Ribosomal RNA homologies and the evolution of the filamentous blue-green bacteria

Summary Ribosomal RNA (rRNA) sequence homology (as determined by comparisons of T1 oligonucleotide catalogs of³²P-labeled 16S rRNAs) has been used to assess phylogenetic relationships within the filamentous and unicellular blue-green bacteria, and to identify regions of evolutionary conservatism within blue-green bacterial 16S rRNAs.Nostoc andFischerella, representatives of two morphologically distinct and highly differentiated orders, are shown to be as closely related (on the basis of RNA sequence homology) as typical members of the non-blue-green bacterial genusBacillus. They are further shown to be (on the same basis) indistinguishable from typical unicellular members of a subgroup of the unicellular blue-green bacterial order Chroococcales. These results have general implications for studies of the origin of differentiated prokaryotes and of evolutionary change in prokaryotic macromolecules. In particular, they provide indirect evidence that the divergences of contemporary major prokaryotic groups are truly ancient ones.     

Evolution of gene orders in genomes of cyanobacteria

Genomes of 23 strains of cyanobacteria were comparatively analyzed using quantitative methods of estimation of gene order similarity. It has been found that reconstructions of phylogenesis of cyanobacteria based on the comparison of the orders of genes in chromosomes and nucleotide sequences appear to be similar. This confirms the applicability of quantitative measures of similarity of gene orders for phylogenetic reconstructions. In the evolution of marine unicellular planktonic cyanobacteria, genome rearrangements are fixed with a low rate (about 3% of gene order changes per 1% of 16S rRNA changes), whereas in other groups of cyanobacteria the gene order can change several times more rapidly. The gene orders in genomes of cyanobacteria and chloroplasts preserve a considerable degree of similarity. The closest relatives of chloroplasts among the analyzed cyanobacteria are likely to be strains from hot springs belonging to the genus Synechococcus. Comparative analysis of gene orders and nucleotide sequences strongly suggests that Synechococcus strains from different environments (sea, fresh waters, hot springs) are not related and belong to evolutionally distant lines.     

Phylogenetic Relationships of Xylella fastidiosa Strains Based on 16S–23S rDNA Sequences

In spite of the lack of resolution of Xylella fastidiosa phylogenetic relationships, parsimony analysis of the 16S–23S rDNA sequence from a wide range of hosts has been evaluated in this research. In order to establish an easier method for sequencing the spacer region completely, a new primer pair was designed. The sequences obtained revealed a higher level of variation than that found in 16S gene sequences, with similarity values ranging from 0.80 to 1.00. The cladogram constructed allowed the clustering of two major clades. From these results it has been possible to recognize the monophyletic grouping of some strains belonging to the same host, possibly representing only one infection process. However, for other hosts there is paraphyletic and polyphyletic grouping. This methodology followed from promising results regarding strain–host clustering. With the parsimony approach, hypothetical genealogical relationship among Xylella strains may be inferred.     

Phylogenetic significance of the pseudoparaphyses in Loculoascomycete taxonomy

The ontogeny of the ascostroma, in particular the centrum structures, has always been regarded as an important criterion in the subdivision of the Loculoascomycetideae (ascomycetous fungi). However, the use of pseudoparaphysis type, cellular or trabeculate, to classify taxa at the ordinal level has been contentious due to the lack of information about their evolution. To determine the phylogenetic significance of the pseudoparaphysis and its variants, DNA sequences of the 18S nuclear rRNA genes from representatives of the orders Pleosporales and Melanommatales were obtained and analyzed. Species with pseudoparaphyses formed a monophyletic group with high statistical confidence. The monophyly of a distinct lineage of species with cellular pseudoparaphyses (the order Pleosporales) is rejected. Likewise, monophyly of a distinct lineage of species with trabeculate pseudoparaphyses (the order Melanommatales) is rejected also. The Pleosporales and Melanommatales are, therefore, not natural orders. The Lophiostomataceae, Phaeosphaeriaceae, and Melanommataceae are most probably polyphyletic, as is the genus Massarina.     

