix, in most respects confirmed the previously suggested tree topology and polyphyly of order Hypermastigida and family Monocercomonadidae but detected one artificial cluster of long branches (Trichonymphidae, Pseudotrichonymphidae, Hexamastix and Tricercomitus). The analyses also indicated that the rooting of the phylum Parabasala on the trichonymphid branch, as widely accepted, is doubtful and that reliable rooting on the basis of current data is likely impossible.

A molecular phylogenetic approach to Longidoridae (Nematoda: Dorylaimida)

Abstract The Longidoridae are a group of ectoparasitic nematodes including two subfamilies and six genera with hundreds of species. Sequences of the D2 and D3 expansion region of the large subunit (LSU) rRNA nuclear gene were amplified and used to reconstruct the phylogeny of longidorids. Phylogenetic analyses with maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) were performed with one outgroup taxon and 62 longidorid sequences. Confidence of inferred clades was assessed by non-parametric bootstrapping for MP and Bayesian posterior probability for ML. All analyses placed Paralongidorus species as an inner group within the otherwise monophyletic genus Longidorus. The genus Xiphinema, except for X. americanum-group species, was placed as the sister group of Longidorus with strong support from the ML and BI analyses. The X. americanum-group was strongly supported as an exclusive clade to other genus Xiphinema species. The position of the Xiphidorus clade was not well resolved and the phylogenetic analyses did not support it as a sister group to Longidorus as previously inferred from morphology. Secondary structure models were constructed for the D2/D3 region of LSU rRNA for all studied species. It was found that sequence-based and structural morphometric rRNA phylogenies were incongruent.

NifH and NifD sequences of heliobacteria: a new lineage in the nitrogenase phylogeny

We determined almost complete nifH and nifD genes from representatives of all recognized genera of heliobacteria, the strictly anaerobic phototrophs belonging to the low GC gram-positive bacteria. The heliobacterial sequences formed a highly supported monophyletic group that is clearly distinct from any known diazotrophs, in both NifH and NifD trees. According to the classification of nitrogenase genes in four major clusters, the clade of heliobacterial sequences belonged to cluster I and did not cluster with any of the Clostridium (cluster III) or Paenibacillus (cluster I) species, the close neighbors of heliobacteria based on the 16S rRNA phylogeny. One partial anfH or alternative nitrogenase sequence was detected from Heliobacterium gestii. Although Heliophilum fasciatum is known to fix nitrogen based on the acetylene reduction test, nifH and/or nifD genes were not detected by either the PCR amplification or Southern hybridization methods.

The basidiomycete genus Polyporus – an emendation based on phylogeny and putative secondary structure of ribosomal RNA molecules

AbstractThe fungal genus Polyporus is an assemblage of white‐rotting lignicolous basidiomycetes. It has undergone considerable expansion and contraction over a period of two and three quarter centuries. Current generic circumscription of Polyporus has kept the genus non‐monophyletic. Species of Polyporus infrageneric group Polyporellus are closely related to some species of Lentinus. We introduce data for ITS2 spacer rRNA secondary structure evolution by quasi‐independent comparison with large subunit rRNA phylogeny, and suggest a fraction of primary nuclear rDNA ITS sequence data as novel taxonomic character. A major taxonomic shift is suggested, supported by molecular and morphological characters, and allowing inclusion of species with gilled hymenophores in Polyporus. Two new names are proposed: Polyporus phyllostipes D.Krüger, nom. nov. and Polyporus gerdai D.Krüger, nom. nov. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Prevalence of Burkholderia sp. nodule symbionts on four mimosoid legumes from Barro Colorado Island, Panama

