Partial sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase and the phylogeny of Prochloron and Prochlorococcus (Prochlorales)

The prochlorophytes, oxygenic photosynthetic prokaryotes having no phycobiliprotein but possessing chlorophylls a and b, have been proposed to have a common ancestry with green chloroplasts, yet this is still controversal. We report here that partial sequence comparisons of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, including sequence data from two prochlorophytes, Prochlorococcus and Prochloron, indicate that Prochlorococcus is more closely related to a photosynthetic bacterium, Chromatium vinosum (-purple bacteria), than to cyanobacteria, while Prochloron is closely related to the prochlorophyte Prochlorothrix and to cyanobacteria. The molecular phylogenetic tree indicates that a common ancestor of Prochlorococcus and -purple bacteria branched off from the land plant lineage earlier than Prochloron, Prochlorothrix, and cyanobacteria.Correspondence to: A. Shimada     

IsProchlorothrix hollandica the best choice as a prokaryotic model for higher plant Chla/b photosynthesis?

We examine the issue of prochlorophyte origins and provide analyses which highlight the limitations of inferring evolutionary trees from anciently diverged sequences that have markedly different GC contents. Under these conditions we have found that current tree reconstruction methods strongly group together sequences with similar GC contents, whether or not the sequences share a common ancestor. We provide 3psbA termini sequence forProchloron didemni and find it does not have the 7 amino acid deletion that occurs in Chla/b chloroplasts andProchlorothrix hollandica. This is consistent with the recent findings of a Chlc like pigment in the light harvesting system in other prochlorophytes but apparently absent inP. hollandica. From these observations we suggest thatP. hollandica is the prochlorophyte most closely related to Chla/b containing chloroplasts and hence the most appropriate prokaryotic model for higher plant Chla/b photosynthesis.     

CYANOBACTERIAL EVOLUTION AND PROCHLOROPHYTE DIVERSITY AS SEEN IN DNA-DEPENDENT RNA POLYMERASE GENE SEQUENCES1

Nucleotide sequence data from DNA-dependent RNA polymerase (rpoC) genes were used to examine the phylogenetic relationships among the phycobiliprotein- and three known chlorophyll b-containing (prochlorophyte) cyanobacteria. The phylogenetic trees obtained confirm the polyphyletic nature of the prochlorophytes. Data from Prochloron cells obtained from six different tunicate host species suggest that at least two closely related groups of Prochloron exist in the same area in Palau, West Caroline Islands. Overall, however, the genetic diversity within the analyzed samples was much smaller than within the nonsymbiotic Prochlorococcus.     

Molecular identification and evolution of the cyclic peptide hepatotoxins, microcystin and nodularin, synthetase genes in three orders of cyanobacteria

The cyanobacterial hepatotoxins, microcystin and nodularin, are produced by a wide range of cyanobacteria. Microcystin production has been reported in the four cyanobacterial orders: Oscillatoriales, Chroococcales, Stigonematales, and Nostocales. The production of nodularin is a distinct characteristic of the Nostocales genus Nodularia. A single rapid method is needed to reliably detect cyanobacteria that are potentially capable of producing these hepatotoxins. To this end, a PCR was designed to detect all potential microcystin and nodularin-producing cyanobacteria from laboratory cultures as well as in harmful algal blooms. The aminotransferase (AMT) domain, which is located on the modules mcyE and ndaF of the microcystin and nodularin synthetase enzyme complexes, respectively, was chosen as the target sequence because of its essential function in the synthesis of all microcystins as well as nodularins. Using the described PCR, it was possible to amplify a 472 bp PCR product from the AMT domains of all tested hepatotoxic species and bloom samples. Sequence data provided further insight into the evolution of the microcystin and nodularin synthetases through bioinformatic analyses of the AMT in microcystin and nodularin synthetases, with congruence between the evolution of 16S rRNA and the AMT domain.     