Molecular characterization of marine Cyanobacteria from the Indian subcontinent deduced from sequence analysis of the phycocyanin operon (cpcb-IGS-cpcA) and 16S-23S ITS region

Molecular characterization of ten marine cyanobacterial isolates belonging to the order Oscillatoriales was carried out using the phycocyanin locus (cpcBA-IGS) and the 16S-23S internally transcribed spacer region. DNA sequences from the phycocyanin operon discriminated ten genotypes, which corresponded to seven morphotypes identified by traditional microscopic analysis. The cpcB coding region revealed 17 % nucleotide variation, while cpcA exhibited 29 % variation across the studied species. Phylogenetic analyses support the conclusion that the Phormidium and Leptolyngbya genera are not monophyletic. The nucleotide variations were heterogeneously distributed with no or minimal informative nucleotides. Our results suggest that the discriminatory power of the phycocyanin region varies across the cyanobacterial species and strains. The DNA sequence analysis of the 16S-23S internally transcribed spacer region also supports the polyphyletic nature of the studied oscillatorian cyanobacteria. This study demonstrated that morphologically very similar strains might differ genotypically. Thus, molecular approaches comprising different gene regions in combination with morphological criteria may provide better taxonomical resolution of the order Oscillatoriales.     

The phylogeny of unicellular, extremely halotolerant cyanobacteria

We examined the morphology, physiology, and 16S rRNA gene sequences of three culture collection strains and of ten novel isolates of unicellular cyanobacteria from hypersaline environments. The strains were morphologically diverse, with average cell widths ranging from 2.8 to 10.3 μm. There were single-celled, colonial, and baeocyte-forming strains. However, morphological traits were markedly variable with culture conditions. In contrast, all strains displayed extreme halotolerance (growing close to optimally at above 12% salinity); all were obligately marine, euryhaline, and moderately thermophilic; and all shared a suite of chemotaxonomic markers including phycobilins, carotenoids, and mycosporine-like amino acids. 16S rRNA gene sequence analysis indicated that the strains were related to each other. Sequence similarity analysis placed the strains in a monophyletic cluster (which we named the Halothece cluster) apart from all cultured or uncultured, not extremely halotolerant cyanobacteria whose 16S rRNA gene sequences are available in public nucleotide sequence databases. This represents the first case in which a phylogenetically coherent group of cyanobacteria can be defined on the basis of physiology. The Halothece cluster contained two subclusters that may be divergent at the generic level, one encompassing 12 strains (spanning 5% 16S rRNA gene sequence divergence and named the Euhalothece subcluster), and a single deep-branching isolate. Phenotypic characterization of the isolates, including morphological, physiological, and chemotaxonomic traits, did not distinguish these subclusters and only weakly suggested the existence of two separate clades, one encompassing strains of small cell size (cell width < 5 m) and another one encompassing strains of larger cell size. Received: 1 September 1997 / Accepted: 13 February 1998     

Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis

We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.     

Salt tolerance and polyphyly in the cyanobacterium Chroococcidiopsis (Pleurocapsales)

Chroococcidiopsis Geitler (Geitler 1933) is a genus of cyanobacteria containing desiccation and radiation resistant strains. Members of the genus live in habitats ranging from hot and cold deserts to fresh and saltwater environments. Morphology and cell division pattern have historically been used to define the genus. To better understand the evolution and ability of the Chroococcidiopsis genus to survive in diverse environments we investigated how salt tolerance varies among 15 strains previously isolated from different locations, and if salt tolerant strains are monophyletic to those isolated from freshwater and land environments. Four markers were sequenced from these 15 strains, the 16S rRNA, rbcL, desC1, and gltX genes. Phylogenetic trees were generated which identified a distinct clade of salt‐tolerant strains. This study demonstrates that the genus is polyphyletic based on saltwater and freshwater phenotypes. To understand the resistance to salt in more details, the strains were grown on a range of sea salt concentrations which demonstrated that the freshwater strains were salt‐intolerant whilst the saltwater strains required salt for growth. This study shows an increased resolution of the phylogeny of Chroococcidiopsis and provides further evidence that the genus is polyphyletic and should be reclassified to improve clarity in the literature.     

