- Volume 53, Issue 6, 2003

Four yeast strains isolated from fermenting botrytized grape musts in the Tokaj wine region of Hungary are shown to represent a new osmotolerant and psychrotolerant species. The new species, Candida zemplinina (type strain 10-372T=CBS 9494T=NCAIM Y016667T), is closely related to Candida stellata, a yeast common on overripe grapes and in sweet fermenting wines. The sequence of the D1/D2 domain of the C. zemplinina 10-372T 26S rDNA shows 8·1 % sequence difference when compared to its counterpart in C. stellata CBS 157T. In the conserved 5·8S gene of the ITS1–5·8S–ITS2 region the difference is 8 %. The D1/D2 domain differs only at two nucleotides from the homologous sequence of a yeast strain isolated from botrytized grapes in California, suggesting that C. zemplinina is a wine yeast that occurs in geographically distant localities.

Six ballistoconidium-forming yeast strains that were isolated from plant leaves collected on Changbai Mountain, north-east China, were assigned to the genus Bensingtonia Ingold emend. Nakase & Boekhout due to the formation of asymmetrical ballistoconidia, cream-coloured colonies and Q-9 as the major ubiquinone. Two separate groups, representing two novel Bensingtonia species, were recognized among these yeasts by 26S rDNA D1/D2 domain, internal transcribed spacer (ITS) region and 18S rDNA sequence analyses. The names Bensingtonia changbaiensis sp. nov. (type strain, CB 346T=AS 2.2310T=CBS 9497T) and Bensingtonia sorbi sp. nov. (type strain, CB 286T=AS 2.2303T=CBS 9498T) are proposed for these two species.

Among ballistoconidium-forming yeast strains isolated from various plant leaves collected in Banna, Yunnan Province, China, five strains that formed pink-coloured colonies and asymmetric ballistoconidia were classified in a single group and assigned to the genus Sporobolomyces by conventional and chemotaxonomic studies. Analyses of the internal transcribed spacer region and 26S rDNA D1/D2 domain sequences indicated that these strains represent a novel species with a close phylogenetic relationship to Sporobolomyces blumeae in the Sporidiobolus lineage, for which the name Sporobolomyces bannaensis sp. nov. is proposed (type strain Y41T=AS 2.2285T=CBS 9204T).

A novel species of the genus Cryptococcus was isolated from sediment collected on the deep-sea floor of Suruga Bay, Japan. Nucleotide sequences of 18S rDNA, internal transcribed spacers, 5·8S rDNA and the D1/D2 region of 26S rDNA of strain SY-260T suggested affinities to a phylogenetic lineage that includes Cryptococcus luteolus. Comparisons of the rDNA sequences of each region clarified that strain SY-260T is related distantly to Bullera coprosmaensis and Bullera oryzae, but is distinct at the species level. As ballistoconidia and sexual reproduction were not observed in strain SY-260T, this strain is described as Cryptococcus surugaensis sp. nov. (type strain, SY-260T=JCM 11903T=CBS 9426T).

The rnpB gene is universally present in bacterial species and encodes the RNA subunit of endoribonuclease P. In this study, rnpB was sequenced in 50 type strains and 29 additional strains of the genus Streptococcus. Putative secondary-structure models and possible interactions in RNase P RNA molecules are discussed. Phylogenetic relationships were studied and Bayesian, maximum-parsimony and minimum-evolution analyses supported six main clades that comprised 22 of the 50 species analysed. Phylogenetic inference was also studied for the 16S rRNA gene; it indicated a similar tree topology, but with weaker support values than for rnpB. Combined analysis of rnpB and 16S resulted in a phylogeny with significantly better support. Variability in the rnpB and 16S genes among all type strains, calculated as Shannon–Wiener information index values, was 0·45 for rnpB and 0·15 for 16S. Intraspecies proximity was assessed by principal coordinate analysis of rnpB for 32 strains of six closely related species (two clades) and showed species-specific clusters, but heterogeneity occurred in two species. It can be concluded that the rnpB gene is suitable for phylogenetic analysis of closely related taxa and has potential as a tool for species discrimination.

Thirty-two protein-encoding genes that are distributed widely among bacterial genomes were tested for the potential usefulness of their DNA sequences in assigning bacterial strains to species. From publicly available data, it was possible to make 49 pairwise comparisons of whole bacterial genomes that were related at the genus or subgenus level. DNA sequence identity scores for eight of the genes correlated strongly with overall sequence identity scores for the genome pairs. Even single-gene alignments could predict overall genome relatedness with a high degree of precision and accuracy. Predictions could be refined further by including two or three genes in the analysis. The proposal that sequence analysis of a small set of protein-encoding genes could reliably assign novel strains or isolates to bacterial species is strongly supported.