Domain structures of chlorophyllide<Emphasis Type="Italic"> a</Emphasis> oxygenase of green plants and<Emphasis Type="Italic"> Prochlorothrix hollandica</Emphasis> in relation to catalytic functions

Chlorophyll b is a photosynthetic antenna pigment found in prochlorophytes and chlorophytes. In chlorophytes, its biosynthesis regulates the photosynthetic antenna size. Chlorophyll b is synthesized from chlorophyll a in a two-step oxygenation reaction by chlorophyllide a oxygenase (CAO). In this study, we first identified the entire sequence of a prochlorophyte CAO gene from Prochlorothrix hollandica to compare it with those from chlorophytes, and we examined the catalytic activity of the gene product. Southern blot analysis showed that the CAO gene is presented in one copy in the P. hollandica genome. The P. hollandica CAO gene (PhCAO) has a coding capacity for 367 amino acids, which is much smaller than that of Arabidopsis thaliana (537 amino acids) and Oryza sativa (542 amino acids) CAO genes. In spite of the small size, PhCAO catalyzed the formation of chlorophyll b. By comparing these sequences, we classified the land-plant sequences into four parts: the N-terminal sequence predicted to be a transit peptide, the successive conserved sequence unique in land plants (A-domain, 134 amino acids), a less-conserved sequence (B-domain, 30 amino acids) and the C-terminal conserved sequence common in chlorophytes and prochlorophytes (C-domain, 337 to 344 amino acids). We demonstrated that the C-domain is sufficient for catalytic activity by transforming the cyanobacterium Synechocystis sp. PCC6803 with the C-domain from A. thaliana. In this paper, the role of the A-domain is discussed in relation to the formation of light-harvesting chlorophyll a/b–protein complexes in land plants.Abbreviations CAO Chlorophyllide a oxygenase - CP Chlorophyll protein - HPLC High-performance liquid chromatography - LHC Light-harvesting complex - PCR Polymerase chain reaction - PS Photosystem     

Molecular phylogeny of Cypripedium (Orchidaceae: Cypripedioideae) inferred from multiple nuclear and chloroplast regions

A molecular analysis was performed on 56 taxa in the orchid genus Cypripedium using nrDNA ITS and five chloroplast regions (trnH-psbA, atpI-atpH, trnS-trnfM, trnL-F spacer, and the trnL intron). The genus Cypripedium was confirmed as monophyletic. Our data provided strong support for monophyletic grouping of eight infrageneric sections (Subtropica, Obtusipetala, Trigonopedia, Sinopedilum, Bifolia, Flabelinervia, Arietinum, and Cypripedium) defined in earlier taxonomic treatments, and paraphyletic grouping of two sections (Irapeana and Retinervi). Within the genus Cypripedium, the first divergent lineage consisted of two Mesomaerican species, and subsequently the Cypripedium debile lineage from eastern Asia was split. Our study did not support the notion that two Asian species (Cypripedium subtropicum and Cypripedium singchii) were closely related to either Mesoamerican Cypripedium irapeanum or North American Cypripedium californicum, as indicated by previous interpretations based on morphological evidences. In addition, one pair of vicariant species, Cypripedium plectrochilum (eastern Asia) and Cypripedium arietinum (North America), unique to section Arietinum, was confirmed. Furthermore, within the monophyletic section Cypripedium two previously recognized subsections, Cypripedium and Macrantha, were shown to be paraphyletic. Our results suggested that this section split into two groups based on distribution (North America vs. Eurasia) instead of such previously used, morphological traits as flower color, and the shape of the lips (labellum) and lateral petals.     

Prochlorococcus marinus nov. gen. nov. sp.: an oxyphototrophic marine prokaryote containing divinyl chlorophyll a and b

Several years ago, prochlorophyte picoplankton were discovered in the N. Atlantic. They have since been found to be abundant within the euphotic zone of the world's tropical and temperate oceans. The cells are extremely small, lack phycobiliproteins, and contain divinyl chlorophyll a and b as their primary photosynthetic pigments. Phylogenies constructed from DNA sequence data indicate that these cells are more closely related to a cluster of marine cyanobacteria than to their prochlorophyte relatives Prochlorothrix and Prochloron. Several strains of this organism have recently been brought into culture, and herewith are given the name Prochlorococcus marinus.     