Phylogenetic study and identification of Vibrio splendidus-related strains based on gyrB gene sequences

Different strains related to Vibrio splendidus have been associated with infection of aquatic animals. An epidemiological study of V. splendidus strains associated with Crassostrea gigas mortalities demonstrated genetic diversity within this group and suggested its polyphyletic nature. Recently 4 species, V. lentus, V. chagasii, V. pomeroyi and V. kanaloae, phenotypically related to V. splendidus, have been described, although biochemical methods do not clearly discriminate species within this group. Here, we propose a polyphasic approach to investigate their taxonomic relationships. Phylogenetic analysis of V. splendidus-related strains was carried out using the nucleotide sequences of 16S ribosomal DNA (16S rDNA) and gyrase B subunit (gyrB) genes. Species delineation based on 16S rDNA-sequencing is limited because of divergence between cistrons, roughly equivalent to divergence between strains. Despite a high level of sequence similarity, strains were separated into 2 clades. In the phylogenetic tree constructed on the basis of gyrB gene sequences, strains were separated into 5 independent clusters containing V. splendidus, V. lentus, V. chagasii-type strains and a putative new genomic species. This phylogenetic grouping was almost congruent with that based on DNA-DNA hybridisation analysis. V. pomeroyi, V. kanaloae and V. tasmaniensis-type strains clustered together in a fifth clade. The gyrB gene-sequencing approach is discussed as an alternative for investigating the taxonomy of Vibrio species.     

Polyphyly in 16S rRNA-based LVTree Versus Monophyly in Whole-genome-based CVTree

We report an important but long-overlooked manifestation of low-resolution power of 16S rRNA sequence analysis at the species level, namely, in 16S rRNA-based phylogenetic trees polyphyletic placements of closely-related species are abundant compared to those in genome-based phylogeny. This phenomenon makes the demarcation of genera within many families ambiguous in the 16S rRNA-based taxonomy. In this study, we reconstructed phylogenetic relationship for more than ten thousand prokaryote genomes using the CVTree method, which is based on whole-genome information. And many such genera, which are polyphyletic in 16S rRNA-based trees, are well resolved as monophyletic clusters by CVTree. We believe that with genome sequencing of prokaryotes becoming a commonplace, genome-based phylogeny is doomed to play a definitive role in the construction of a natural and objective taxonomy.     

Relationships between the Arctic and the Antarctic cyanobacteria; three Phormidium-like strains evaluated by a polyphasic approach

Selected strains of filamentous Phormidium-like cyanobacteria isolated from two Arctic regions (Ellesmere Island, High Canadian Arctic and Svalbard) and from Antarctica (Antarctic peninsula, South Shetland Islands and South Orkney Islands) were studied. The polyphasic approach used included phenotypic observations of morphological features and genotypic analyses (restriction fragment length polymorphism of 16S rRNA gene, internal transcribed space, 16S rRNA gene sequence analysis). Although genotypes generally correspond to observed morphotypes, the genetic analyses revealed a high degree of biodiversity that could not be unveiled using solely morphological evaluations. According to the phylogenetic analysis, the three clones were divided into two major clades, indicating that the phylogenetic distance between Arct-Ph5/Ant-Ph68 and Ant-Ph58 was so large they belonged to different genera. The polyphyletic position of strains of the genus Phormidium was confirmed by this study, attesting the need to entirely revise classification in this taxon in the future.     