The nucleotide sequences of 16S and 23S rRNA genes (rDNA) were determined for 11 isolates of Streptococcus dysgalactiae subsp. equisimilis from slaughtered pigs with endocarditis, arthritis or lymphadenitis and strain ATCC 35666, designated as a strain of subspecies equisimilis. The sequences of each of the genes were compared phylogenetically with the corresponding sequences of S. dysgalactiae subsp. dysgalactiae ATCC 43078T and ATCC 27957, which were also determined in this study. Based on the 16S rDNA analysis, the isolates of S. dysgalactiae subsp. equisimilis were divided into two distinct groups, designated groups 1 and 2. S. dysgalactiae subsp. equisimilis ATCC 35666 was closely related to the group 2 strains. The S. dysgalactiae subsp. dysgalactiae strains seemed to be associated with the group 1 strains, which was not consistent with the conventional subspecific classification of S. dysgalactiae. In contrast, the 23S rDNA analysis distinguished S. dysgalactiae subsp. dysgalactiae strains from subsp. equisimilis strains. This inconsistency between phylogenies based on 16S and 23S rDNA indicates that 23S rDNA is a more rigid marker for determining the phylogenetic relationships and taxonomic position of these organisms than is 16S rDNA.

The family Vibrionaceae is considered to be one of the most diverse and well-studied groups of bacteria. Here, evolution is assessed within the Vibrionaceae to determine whether multiple origins of eukaryotic associations have occurred within this diverse group of bacteria. Analyses were based on a large molecular dataset, along with a matrix that consisted of 100 biochemical and restriction digest characters. By using direct optimization methods to analyse both datasets individually and in combination, a total-evidence cladogram has been produced, which supports the hypothesis that several important symbionts (both mutualistic and pathogenic) within the Vibrionaceae are not monophyletic. This leads us to consider that symbiosis (and subsequently, associations with Eukarya) has evolved multiple times within the Vibrionaceae lineage.

Phylogenetic analyses of the nuclear-encoded small-subunit rDNA sequences from taxa representing all of the major lineages of green algae, including new sequences for the Trentepohliales, consistently indicated that the subaerial Trentepohliales are closely related to ulvophycean marine green algae, particularly to the siphonous and hemisiphonous orders. The presence of phragmoplast-type cytokinesis in the order Trentepohliales remains enigmatic, and it is interesting that this type of cell division is associated with terrestrial (subaerial) habits.

With reference to the first Principle of the International Code of Nomenclature of Bacteria, which emphasizes stability of names, it is proposed that the original adjectival form of the specific epithet be conserved in the reclassification of Bacteroides forsythus to the new genus Tannerella. Thus, Tannerella forsythensis Sakamoto et al. 2002 should be Tannerella forsythia Sakamoto et al. 2002 corrig., gen. nov., comb. nov., and we put forward a Request for an Opinion to the Judicial Commission regarding this correction.

Phragmoplast-mediated cell division characterizes the land plants in the streptophyte lineage and some species of the green algal orders Coleochaetales, Charales and Zygnematales that are basal to that lineage. This type of cell division is generally not found in the other green plant lineage, the chlorophyte algae. A well-developed phragmoplast-type cell division has been documented, however, in two subaerial green algae (Cephaleuros parasiticus and Trentepohlia odorata) belonging to the order Trentepohliales – an order that molecular sequence data place unequivocally within the chlorophytes rather than streptophytes. Is the phragmoplast-mediated cell division of the Trentepohliales a case of homology or non-homology? To gain more insight into this question, we are exploring the potential phylogenetic information inferred from gene sequences of phragmoplastin, a dynamin-like protein that has been associated with cell-plate formation during phragmoplast-mediated cytokinesis in land plants. Primers for green algae were designed based on an available phragmoplastin sequence from soybean and yielded PCR amplifications from the trentepohlialean green algae Trentepohlia and Cephaleuros and the leafy liverwort Bazzania. These are the first published data for phragmoplastins in algae and liverworts. Analysis of phragmoplastin gene sequences in chlorophyte and streptophyte green algae may help to chart the evolution of the development of the phragmoplast.

The Helicosporidia are invertebrate pathogens that have recently been identified as non-photosynthetic green algae (Chlorophyta). In order to confirm the algal nature of the genus Helicosporidium, the presence of a retained chloroplast genome in Helicosporidia cells was investigated. Fragments homologous to plastid 16S rRNA (rrn16) genes were amplified successfully from cellular DNA extracted from two different Helicosporidium isolates. The fragment sequences are 1269 and 1266 bp long, are very AT-rich (60·7 %) and are similar to homologous genes sequenced from non-photosynthetic green algae. Maximum-parsimony, maximum-likelihood and neighbour-joining methods were used to infer phylogenetic trees from an rrn16 sequence alignment. All trees depicted the Helicosporidia as sister taxa to the non-photosynthetic, pathogenic alga Prototheca zopfii. Moreover, the trees identified Helicosporidium spp. as members of a clade that included the heterotrophic species Prototheca spp. and the mesotrophic species Chlorella protothecoides. The clade is always strongly supported by bootstrap values, suggesting that all these organisms share a most recent common ancestor. Phylogenetic analyses inferred from plastid 16S rRNA genes confirmed that the Helicosporidia are non-photosynthetic green algae, close relatives of the genus Prototheca (Chlorophyta, Trebouxiophyceae). Such phylogenetic affinities suggest that Helicosporidium spp. are likely to possess Prototheca-like organelles and organelle genomes.

