Phylogenetic relationships among eukaryotic kingdoms inferred from ribosomal RNA sequences

Summary Phylogenetic trees among eukaryotic kingdoms were inferred for large- and small-subunit rRNAs by using a maximum-likelihood method developed by Felsenstein. Although Felsenstein's method assumes equal evolutionary rates for transitions and transversions, this is apparently not the case for these data. Therefore, only transversiontype substitutions were taken into account. The molecules used were large-subunit rRNAs fromXenopus laevis (Animalia), rice (Plantae),Saccharomyces cerevisiae (Fungi),Dictyostelium discoideum (Protista), andPhysarum polycephalum (Protista); and small-subunit rRNAs from maize (Plantae),S. cerevisiae, X. laevis, rat (Animalia), andD. discoideum. Only conservative regions of the nucleotide sequences were considered for this study. In the maximum-likelihood trees for both large- and small-subunit rRNAs, Animalia and Fungi were the most closely related eukaryotic kingdoms, and Plantae is the next most closely related kingdom, although other branching orders among Plantae, Animalia, and Fungi were not excluded by this work. These three eukaryotic kingdoms apparently shared a common ancestor after the divergence of the two species of Protista,D. discoideum andP. polycephalum. These two species of Protista do not form a clade, andP. polycephalum diverged first andD. discoideum second from the line leading to the common ancestor of Plantae, Animalia, and Fungi. The sequence data indicate that a drastic change occurred in the nucleotide sequences of rRNAs during the evolutionary separation between prokaryote and eukaryote.     

The Mesorhizobium loti purB gene is involved in infection thread formation and nodule development in Lotus japonicus

The purB and purH mutants of Mesorhizobium loti exhibited purine auxotrophy and nodulation deficiency on Lotus japonicus. In the presence of adenine, only the purH mutant induced nodule formation and the purB mutant produced few infection threads, suggesting that 5-aminoimidazole-4-carboxamide ribonucleotide biosynthesis catalyzed by PurB is required for the establishment of symbiosis.     

Nitric oxide production induced in roots of Lotus japonicus by lipopolysaccharide from Mesorhizobium loti

Lipopolysaccharide (LPS) is a bacterial molecule that induces nitric oxide (NO) production and triggers defense systems in plant-pathogen interactions. NO production is induced in the roots of Lotus japonicus after inoculation of the roots with its microsymbiont Mesorhizobium loti. However, the rhizobial molecule that induces NO production has not yet been identified. We investigated NO production in the roots of L. japonicus by treatment with LPS of M. loti. LPS was prepared by phenol-hot water extraction and separated into several fractions: polysaccharide, lipooligosaccharide, oligosaccharide and lipid A. In the roots of L. japonicus, NO production was observed with an NO-specific fluorescent dye 4, 10 and 24 h after treatment with each fraction of LPS. NO production was detected 4 h after treatment with all fractions. NO production was also detectable 24 h after treatment, except after treatment with the polysaccharide and oligosaccharide fractions. Expression of a class 1 hemoglobin gene and application of an NO scavenger showed that the treatment with LPS and LOS induced a similar response to inoculation with M. loti. These data suggest that LPS of M. loti induces NO production after inoculation with M. loti.     

Expressed sequence tags from roots and nodule primordia of Lotus japonicus infected with Mesorhizobium loti

Messenger RNA from young Lotus japonicus roots carrying root nodule primordia appearing after inoculation with Mesorhizobium loti bacteria were used to construct a cDNA expression library. Single-pass sequencing employing colony-polymerase chain reaction (PCR) and analysis of PCR products established a total of 2,397 new expressed sequence tags (ESTs). We have putatively identified 1,236 known and 484 hypothetical proteins coded by the corresponding mRNAs. The remaining cDNAs are unknown (316) or redundant overlapping cDNAs (361). We hope that this batch of ESTs will assist in the recognition of plant genes involved during development of nitrogen-fixing root nodules.     

The trehalose utilization gene thuA ortholog in Mesorhizobium loti does not influence competitiveness for nodulation on Lotus spp.

Competitiveness for nodulation is a desirable trait in rhizobia strains used as inoculant. In Sinorhizobium meliloti 1021 mutation in either of the trehalose utilization genes thuA or thuB influences its competitiveness for root colonization and nodule occupancy depending on the interacting host. We have therefore investigated whether mutation in the thuA ortholog in Mesorhizobium loti MAFF303099 also leads to a similar competitive phenotype on its hosts. The results show that M. loti thuA mutant Ml7023 was symbiotically effective and was as competitive as the wild type in colonization and nodule occupancy on Lotus corniculatus and Lotus japonicus. The thuA gene in M. loti was not induced during root colonization or in the infection threads unlike in S. meliloti, despite its induction by trehalose and high osmolarity in in vitro assays.     