Elainella gen. nov.: a new tropical cyanobacterium characterized using a complex genomic approach

Cyanobacteria represent an ancient, monophyletic lineage of bacteria with the ability to undertake oxygenic photosynthesis. Although they possess a relatively high degree of morphological variability compared with other prokaryotes and there is a wealth of molecular data, there are still significant gaps in our knowledge of cyanobacterial diversity, especially in tropical areas. Here, we present a novel, filamentous, tropical cyanobacterium, which could be classified as Pseudophormidium based on morphological criteria. A total evidence investigation employing ecological, morphological and genomic data, indicated that our strains form a new and ancient evolutionary lineage among cyanobacteria unrelated to Pseudophormidium. Based on this polyphasic assessment, our strains represent a novel, monospecific genus: Elainella. This new genus represents an example of phenotypic convergence, which seems to be a prevalent macroevolutionary pattern in cyanobacteria, a likely cause of the frequently cited polyphyly within a majority of genera.     

Evidence for polyphyletic origin of the members of the orders of Oscillatoriales and Pleurocapsales as determined by 16S rDNA analysis

Phylogenetic analyses were carried out on six pleurocapsalean strains and 12 oscillatorialean strains by sequence determination of the 16S rDNA (16S rRNA gene). Although heterocyst-forming strains of the orders Nostocales and Stigonematales were shown to be monophyletic, unicellular strains of the orders Chroococcales and filamentous Oscillatoriales were shown to be polyphyletic, as reported earlier. Moreover, unicellular and baeocyte-forming strains of the order Pleurocapsales, which were thought to be monophyletic, were newly found to be polyphyletic. The results strongly indicate that even morphology was not necessarily reflected in the phylogenetic relationships at the order level. A need exists to reconstruct the taxonomy of cyanobacteria at the order level.     

The occurrence of cyanobacteria in pulp and paper waste-treatment systems

Pulp and paper secondary waste-treatment systems in Brazil, Canada, New Zealand, and the U.S.A. contained dynamic cyanobacterial communities, some of which exceeded heterotrophic bacterial biomass. No other viable photoautotrophic populations were detected in the ponds. Regardless of geographical location, Oscillatoriales including Phormidium, Geitlerinema, and Pseudanabaena were the dominant taxa. As well, Chroococcus (Chroococcales) was an important genus in Brazil and New Zealand. The possible impact of cyanobacteria on waste-treatment efficiency deserves further study given their large biomass and diverse metabolic characteristics.     

Distribution patterns of marine planktonic cyanobacterial assemblages in transitional marine habitats using 16S rRNA phylogeny

The community composition of marine planktonic cyanobacteria in transitional marine habitats can influence its overall contribution to aquatic primary production. To understand distribution patterns of marine planktonic cyanobacterial assemblages, phylogenetic and statistical analyses were undertaken on planktonic cyanobacterial 16S rRNA gene sequences from four transitional marine habitats [Baltic Sea (BL), Monterey Bay (MB), South China Sea (SCS) and Sundarbans (SB)]. Out of 3255 sequences analyzed, only 546 sequences were found to be planktonic cyanobacteria and were considered in this study. Among these, 338 sequences representative of Sundarbans, the world's largest mangrove were generated based on Sanger and Illumina sequencing approaches. Based on 16S rRNA phylogeny, four major taxonomic orders of marine planktonic cyanobacteria were recovered in varying proportions with several novel 16S rRNA sequences in each of the four targeted sites. Members of the order Synechococcales were dominant in all the sites (?94% sequences) while the orders Chroococcales and Oscillatoriales were only detected in SB and SCS sites, respectively. In the phylogenetic tree, sequences representing the major marine picocyanobacterial genus Synechococcus showed overwhelming dominance in SB and they were found in three other sites. Prochlorococcus ‐like sequences were found in sizeable number in MB and SCS but were absent in SB and coastal BL. Synechococcus ‐like sequences were represented by three major marine clusters (5.1, 5.2, and 5.3). Three novel clades as part of Synechococcus cluster were detected only in SB and one novel clade in BL. The majority of OTUs were found to be exclusive to each site, whereas some were shared by two or more sites as revealed by beta‐diversity analysis.     