Molecular Evidence for Polyphyletic Origin of the Primary Symbionts of Sucking Lice (Phthiraptera,Anoplura)

Based on 16S rDNA analyses, the primary symbionts of sucking lice were found to form a polyphyletic assemblage of several distant lineages that have arisen several times within Enterobacteriaceae and at least once within Legionellaceae. Another independent lineage of endosymbiotic enterobacteria inhabits a sister group of the sucking lice, Rhynchophthirina. The inspection of 16S rDNA supports the symbiotic nature of the investigated bacteria; they display a typical trait of degenerative processes, an increased AT content (Adenine–Thymine content) in comparison with free-living bacteria. The calculation of divergence time between the closest anopluran and rhynchophthirine symbionts further support their independent origin. The results shown here, together with evidence from other groups, indicate that the significance of primary symbionts for blood-feeding insects should be reconsidered.     

Phylogenetic relationships of the downy mildews (Peronosporales) and related groups based on nuclear large subunit ribosomal DNA sequences

In order to investigate phylogenetic relationships of the Peronosporomycetes (Oomycetes), nuclear large subunit ribosomal DNA sequences containing the D1 and D2 region were analyzed of 92 species belonging to the orders Peronosporales, Pythiales, Leptomitales, Rhipidiales, Saprolegniales and Sclerosporales. The data were analyzed applying methods of neighbor-joining as well as maximum parsimony, both statistically supported using the bootstrap method. The results confirm the major division between the Pythiales and Peronosporales on the one hand and the Saprolegniales, Leptomitales, and Rhipidiales on the other. The Sclerosporales were shown to be polyphyletic; while Sclerosporaceae are nested within the Peronosporaceae, the Verrucalvaceae are merged within the Saprolegniales. Within the Peronosporomycetidae, Pythiales as well as Peronosporales as currently defined are polyphyletic. The well supported Albugo clade appears to be the most basal lineage, followed by a Pythium-Lagenidium clade. The third, highly supported clade comprises the Peronosporaceae together with Sclerospora, Phytophthora, and Peronophythora. Peronophythora is placed within Phytophthora, indicating that both genera should be merged. Bremiella seems to be polyphyletic within the genus Plasmopara, suggesting a transfer to Plasmopara. The species of Peronospora do not appear as a monophyletic group. Peronospora species growing on Brassicaceae form a highly supported clade.     

PCR analysis of the distribution of unicellular cyanobacterial diazotrophs in the Arabian Sea

An oligonucleotide primer, NITRO821R, targeting the 16S rRNA gene of unicellular cyanobacterial N2 fixers was developed based on newly derived sequences from Crocosphaera sp. strain WH 8501 and Cyanothece sp. strains WH 8902 and WH 8904 as well as several previously described sequences of Cyanothece sp. and sequences of intracellular cyanobacterial symbionts of the marine diatom Climacodium frauenfeldianum. This oligonucleotide is specific for the targeted organisms, which represent a well-defined phylogenetic lineage, and can detect as few as 50 cells in a standard PCR when it is used as a reverse primer together with the cyanobacterium- and plastid-specific forward primer CYA359F (U. Nubel, F. Garcia-Pichel, and G. Muyzer, Appl. Environ. Microbiol. 63:3327-3332, 1997). Use of this primer pair in the PCR allowed analysis of the distribution of marine unicellular cyanobacterial diazotrophs along a transect following the 67 degrees E meridian from Victoria, Seychelles, to Muscat, Oman (0.5 degrees S to 26 degrees N) in the Arabian Sea. These organisms were found to be preferentially located in warm (>29 degrees C) oligotrophic subsurface waters between 0 and 7 degrees N, but they were also found at a station north of Oman at 26 degrees N, 56 degrees 35'E, where similar water column conditions prevailed. Slightly cooler oligotrophic waters (<29 degrees C) did not contain these organisms or the numbers were considerably reduced, suggesting that temperature is a key factor in dictating the abundance of this unicellular cyanobacterial diazotroph lineage in marine environments.     