Phylogenetic relationships among Syndermata inferred from nuclear and mitochondrial gene sequences

Phylogenetic relationships among Syndermata have been extensively debated, mainly because the sister-group of the Acanthocephala has not yet been clearly identified from analyses of morphological and molecular data. Here we conduct phylogenetic analyses on samples from the 4 classes of Acanthocephala (Archiacanthocephala, Eoacanthocephala, Polyacanthocephala, and Palaeacanthocephala) and the 3 Rotifera classes (Bdelloidea, Monogononta, and Seisonidea). We do so using small-subunit (SSU) and large-subunit (LSU) ribosomal DNA and cytochrome c oxidase subunit 1 (cox 1) sequences. These nuclear and mitochondrial DNA sequences were obtained for 27 acanthocephalans, 9 rotifers, and representatives of 6 phyla that were used as outgroups. Maximum parsimony (MP), maximum likelihood (ML), and Bayesian analyses were conducted on the nuclear rDNA(SSU+LSU) and the combined sequence dataset(SSU+LSU+cox 1 genes). Phylogenetic analyses of the combined rDNA and cox 1 data uniformly provided strong support for a clade including rotifers plus acanthocephalans (Syndermata). Strong support was also found for monophyly of Acanthocephala in analyses of the combined dataset or rDNA sequences alone. Within the Acanthocephala the monophyletic grouping of the representatives of each class was strongly supported. Our results depicted Archiacanthocephala as the sister-group to the remaining acanthocephalans. Analyses of the combined dataset recovered a sister-group relationship between Acanthocephala and Bdelloidea by parsimony, likelihood, and Bayesian methods. Support for this clade was generally strong. Alternative topologies that depicted a different rotifer sister-group of Acanthocephala (or monophyly of Rotifera) were significantly worse. In this paraphyletic assemblage of rotifers, the relative positions of Seisonidea and Monogononta to the clade Bdelloidea+Acanthocephala were inconsistent among trees based on different inference methods. These results indicate that Bdelloidea is the free-living sister-group to acanthocephalans, which should prove key for comparative investigations of the morphological, molecular, and ecological changes accompanying the evolution of parasitism.     

Flavonoids in seed and root exudates of Lotus pedunculatus and their biotransformation by Mesorhizobium loti

Analysis of the flavonoid content of seed and root exudates of Lotus pedunculatus was undertaken using multiple coupled analytical techniques: capillary zone electrophoresis coupled to a UV spectral array detector (CZE-UV), high performance thin-layer chromatography with densitometry (HPTLC-UV) and gas chromatography with mass spectrometry (GC-MS). These procedures provided separation, identification and structural confirmation of the previously unidentified flavonoids in this plant's seed and root exudates and were particularly applicable to samples from a small seeded legume. Catechin, naringenin, kaempferol, quercetin aglycone and 3 different glycosides of quercetin were detected in seed exudate. Sterile root exudates contained catechin, naringenin and quercetin in addition to apigenin, kaempferol and other flavones and flavanones for which partial identifications were obtained. When sterile root exudate was incubated with Mesorhizobium loti , changes in its flavonoid content were detected. Analysis of bacterial cells after incubation revealed the presence of quercetin, kaempferol and one other flavone. The monocyclic aromatics protocatechuic acid and phloroglucinol were detected in both the incubated root exudate and its bacterial cells.     

Genetic diversity and relationships among wild and cultivated Stenocereus queretaroensis populations in western Mexico

Stenocereus queretaroensis is an endemic, chiropterophilous, columnar cactus of economic importance in Mexico. To investigate the effect of artificial selection on genetic diversity, inter-simple sequence repeat (ISSR) markers were used to estimate the genetic variation of wild, orchard, and backyard populations of S. queretaroensis from two regions in western Mexico. Six primers were used to generate 62 bands, of which 39 were polymorphic (62.9%). The total genetic diversity was similar in the wild (H_T = 0.296) and orchard (H_T = 0.291) groups, and slightly lower in the backyard group (H_T = 0.281). Wild populations (F_ST = 0.13) were less differentiated than backyard (F_ST = 0.17) and orchard (F_ST = 0.21) populations. The wild and backyard groups were genetically closer (0.015) than the wild and orchard (0.018) groups. A Mantel test revealed a positive correlation between genetic and geographic distances (r = 0.433, p = 0.002). In conclusion, gene flow and the prevailing management system have efficiently maintained genetic diversity and facilitated inter-population differentiation in S. queretaroensis.     