Ulocladium systematics revisited: phylogeny and taxonomic status

The genus Ulocladium represents phaeodictyosporic Hyphomycetes that produce conidia that are essentially obovoid in shape. Previous molecular studies that included Ulocladium and related taxa in Alternaria, Embellisia, and Stemphylium revealed a conflict between morphology and phylogeny, and Ulocladium was supported as polyphyletic with a paraphyletic core group. Moreover, the genus consistently resolved within a larger Alternaria/Ulocladium clade, resulting in paraphyly of Alternaria and questions as to the taxonomic status of Ulocladium. In the present study, 13 Ulocladium species and three genetic loci were included for a more comprehensive systematic analysis of the genus than had previously been conducted. Total genomic DNA was extracted from representative taxa and sequences were determined for the nuclear internal transcribed spacer region, including the 5.8S rDNA gene, and the protein-coding genes glyceraldehyde-3-phosphate dehydrogenase and Alt a1. Subsequent phylogenetic analyses based on maximum parsimony and Bayesian methods included related Alternaria, Embellisia, and Stemphylium spp. Results supported previous findings of polyphyletic and paraphyletic relationships of Ulocladium among other taxa. Ten Ulocladium species clustered into a core Ulocladium clade and all taxa possessed the key diagnostic feature of Ulocladium, namely, conidia essentially obovoid in shape. However, A. cheiranthi and E. indefessa also clustered within this group with high bootstrap support but did not posses this diagnostic feature. This paraphyletic clade resolved basal to the core Alternaria clade with high bootstrap support, unlike previous studies in which its position was imbedded within the primary Alternaria clade. Thus, the status of the genus as an independent lineage and a unique taxon is strongly supported. As previously reported, U. alternariae and U. oudemansii, which posses the key conidium characteristics of Ulocladium, clustered as a separate clade sister to the core Ulocladium clade. Further studies are necessary to determine if these taxa represent an independent lineage or share a common ancestor with other Ulocladium species. Obovoid conidia were poorly represented in the isolate of U. lanuginosum that was included in these analyses (the only U. lanuginosum isolate currently available), and the isolate resolved as A. radicina based upon all three loci sequenced. Based upon these data and the origin of the isolate, which was originally deposited as A. malvae, a reassessment of its identity is supported.     

Sequence analysis and phylogenetic reconstruction of the genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase from the chlorophyllb-containing prokaryoteProchlorothrix hollandica

Summary Prochlorophytes similar toProchloron sp. andProchlorothrix hollandica have been suggested as possible progenitors of the plastids of green algae and land plants because they are prokaryotic organisms that possess chlorophyllb (chlb). We have sequenced theProchlorothrix genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco),rbcL andrbcS, for comparison with those of other taxa to assess the phylogenetic relationship of this species. Length differences in the large subunit polypeptide among all sequences compared occur primarily at the amino terminus, where numerous short gaps are present, and at the carboxy terminus, where sequences ofAlcaligenes eutrophus and non-chlorophyllb algae are several amino acids longer. Some domains in the small subunit polypeptide are conserved among all sequences analyzed, yet in other domains the sequences of different phylogenetic groups exhibit specific structural characteristics. Phylogenetic analyses ofrbcL andrbcS using Wagner parsimony analysis of deduced amino acid sequences indicate thatProchlorothrix is more closely related to cyanobacteria than to the green plastid lineage. The molecular phylogenies suggest that plastids originated by at least three separate primary endosymbiotic events, i.e., once each leading to green algae and land plants, to red algae, and toCyanophora paradoxa. TheProchlorothrix rubisco genes show a strong GC bias, with 68% of the third codon positions being G or C. Factors that may affect the GC content of different genomes are discussed.     