Structural features of the plastid ribosomal RNA operons of two red algae: Antithamnion sp. and Cyanidium caldarium

The nucleotide sequences of the plastid 16S rDNA of the multicellular red alga Antithamnion sp. and the 16S rDNA/23S rDNA intergenic spacers of the plastid DNAs of the unicellular red alga Cyanidium caldarium and of Antithamnion sp. were determined. Sequence comparisons support the idea of a polyphyletic origin of the red algal and the higher-plant chloroplasts. Both spacer regions include the unsplit tRNA^Ile (GAU) and tRNA^Ala (UGC) genes and so the plastids of both algae form a homogeneous group with those of chromophytic algae and Cyanophora paradoxa characterized by small-sized rDNA spacers in contrast to green algae and higher plants. Nevertheless, remarkable sequence differences within the rRNA and the tRNA genes give the plastids of Cyanidium caldarium a rather isolated position.     

Effect of temperature and light on growth of and photosynthesis by Synechococcus isolates typical of those predominating in the octopus spring microbial mat community of Yellowstone National Park

Previous molecular analysis of the Octopus Spring cyanobacterial mat revealed numerous genetically distinct 16S rRNA sequences from predominant Synechococcus populations distantly related to the readily cultivated unicellular cyanobacterium Synechococcus lividus. Patterns in genotype distribution relative to temperature and light conditions suggested that the organisms contributing these 16S rRNA sequences may fill distinct ecological niches. To test this hypothesis, Synechococcus isolates were cultivated using a dilution and filtration approach and then shown to be genetically relevant to natural mat populations by comparisons of similarities of 16S rRNA genes and 16S-23S internal transcribed spacer (ITS) regions. Most isolates were identical or nearly identical at both loci to predominant mat genotypes; others showed 1- to 2-nucleotide differences at the 16S rRNA locus and even greater difference in ITS sequences. Isolates with predominant mat genotypes had distinct temperature ranges and optima for growth that were consistent with their distributions in the mat. Isolates with genotypes not previously detected or known to be predominant in the mat exhibited temperature ranges and optima that were not representative of predominant mat populations and also grew more slowly. Temperature effects on photosynthesis did not reflect temperature relations for growth. However, the isolate with the highest temperature optimum and upper limit was capable of performing photosynthesis at a higher temperature than other isolates. Growth rate and photosynthetic responses provided evidence for light acclimation but evidence of, at best, only subtle light adaptation.     

Cryptic diversity of the symbiotic cyanobacterium Synechococcus spongiarum among sponge hosts

Cyanobacteria are common members of sponge-associated bacterial communities and are particularly abundant symbionts of coral reef sponges. The unicellular cyanobacterium Synechococcus spongiarum is the most prevalent photosynthetic symbiont in marine sponges and inhabits taxonomically diverse hosts from tropical and temperate reefs worldwide. Despite the global distribution of S. spongiarum , molecular analyses report low levels of genetic divergence among 16S ribosomal RNA (rRNA) gene sequences from diverse sponge hosts, resulting either from the widespread dispersal ability of these symbionts or the low phylogenetic resolution of a conserved molecular marker. Partial 16S rRNA and entire 16S–23S rRNA internal transcribed spacer (ITS) genes were sequenced from cyanobacteria inhabiting 32 sponges (representing 18 species, six families and four orders) from six geographical regions. ITS phylogenies revealed 12 distinct clades of S. spongiarum that displayed 9% mean sequence divergence among clades and less than 1% sequence divergence within clades. Symbiont clades ranged in specificity from generalists to specialists, with most (10 of 12) clades detected in one or several closely related hosts. Although multiple symbiont clades inhabited some host sponges, symbiont communities appear to be structured by both geography and host phylogeny. In contrast, 16S rRNA sequences were highly conserved, exhibiting less than 1% sequence divergence among symbiont clades. ITS gene sequences displayed much higher variability than 16S rRNA sequences, highlighting the utility of ITS sequences in determining the genetic diversity and host specificity of S. spongiarum populations among reef sponges. The genetic diversity of S. spongiarum revealed by ITS sequences may be correlated with different physiological capabilities and environmental preferences that may generate variable host–symbiont interactions.