Sequences of 16S rRNA and partial 23S rRNA genes and PCR assays with genotype-specific primers indicated that bacteria in the genus Burkholderia were the predominant root nodule symbionts for four mimosoid legumes ( Mimosa pigra, M. casta, M. pudica, and Abarema macradenia) on Barro Colorado Island, Panama. Among 51 isolates from these and a fifth mimosoid host ( Pithecellobium hymenaeafolium), 44 were Burkholderia strains while the rest were placed in Rhizobium, Mesorhizobium, or Bradyrhizobium. The Burkholderia strains displayed four distinct rRNA sequence types, ranging from 89% to 97% similarity for 23S rRNA and 96.5–98.4% for 16S rRNA. The most common genotype comprised 53% of all isolates sampled and was associated with three legume host species. All Burkholderia genotypes formed nodules on Macroptilium atropurpureum or Mimosa pigra, and sequencing of rRNA genes in strains re-isolated from nodules verified identity with inoculant strains. Sequence analysis of the nitrogenase α-subunit gene ( nifD) in two of the Burkholderia genotypes indicated that they were most similar to a partial sequence from the nodule-forming strain Burkholderia tuberum STM 678 from South Africa. In addition, a PCR screen with primers specific to Burkholderia nodB genes yielded the expected amplification product in most strains. Comparison of 16S rRNA and partial 23S rRNA phylogenies indicated that tree topologies were significantly incongruent. This implies that relationships across the rRNA region may have been altered by lateral gene transfer events in this Burkholderia population.

Nonhomogeneous Model of Sequence Evolution Indicates Independent Origins of Primary Endosymbionts Within the Enterobacteriales (γ-Proteobacteria)

Standard methods of phylogenetic reconstruction are based on models that assume homogeneity of nucleotide composition among taxa. However, this assumption is often violated in biological data sets. In this study, we examine possible effects of nucleotide heterogeneity among lineages on the phylogenetic reconstruction of a bacterial group that spans a wide range of genomic nucleotide contents: obligately endosymbiotic bacteria and free-living or commensal species in the gamma-Proteobacteria. We focus on AT-rich primary endosymbionts to better understand the origins of obligately intracellular lifestyles. Previous phylogenetic analyses of this bacterial group point to the importance of accounting for base compositional variation in estimating relationships, particularly between endosymbiotic and free-living taxa. Here, we develop an approach to compare susceptibility of various phylogenetic reconstruction methods to the effects of nucleotide heterogeneity. First, we identify candidate trees of gamma-Proteobacteria groEL and 16S rRNA using approaches that assume homogeneous and stationary base composition, including Bayesian, maximum likelihood, parsimony, and distance methods. We then create permutations of the resulting candidate trees by varying the placement of the AT-rich endosymbiont Buchnera. These permutations are evaluated under the nonhomogeneous and nonstationary maximum likelihood model of Galtier and Gouy, which allows equilibrium base content to vary among examined lineages. Our results show that commonly used phylogenetic methods produce incongruent trees of the Enterobacteriales, and that the placement of Buchnera is especially unstable. However, under a nonhomogeneous model, various groEL and 16S rRNA phylogenies that separate Buchnera from other AT-rich endosymbionts (Blochmannia and Wigglesworthia) have consistently and significantly higher likelihood scores. Blochmannia and Wigglesworthia appear to have evolved from secondary endosymbionts, and represent an origin of primary endosymbiosis that is independent from Buchnera. This application of a nonhomogeneous model offers a computationally feasible way to test specific phylogenetic hypotheses for taxa with heterogeneous and nonstationary base composition.