The chloroplast genes of dinoflagellates are distributed among small, circular dsDNA molecules termed minicircles. In this paper, we describe the structure of the non-coding region of the psbA minicircle from Symbiodinium. DNA sequence was obtained from five Symbiodinium strains obtained from four different coral host species (Goniopora tenuidens, Heliofungia actiniformis, Leptastrea purpurea and Pocillopora damicornis), which had previously been determined to be closely related using LSU rDNA region D1/D2 sequence analysis. Eight distinct sequence blocks, consisting of four conserved cores interspersed with two metastable regions and flanked by two variable regions, occurred at similar positions in all strains. Inverted repeats (IRs) occurred in tandem or ‘twin’ formation within two of the four cores. The metastable regions also consisted of twin IRs and had modular behaviour, being either fully present or completely absent in the different strains. These twin IRs are similar in sequence to double-hairpin elements (DHEs) found in the mitochondrial genomes of some fungi, and may be mobile elements or may serve a functional role in recombination or replication. Within the central unit (consisting of the cores plus the metastable regions), all IRs contained perfect sequence inverses, implying they are highly evolved. IRs were also present outside the central unit but these were imperfect and possessed by individual strains only. A central adenine-rich sequence most closely resembled one in the centre of the non-coding part of Amphidinium operculatum minicircles, and is a potential origin of replication. Sequence polymorphism was extremely high in the variable regions, suggesting that these regions may be useful for distinguishing strains that cannot be differentiated using molecular markers currently available for Symbiodinium.

Phylogenetic analysis of small and large subunits of rDNA genes suggested that Foraminifera originated early in the evolution of eukaryotes, preceding the origin of other rhizopodial protists. This view was recently challenged by the analysis of actin and ubiquitin protein sequences, which revealed a close relationship between Foraminifera and Cercozoa, an assemblage of various filose amoebae and amoeboflagellates that branch in the so-called crown of the SSU rDNA tree of eukaryotes. To further test this hypothesis, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1) from five foraminiferans, two cercozoans and the testate filosean Gromia oviformis. Analysis of our data confirms a close relationship between Foraminifera and Cercozoa and points to Gromia as the closest relative of Foraminifera.

It is argued here that the anaerobic protozoan zooflagellate Parabasalia, Carpediemonas and Eopharyngia (diplomonads, enteromonads, retortamonads) constitute a holophyletic group, for which the existing name Trichozoa is adopted as a new subphylum. Ancestrally, Trichozoa probably had hydrogenosomes, stacked Golgi dictyosomes, three anterior centrioles and one posterior centriole: the typical tetrakont pattern. It is also argued that the closest relatives of Trichozoa are Anaeromonada (Trimastix, oxymonads), and the two groups are classified as subphyla of a revised phylum Metamonada. Returning Parabasalia and Anaeromonadea to Metamonada, as in Grassé's original classification, simplifies classification of the kingdom Protozoa by reducing the number of phyla within infrakingdom Excavata from five to four. Percolozoa (Heterolobosea plus Percolatea classis nov.) and Metamonada are probably both ancestrally quadriciliate with a kinetid of four centrioles attached to the nucleus; the few biciliates among them are probably secondarily derived. Metamonada ancestrally probably had two divergent centriole pairs, whereas, in Percolozoa, all four centrioles are parallel. It is suggested that Discicristata (Percolozoa, Euglenozoa) are holophyletic, ancestrally with two parallel centrioles. In the phylum Loukozoa, Malawimonadea classis nov. is established for Malawimonas (with a new family and order also) and Diphyllatea classis nov., for Diphylleida (Diphylleia, Collodictyon), is transferred back to Apusozoa. A new class, order and family are established for the anaerobic, biciliate, tricentriolar Carpediemonas, transferring it from Loukozoa to Trichozoa because of its triply flanged cilia; like Retortamonas, it may be secondarily biciliate – its unique combination of putative hydrogenosomes and flanged cilia agree with molecular evidence that Carpediemonas is sister to Eopharyngia, diverging before their ancestor lost hydrogenosomes and acquired a cytopharynx. Removal of anaeromonads and Carpediemonas makes Loukozoa more homogeneous, being basically biciliate, aerobic and free-living, in contrast to Metamonada. A new taxon-rich rRNA tree supports holophyly of Discicristata and Trichozoa strongly, holophyly of Metamonada and Excavata and paraphyly of Loukozoa weakly. Mitochondria were probably transformed into hydrogenosomes independently in the ancestors of lyromonad Percolozoa and Metamonada and further reduced in the ancestral eopharyngian. Evidence is briefly discussed that Metamonada and all other excavates share a photosynthetic ancestry with Euglenozoa and are secondarily non-photosynthetic, as predicted by the cabozoan hypothesis for a single secondary symbiogenetic acquisition of green algal plastids by the last common ancestor of Euglenozoa and Cercozoa. Excavata plus core Rhizaria (Cercozoa, Retaria) probably form an ancestrally photophagotrophic clade. The origin from a benthic loukozoan ancestor of the characteristic cellular features of Percolozoa and Euglenozoa through divergent adaptations for feeding on or close to surfaces is also discussed.