Phylogeny of Rhododendron section Vireya (Ericaceae) based on two non-coding regions of cpDNA

Section Vireya (Blume) Copel.f. is one of the most morphologically diverse groups of the genus Rhododendron. Vireyas have a unique distribution for the genus, being predominantly found throughout the Malesian Archipelago. The alpha taxonomy of section Vireya is relatively well understood and taxa are easily distinguished from other rhododendrons by their general appearance. Defining characteristics of the section the possession of seeds with tailed appendages at both ends, the twisting of capsule valves after opening, placentas that separate as thread-like structures from the central axis as the capsule opens are all subject to exceptions. Phylogenetic analyses of two cpDNA regions, psbA-trnH and trnT-trnL intergenic spacers are reported. The results of each analysis were generally congruent, with clades relating strongly to geographic areas. Section Vireya is monophyletic with the inclusion of at least one species of section Rhododendron. Only two of the seven subsections currently circumscribed are monophyletic: Malayovireya Sleumer and Siphonovireya Sleumer. ``Euvireya', all subsections excluding the paraphyletic subsection Pseudovireya, is monophyletic and includes two major clades, one restricted to eastern Malesia and the other to the western and middle Malesia.     

Molecular detection of potentially toxic cyanobacteria and their associated bacteria in lake water column and sediment

We investigated the molecular diversity of cyanobacteria and bacteria during a water bloom in a lake with a long history of toxic cyanobacterial blooms (Lake Kastoria, Greece). We also tested the hypothesis whether bloom-forming cyanobacteria are preserved in the lake’s sediment 2 years after the bloom. The dominant cyanobacteria during the bloom included the potentially toxin-producing Microcystis aeruginosa and several other Chroococcales forms closely related to the genus Microcystis. This suggests that the use of cyanobacterial-specific primers seems to be very informative in describing the cyanobacteria during the water blooms. The bacterial community showed high diversity, consisting mostly of singleton and doubleton phylotypes. The majority of the phylotypes were typical lake bacteria including some potential pathogens and toxin metabolising bacteria, suggesting that the dominant toxic cyanobacteria did not have any significant effect on the bacterial community structure. In the sediment, 2 years after the water bloom, no bloom-forming cyanobacteria were retrieved, suggesting that they cannot be preserved in the sediment. Similar to the water column, sediment bacterial diversity was also high, consisting mostly of yet-uncultured bacteria that are related to environments where organic matter degradation takes place.     

The phylogenetic relationships of Cynoglottis (Boraginaceae- Boragineae) inferred from ITS, 5.8S and trnL sequences

A molecular phylogenetic analysis of Cynoglottis was performed to evaluate previous hypotheses based on non-molecular evidence concerning the position of this genus within Boraginaceae tribe Boragineae. ITS-5.8S and trnL_UAA sequences from the nuclear and chloroplast non-coding genomes were obtained for four Cynoglottis taxa and selected members of the related genera Anchusa, Anchusella, Gastrocotyle, Brunnera and Pentaglottis. Cynoglottis is monophyletic, but neither trnL nor ITS support a close relationship with Brunnera, unlike previously supposed on morphological grounds. Brunnera is, instead, related to the southwestern European monotypic genus Pentaglottis, with which it forms a basal clade. ITS-5.8S sequences show that Anchusa thessala, a southeastern European annual species of Anchusa subg. Buglossellum, is sister to Cynoglottis and that the two taxa form a clade which also includes the Balkan endemic Gastrocotyle macedonica. Species of Anchusa subg. Anchusa form a separate lineage with high bootstrap support, suggesting that this heterogeneous genus is paraphyletic with respect to Cynoglottis. ITS sequences also discriminate between the Balkan-Apenninic diploid C. barrelieri and the Anatolian tetraploid C. chetikiana, albeit with low support. The molecular results are discussed in the light of karyological, morphological and chorological aspects.This work has been supported by M.I.U.R. 40% 2003 and the University of Firenze.     