Phylogenetic analyses of Nitella subgenus Tieffallenia (Charales, Charophyceae) using nuclear ribosomal DNA internal transcribed spacer sequences

Nucleotide sequences from the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal RNA (rRNA) gene were obtained for 13 Nitella species belonging to the subgenus Tieffallenia in order to test the chloroplast gene phylogeny previously used for taxonomic studies. The phylogenetic relationships of the 13 Nitella species based on nuclear DNA markers were congruent with those in the chloroplast gene phylogeny using the combined sequence data set for the genes encoding the beta subunit of ATP synthase (atpB) and the large subunit of Rubisco (rbcL). Therefore, the chloroplast gene phylogeny at the species level within the subgenus Tieffallenia seems to be unaffected by the maternal inheritance of chloroplast genes and can be used to obtain a natural taxonomy at the species level. This may result from the monoecious sexual reproduction that occurs in all the Nitella species examined. Phylogenetic analyses based on the concatenated chloroplast atpB and rbcL and nuclear 5.8S rRNA and ITS sequences generally gave higher resolution at or above species level within the subgenus Tieffallenia than the ITS–5.8S rRNA phylogeny or the rbcL–alpB analyses.

Evolution of plant parasitism among nematodes.

Despite extraordinary diversity of free-living species, a comparatively small fraction of nematodes are parasites of plants. These parasites represent at least three disparate clades in the nematode tree of life, as inferred from rRNA sequences. Plant parasites share functional similarities regarding feeding, but many similarities in feeding structures result from convergent evolution and have fundamentally different developmental origins. Although Tylenchida rRNA phylogenies are not fully resolved, they strongly support convergent evolution of sedentary endoparasitism and plant nurse cells in cyst and root-knot nematodes. This result has critical implications for using model systems and genomics to identify and characterize parasitism genes for representatives of this clade. Phylogenetic studies reveal that plant parasites have rich and complex evolutionary histories that involve multiple transitions to plant parasitism and the possible use of genes obtained by horizontal transfer from prokaryotes. Developing a fuller understanding of plant parasitism will require integrating more comprehensive and resolved phylogenies with appropriate choices of model organisms and comparative evolutionary methods.

A mitochondrial 12S and 16S rRNA phylogeny of critical genera of Phoridae (Diptera) and related families of Aschiza

Phylogenetic analysis of mitochondrial 12S and 16S rRNA gene sequences supports the monophyly of the Phoridae. Within this family the Phorinae clade includes two aberrant termitophilous subfamilies, the Thaumatoxeninae and the Termitoxeniinae, which cluster with Dohrniphora and Diplonevra. These two genera include termitophiles and parasitoids of termites, so we hypothesize that these termitophilous phorids are a monophyletic group. While the data neither refute nor support the assumed monophyly of the Metopininae, the genera of this subfamily were not monophyletic in our analysis, but fell into two subclades that correspond with the tribes Metopinini and Gymnophorini.

Mitochondrial 16S rRNA sequence variations and phylogeny of the Chinese sisorid catfishes

Partial sequences of mitochondrial 16S rRNA gene were obtained by PCR amplification for comparisons among nine species of glyptosternoid fishes and six species of non-glyptosternoids representing 10 sisorid genera. There are compositional biases in the A-rich impaired regions and G-rich paired regions. A-G transitions are primarily responsible for the Ts/Tv bias in impaired regions. The overall substitution rate in impaired regions is almost two times higher than that in the paired regions. Saturation plots at comparable levels of sequence divergence demonstrate no saturation effects. Phylogenetic analyses using both maximum likelihood and Bayesian methods support the monophyly of Sisoridae. Chinese sisorid catfishes are composed of two major lineages, one represented by (Gagata (Bagarius, Glyptothorax)) and the other by "glyptosternoids + Pseudecheneis". The glyptosternoids may not be a monophyletic group. A previous hypothesis referring to Pseudecheneis as the sister group of monophyletic glyptosternoids, based on morphological evidence, is not supported by the molecular data. Pseudecheneis is shown to be a sister taxon of Glaridoglanis. Pareuchiloglanis might be paraphyletic with Pseudexostoma and Euchiloglanis. Our results also support the hypothesis that Pareuchiloglanis anteanalis might be considered as the synonyms of Pareuchiloglanis sinensis, and genus Euchiloglanis might have only one valid species, Euchiloglanis davidi.