The origin of land plants: Phylogenetic relationships among charophytes,bryophytes, and vascular plants inferred from complete small-subunit ribosomal RNA gene sequences

Complete nuclear-encoded small-subunit 18S rRNA (=SSU rRNA) gene sequences were determined for the prasinophyte green alga Mantoniella squamata; the charophycean green algae Chara foetida, Coleochaete scutata, Klebsormidium flaccidum, and Mougeotia scalaris; the bryophytes Marchantia polymorpha, Fossombronia pusilla, and Funaria hygrometrica; and the lycopod Selaginella galleottii to get a better insight into the sequential evolution from green algae to land plants. The sequences were aligned with several previously published SSU rRNA sequences from chlorophytic and charophytic algae as well as from land plants to infer the evolutionary relationships for major evolutionary lineages within the Chlorobionta by distance matrix, maximum parsimony, and maximum likelihood analyses. Phylogenetic trees created by the different methods consistently placed the Charophyceae on the branch leading to the land plants. The Charophyceae were shown to be polyphyletic with the Charales (charalean algae) diverging earlier than the Coleochaetales, Klebsormidiales, Chlorokybales, and Zygnematales (charophycean algae) which branch from a point closer to the land plants in most analyses. Maximum parsimony and maximum likelihood analyses imply a successive evolution from charophycean algae, particularly Coleochaetales, to bryophytes, lycopods, and seed plants. In contrast, distance matrix methods group the bryophytes together with the charophycean algae, suggesting a separate evolution of these organisms compared with the club moss and the seed plants. Correspondence to.: V.A.R. Huss     

Phylogenetic relationships of Empetraceae inferred from sequences of chloroplast gene matK and nuclear ribosomal DNA ITS region

We used sequences of nrDNA ITS and chloroplast gene matK to evaluate the monophyly of Empetrum and Corema and to examine phylogenetic relationships of the Empetraceae. Sequences of these two DNA markers were obtained for 11 plant samples, representing species of Empetrum from both the Southern and Northern Hemispheres, species and subspecies of Corema, and the monotypic Ceratiola. Sequences of four species of Rhododendron were used for rooting purposes. Our results show that species of Empetrum form a clade sister to the clade containing both Corema and Ceratiola. These two clades are strongly supported in both the matK and ITS trees, suggesting that Ceratiola is more closely related to Corema than to Empetrum, and is not of a hybrid origin between the ancestors of the latter two genera. In the matK tree, Corema conradii is more closely related to Ceratiola than to Corema album and C. album subsp. azoricum, whereas in the ITS tree, Ceratiola is allied with Corema album and C. album subsp. azoricum. This suggests that C. conradii might be a hybrid between ancestral populations of Ceratiola and C. album. The monophyly of Empetrum rejects the hypothesis of its independent origin in the two Hemispheres. Our trees also suggest the fact that the modern amphitropical distribution of Empetrum is the result of long distance dispersal, not of the vicarious events.     

Structure-function relationships in the alpha subunit of Klebsiella pneumoniae nitrogenase MoFe protein from analysis of nifD mutants.

Crude extracts of wild-type, nitrogenase-derepressed Klebsiella pneumoniae fractionated by nondenaturing gel electrophoresis contain, in addition to the major form of the MoFe protein, two minor variants of lower electrophoretic mobility. Of seven Nif- mutants of K. pneumoniae with nonpolar point mutations in nifD (encoding the alpha subunit of Kp1), three exhibit a wild-type-like electrophoretic pattern, whereas in the remaining four, the slowest-migrating form becomes the predominant species. Amino acid substitutions in mutants of the first type are located in the N terminus of NifD and include Gly-85 to Arg (UN1661), Glu-121 to Lys (UN1649), and Gly-161 to Asp (UN1683). Mutations of the second type are Gly-186 to Asp (UN1648), Gly-195 to Glu (UN1680), Ser-443 to Pro (UN1793), and Gly-455 to Asp (UN1650). Six of the mutated residues show interspecies conservation, three are close to conserved cysteines, and two are located next to conserved histidines. Based on evidence pointing to the possibility that the lowest-mobility form lacks the iron-molybdenum cofactor, these results provide insights into the functional significance of specific sites in the alpha subunit of the MoFe protein.     

Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences.

The families within the class Mollicutes are distinguished by their morphologies, nutritional requirements, and abilities to metabolize certain compounds. Biosystematic classification of the plant-pathogenic mycoplasmalike organisms (MLOs) has been difficult because these organisms have not been cultured in vitro, and hence their nutritional requirements have not been determined nor have physiological characterizations been possible. To investigate the evolutionary relationship of the MLOs to other members of the class Mollicutes, a segment of a ribosomal protein operon was cloned and sequenced from an aster yellows-type MLO which is pathogenic for members of the genus Oenothera and from Acholeplasma laidlawii. The deduced amino acid sequence data from the rpl22 and rps3 genes indicate that the MLOs are more closely related to A. laidlawii than to animal mycoplasmas, confirming previous results from 16S rRNA sequence comparisons. This conclusion is also supported by the finding that the UGA codon is not read as a tryptophan codon in the MLO and A. laidlawii, in contrast to its usage in Mycoplasma capricolum.     

Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa,Argentina

Three symbiotic nitrogen-fixing bacteria (BD68^T, BD66 and BD73) isolated from root nodules of Lotus tenuis in lowland soils of the Flooding Pampa (Argentina), previously classified as members of the Mesorhizobium genus, were characterized in this study. Phylogenetic analysis of their 16S rRNA gene sequences showed a close relationship to M. japonicum MAFF 303099^T, M. erdmanii USDA 3471^T, M. carmichaelinearum ICMP 18942^T, M. opportunistum WSM 2975^T and M. jarvisii ATCC 33699^T, with sequence identities of 99.72%–100%. Multilocus sequence analysis of other housekeeping genes revealed that the three isolates belonged to a phylogenetically distinct clade within the genus Mesorhizobium. Strain BD68^T was designated as the group representative and its genome was fully sequenced. The average nucleotide identity and in silico DNA-DNA hybridization comparisons between BD68^T and the most related type strains showed values below the accepted threshold for species discrimination. Phenotypic and chemotaxonomic features were also studied.Based on these results, BD68^T, BD66 and BD73 could be considered to represent a novel species of the genus Mesorhizobium, for which the name Mesorhizobium intechi sp. nov. is hereby proposed. The type strain of this species is BD68^T (=CECT 9304^T = LMG 30179^T).     

Tribal delimitation and phylogenetic relationships of Loteae and Coronilleae (Faboideae: Fabaceae) with special reference to Lotus: evidence from nuclear ribosomal ITS sequences

The temperate herbaceous tribes Loteae and Coronilleae have traditionally been regarded as taxonomically distinct entities. More recent morphological assessments, however, have challenged this view and suggest combining the two tribes under Loteae. Two key features used to distinguish the Coronilleae from Loteae include jointed fruits and branched root nodules. We evaluate the taxonomic utility of these characters using information derived from phylogenetic analyses of the internal transcribed spacers ITS1 + 2, and the intervening 5.8S region of nuclear ribosomal DNA. Results from this study show that neither the Loteae nor Coronilleae form individual monophyletic groups, and that key fruit and root nodule characters used to distinguish the Coronilleae are homoplastic. Given these data, we support the recognition of a single tribe, Loteae. We also find that Lotus, the largest and most morphologically complex genus in either tribe, is not monophyletic. Rather, it consists of two geographically distinct lineages, Old and New World, each of which are more closely related to other Loteae genera: Old World Lotus are more closely related to Old World Anthyllis, while New World Lotus show closer affinities to Old World Coronilla. These data also have important implications for the biogeography of New World Lotus: equally most parsimonious reconstructions suggest a complex scenario of intercontinental dispersals that involve not only Old World Lotus but Coronilla as well.     

Phylogenetic relationships of the Raphidophyceae and Xanthophyceae as inferred from nucleotide sequences of the 18S ribosomal RNA gene

Some earlier studies suggested an evolutionary relationship between the Raphidophyceae (chloromonads) and Xanthophyceae (yellow-green algae), whereas other studies suggested relationships with different algal classes or the öomycete fungi. To evaluate the relationships, we determined the complete nucleotide sequences of the 18S ribosomal RNA gene from the raphidophytes Vacuolaria virescens, Chattonella subsalsa, and Heterosigma carterae, and the xanthophytes Vaucheria bursata, Botrydium stoloniferum, Botrydiopsis intercedens, and Xanthonema debile. The results showed that the Xanthophyceae were most closely related to the Phaeophyceae. A cladistic analysis of combined data sets (nucleotide sequences, ultrastructure, and pigments) suggested the Raphidophyceae are the sister taxon to the Phaeophyceae-Xanthophyceae clade, but the bootstrap value was low (40%). The raphidophyte genera were united with high (100%) bootstrap values, supporting a hypothesis based upon ultrastructural features that marine and freshwater raphidophytes form a monophyletic group. We examined the relationship between Vaucheria, a siphoneous xanthophyte alga, and the öomycetes, and we confirmed that Vaucheria is a member of the class Xanthophyceae. Partial nucleotide sequences of the 18S rRNA gene from eight xanthophytes (including Bumillariopsis filiformis, Heterococcus caespitiosus, and Mischococcus sphaerocephalus) produce a phylogeny that is not congruent with the current morphology-based classification scheme.     

Bradyrhizobia from wild Phaseolus, Desmodium, and Macroptilium species in northern Mexico

rRNA genetic markers were analyzed in 97 isolates of nodule bacteria from six legume species in Chihuahua, Mexico. The most common genotypes were widely shared across host species and had 16S rRNA sequences identical to those of strains from an eastern North American legume (Amphicarpaea) that are closely related to Bradyrhizobium elkanii.