Taxonomic revision of Chloromonas nivalis (Volvocales, Chlorophyceae) strains, with the new description of two snow-inhabiting Chloromonas species.

Chloromonas nivalis (Volvocales, Chlorophyceae) is considered a cosmopolitan species of a snow-inhabiting microalga because cysts morphologically identifiable as zygotes of the species are distributed worldwide. However, recent molecular data demonstrated that field-collected cysts identified as the zygotes consist of multiple species. Recently, we demonstrated that species identification of snow-inhabiting Chloromonas species is possible based on light and electron microscopy of asexual life cycles in strains and molecular phylogenetic analyses. Vegetative cells without eyespots and of inverted-teardrop shape have been reported once in North American material of C. nivalis; however, strains with such vegetative cells in snow-inhabiting species of Chloromonas have not been examined taxonomically in detail. Here, we used light and transmission electron microscopy together with molecular analyses of multiple DNA sequences to examine several C. nivalis strains. The morphological data demonstrated that one North American strain could be identified as C. nivalis, whereas three other strains should be re-classified as C. hoshawii sp. nov. and C. remiasii sp. nov. based on vegetative cell morphology, the number of zoospores within the parental cell wall during asexual reproduction, and whether cell aggregates (resulting from repeated divisions of daughter cells retained within a parental cell wall) were observed in the culture. This taxonomic treatment was supported by multigene phylogeny and comparative molecular analyses that included a rapidly evolving DNA region. Our molecular phylogenetic analyses also demonstrated that the North American strain of C. nivalis was phylogenetically separated from the Austrian and Japanese specimens previously identified as C. nivalis based on zygote morphology.

Chloromonas arctica sp. nov., a psychrotolerant alga from snow in the High Arctic (Chlamydomonadales, Chlorophyta).

With the advent of molecular phylogenetic methods, it has become possible to assess the bioversity of snow algae more accurately. In this study, we focused on a morphological, ultrastructural and taxonomic description of a new Chloromonas-like alga isolated from snow in the High Arctic (Svalbard). Light and transmission electron microscopy revealed broad ellipsoidal or ellipsoidal-cylindrical, occasionally spherical cells with a chloroplast without a pyrenoid, an inconspicuous eyespot and a papilla. The size difference and the aforementioned morphological traits clearly distinguished the alga from its closest counterparts within the genus Chloromonas. Moreover, we were able to cultivate the alga at both 5 and 20 °C, revealing the psychrotolerant nature of the strain. Phylogenetic analyses of the plastid rbcL and nuclear 18S rRNA gene showed that the alga is nested within a clade containing a number of psychrotolerant strains within the Chloromonadinia phylogroup (Chlorophyceae). In the rbcL phylogeny, the alga formed an independent lineage, sister to the freshwater species Chloromonas paraserbinowii. Comparisons of secondary structure models of a highly variable ITS2 rDNA marker showed support for a distinct species identity for the new strain. The ITS2 secondary structure of the new isolate differed from the closest matches 'Chlamydomonas' gerloffii and Choloromonas reticulata by three and five compensatory base changes, respectively. Considering the morphological and molecular differences from its closest relatives, a new psychrotolerant species from the Arctic, Choromonas arctica sp. nov., is proposed.

Molecular phylogeny, systematics, and revision of the type species of Lobomonas, L. francei (Volvocales, Chlorophyta) and closely related taxa.

In the present study, three new strains of the rare volvocalean green alga Lobomonas were isolated from field-collected samples, one from Sardinia (Italy) and two from Argentina, and comparatively studied. The Sardinian and one of the Argentinian strains were identified as Lobomonas francei, the type species of the genus, whereas the second Argentinian strain corresponded to L. panduriformis. Two additional nominal species of Lobomonas from culture collections (L. rostrata and L. sphaerica) were included in the analysis and shown to be morphologically and molecularly identical to the L. francei strains. The presence, number, and shapes of cell wall lobes, the diagnostic criterion of Lobomonas, were shown to be highly variable depending on the chemical composition of the culture medium used. The analyses by SEM gave evidence that the cell wall lobes in Lobomonas originate at the junctions of adjacent cell wall plates by extrusion of gelatinous material. The four L. francei strains had identical nrRNA gene sequences and differed by only one or two substitutions in the ITS1 + ITS2 sequences. In the phylogenetic analyses, L. francei and L. panduriformis were sister taxa; however, another nominal Lobomonas species (L. monstruosa) did not belong to this genus. Lobomonas, together with taxa designated as Vitreochlamys, Tetraspora, and Paulschulzia, formed a monophyletic group that in the combined analyses was sister to the "Chlamydomonas/Volvox-clade." Based on these results, Lobomonas was revised, the diagnosis of the type species emended, a lectotype and an epitype designated, and several taxa synonymized with the type species.

Morphology and phylogeny of a new wall-less freshwater volvocalean flagellate, Hapalochloris nozakii gen. et sp. nov. (Volvocales, Chlorophyceae).