Identification and characterization of traE genes of Spiroplasma kunkelii

Four traE homologs, designated traE1, traE2, traE3 and traE4, were identified and amplified from the genome of the leafhopper-transmitted corn stunt pathogen Spiroplasma kunkelii and were predicted to encode membrane-bound adenine tri-phosphatases (ATPases). Deduced proteins of all traE genes have 62.3% to 89.9% similarity to the conserved VirB4 domain that is frequently a component of type IV secretory pathways involved in intracellular trafficking and secretion of DNA and proteins. In phylogenetic analysis, TraE homologs of S. kunkelii, Mycoplasma pulmonis and Mycoplasma fermentans cluster together and are more similar to TraE proteins of Gram-positive bacteria than to those of Gram-negative bacteria, thereby resembling the 16S rRNA phylogeny. Gene traE2 was most conserved whereas the presence of the three other traE genes varied among S. kunkelii strains, M2, CS-2B, FL-80 and PU8-17. Further, traE1 and traE2 appeared to be located on the chromosome, and traE3 and traE4 genes on plasmids of S. kunkelii strain M2. Transcripts of the spiralin gene and traE2 genes were detected on Northern blots containing total ribonucleic acids (RNA) of S. kunkelii cultures and S. kunkelii-infected plants and insects, in which traE2 appeared to be of a larger transcription unit. Full-length expression products of the other traE genes were not detected. S. kunkelii traE genes could be involved in S. kunkelii cell morphogenesis, adhesion and DNA recombination.

Critical analysis of the topology and rooting of the parabasalian 16S rRNA tree

The morphological classification of the protozoan phylum Parabasala is not in absolute agreement with the 16S rRNA phylogeny. However, there are strong indications that tree-construction artifacts play a considerable role in the shaping of the 16S rRNA tree. We have performed rigorous analyses designed to minimize such artifacts using the slow–fast and taxa-exclusion methods. The analyses, which included new sequences from the genera Monocercomonas and Hexamastix, in most respects confirmed the previously suggested tree topology and polyphyly of Hypermastigida and Monocercomonadidae but detected one artificial cluster of long branches (Trichonymphidae, Pseudotrichonymphidae, Hexamastix, and Tricercomitus). They also indicated that the rooting of the phylum on the trichonymphid branch is probably wrong and that reliable rooting on the basis of current data is likely impossible. We discuss the tree topology in the view of anagenesis of cytoskeletal and motility organelles and suggest that a robust taxonomic revision requires extensive analysis of other gene sequences.

Patterns of bacterial gene movement.

Lateral gene transfer has emerged as an important force in bacterial evolution. A substantial number of genes can be inserted into or deleted from genomes through the process of lateral transfer. In this study, we looked for atypical occurrence of genes among related organisms to detect laterally transferred genes. We have analyzed 50 bacterial complete genomes from nine groups. For each group we use a 16s rRNA phylogeny and a comparison of protein similarity to map gene insertions/deletions onto their species phylogeny. The results reveal that there is poor correlation of genes inserted, deleted, and duplicated with evolutionary branch length. In addition, the numbers of genes inserted, deleted, or duplicated within the same branch are not always correlated with each other. Nor is there any similarity within groups. For example, in the Rhizobiales group, the ratio of insertions to deletions in the evolutionary branch leading to Agrobacterium tumefaciens str. C58 (Cereon) is 0.52, but it is 39.52 for Mesorhizobium loti. Most strikingly, the number of insertions of foreign genes is much larger in the external branches of the trees. These insertions also greatly outnumber the occurrence of deletions, and yet the genome sizes of these bacteria remain roughly constant. This indicates that many of the insertions are specific to each organism and are lost before related species can evolve. Simulations of the process of insertion and deletion, tailored to each phylogeny, support this conclusion.

Mitochondrial 16S rRNA se-quence variations and phy-logeny of the Chinese sisorid catfishes

Mitochondrial 16S rRNA se-quence variations and phy-logeny of the Chinese sisorid catfishes