Phylogeny of Castanea (Fagaceae) based on chloroplast trnT-L-F sequence data

Species in the genus Castanea are widely distributed in the deciduous forests of the Northern Hemisphere from Asia to Europe and North America. They show floristic similarity but differences in chestnut blight resistance especially among eastern Asian and eastern North American species. Phylogenetic analyses were conducted in this study using sequences of three chloroplast noncoding trnT-L-F regions. The trnT-L region was found to be the most variable and informative region. The highest proportion of parsimony informative sites, more and larger indels, and higher pairwise distances between taxa were obtained at trnT-L than at the other two regions. The high A+T values (74.5%) in the Castanea trnT-L region may explain the high proportion of transversions found in this region where as comparatively lower A+T values were found in the trnL intron (68.35%) and trnL-F spacer (70.07%) with relatively balanced numbers of transitions and transversions. The genus Castanea is supported as a monophyletic clade, while the section Eucastanon is paraphyletic. C. crenata is the most basal clade and sister to the remainder of the genus. The three Chinese species of Castanea are supported as a single monophyletic clade, whose sister group contains the North American and European species. There is consistent but weak support for a sister–group relationship between the North American species and European species.     

Toxic cyanobacteria (blue-green algae) in Finnish fresh and coastal waters

A survey of the occurrence of toxic blooms of cyanobacteria in Finnish fresh and coastal waters was made during 1985 and 1986. Toxicity of the freeze-dried water bloom samples was tested by mouse-bioassay (i.p.). Forty-four per cent (83/188) of the bloom samples were found to be lethally toxic. Hepatotoxic blooms (54) were almost twice as common as neurotoxic ones (29). Anabaena was the most frequently found genus in toxic and non-toxic blooms and it was present in all neurotoxic samples. Statistical associations were found between hepatotoxicity and incidence of Microcystis aeruginosa, M. viridis, M. wesenbergii, Anabaena flos-aquae and Anabaena spiroides. Neurotoxicity was statistically associated with Anabaena lemmermannii, Anabaena flos-aquae and Gomphosphaeria naegeliana. Isolation of strains of cyanobacteria confirmed the occurrence of hepatotoxic and neurotoxic strains of Anabaena, as well as hepatotoxic strains of Microcystis and Oscillatoria species.Toxic blooms caused cattle poisonings at three different lakes during the study period. Toxic blooms also occurred in drinking water sources. Our study shows that toxic cyanobacteria are more common in Finnish lakes than would be expected on the basis of animal poisonings. The results of this study show the existence of toxic cyanobacteria in Finnish water supplies and the need for their continued study as agents of water based disease.     

Evolutionary Relationships in the Sand-Dwelling Cichlid Lineage of Lake Tanganyika Suggest Multiple Colonization of Rocky Habitats and Convergent Origin of Biparental Mouthbrooding

The cichlid species flock of Lake Tanganyika is comprised of seven seeding lineages that evolved in step with changes of the lake environment. One seeding lineage diversified into at least six lineages within a short period of time. Our study focuses on the diversification of one of these lineages, the Ectodini, comprising highly specialized, sand- and rock-dwelling species. They display two distinct breeding styles: maternal and biparental mouthbrooding. By analyzing three mtDNA gene segments in 30 species representing all 13 described genera, we show that the Ectodini rapidly diversified into four clades at the onset of their radiation. The monotypic genus Grammatotria is likely to represent the most ancestral split, followed by the almost contemporary origin of three additional clades, the first comprising the benthic genus Callochromis, the second comprising the benthic genera Asprotilapia, Xenotilapia, Enantiopus, and Microdontochromis, and the third comprising the semi-pelagic genera Ophthalmotilapia, Cardiopharynx, Cyathopharynx, Ectodus, Aulonocranus, Lestradea, and Cunningtonia. Our study confirms the benthic and sand-dwelling life-style as ancestral. Rocky habitats were colonized independently in the Xenotilapia- and Ophthalmotilapia-clade. The Xenotilapia-clade comprises both maternal and biparental mouthbrooders. Their mode of breeding appears to be highly plastic: biparental mouthbrooding either evolved once in the common ancestor of the clade, to be reverted at least three times, or evolved at least five times independently from a maternally mouthbrooding ancestor. Furthermore, the genera Xenotilapia, Microdontochromis, Lestradea, and Ophthalmotilapia appeared paraphyletic in our analyses, suggesting the need of taxonomic revision.