New strains of a wall-less unicellular volvocalean flagellate were isolated from a freshwater environment in Japan. Observations of the alga, described here as Hapalochloris nozakii Nakada, gen. et sp. nov., were made using light, fluorescence, and electron microscopy. Each vegetative cell had two flagella, four contractile vacuoles, and a spirally furrowed cup-shaped chloroplast with an axial pyrenoid, and mitochondria located in the furrows. Based on the morphology, H. nozakii was distinguished from other known wall-less volvocalean flagellates. Under electron microscopy, fibrous material, instead of a cell wall and dense cortical microtubules, was observed outside and inside the cell membrane, respectively. Based on the phylogenetic analyses of 18S rRNA gene sequences, H. nozakii was found to be closely related to Asterococcus, Oogamochlamys, Rhysamphichloris, and "Dunaliella" lateralis and was separated from other known wall-less flagellate volvocaleans, indicating independent secondary loss of the cell wall in H. nozakii. In the combined 18S rRNA and chloroplast gene tree, H. nozakii was sister to Lobochlamys.

A survey of Polytomella (Chlorophyceae, Chlorophyta) strains in public culture collections.

Polytomella is a genus of colorless green algae in the Reinhardtinia clade of the Chlamydomonadales, which has proven useful for a broad range of studies particularly those exploring the evolutionary loss of photosynthesis and mitochondrial genomics/biochemistry. Although 13 Polytomella strain accessions are currently available from public culture collections, the taxonomic status and redundancy of many of these strains is not clear because of possible mix-ups, deficient historical records, and incomplete molecular data. This study therefore considers previously available and/or new cox1 and mitochondrial DNA telomere sequences from all 13 Polytomella strain accessions. Among four of these, namely P. parva SAG 63-3, P. piriformis SAG 63-10, P. capuana SAG 63-5, and P. magna SAG 63-9, cox1 and mitochondrial telomere regions are both highly divergent between strains. All of the remaining nine Polytomella strain accessions have cox1 sequences that are identical to that of P. parva SAG 63-3 and although five of these have a mitochondrial telomere haplotype that is identical to that of P. parva SAG 63-3, the remaining four have one of three different haplotypes. Among the 10 strains with identical cox1 sequences, we suggest that three of the telomere haplotypes are associated with distinct geographical isolates of Polytomella and the fourth evolved from one of these isolates during 50 years of active culture.

Phylogenetic Position and Molecular Chronology of a Colonial Green Flagellate, Stephanosphaera pluvialis (Volvocales, Chlorophyceae), among Unicellular Algae.

The genus Balticola comprises a group of unicellular green flagellate algae and is composed of four species formerly classified in the genus Haematococcus. Balticola is closely related to a colonial green flagellate, Stephanosphaera pluvialis. Although the phylogeny among these genera was previously investigated based on two nuclear gene sequences, the phylogenetic sister of S. pluvialis has yet to be determined. In the present study, the species diversity of Balticola and Stephanosphaera was investigated using 18S rRNA gene sequences, and phylogenetic analyses of combined nuclear and chloroplast gene sequences were performed to understand the evolutionary origin of coloniality in Stephanosphaera. The divergence times of four colonial volvocalean flagellates from their respective unicellular sisters were also estimated. Six Balticola genotypes and a single Stephanosphaera genotype were recognized, and one Balticola genotype was resolved as the sister of S. pluvialis, showing that Balticola is a nonmonophyletic genus. The divergence time of Stephanosphaera from its nearest Balticola relative was estimated to be 4-63 million years ago, and these genera represent the most recently diverged pair of unicellular and colonial flagellates among the Volvocales.

Diversity of bacteria, archaea and protozoa in a perchlorate treating bioreactor.

A microbial consortium reducing high level of perchlorate was developed and in a fed batch bioreactor using acetate as substrate perchlorate was reduced at 0.25 g/g vss. day. Under stable performance, the microbial community structure of the reactor was analyzed through molecular and phenotypic methods. The diversity of bacteria and archaea were analyzed through whole cell Fluorescence In-Situ Hybridization (FISH) and PCR-Denaturing Gradient Gel Electrophoresis (DGGE), whereas higher trophic community was analyzed phenotypically. FISH analysis revealed the presence of alpha, beta, gamma and delta proteobacteria in the sludge, dominated by beta proteobacteria (68.7%). DGGE analysis of bacteria revealed the presence of a single known perchlorate reducing bacterium-Dechloromonas, nitrate reducers like Thaeura and Azoarcus and a number of other genera so far not reported as perchlorate or nitrate reducing. The archaea community was represented by an acetoclastic methanogen, Methanosaeta harundinacea. We have also observed the presence of an acetate consuming flagellate, Polytomella sp. in significant number in the reactor. Archaea and protozoa community in perchlorate treating bioreactor is reported first time in this study and point out further the significance of non perchlorate reducing but acetate scavenging microbial groups in acetate fed perchlorate treating reactors.

Validation of Polytomella piriformis nomen nudum (Chlamydomonadaceae): a Distinct Lineage Within a Genus of Nonphotosynthetic Green Algae.

Polytomella strain SAG 63-10 was first described by Pringsheim (1963) as Polytomella piriformis nomen nudum. The current study validates the name Polytomella piriformis following the International Code of Nomenclature for algae, fungi, and plants (ICN). We present 18S rRNA sequences of SAG 63-10 and several other Polytomella strains, which, along with existing mitochondrial DNA sequences, clearly distinguishes P. piriformis n. sp. from other available Polytomella species. The first type material of the species is presented, as well as an illustration and micrographs. Our own observations of P. piriformis SAG 63-10 are compared to Pringsheim's description and to descriptions of other valid Polytomella spp.

Multiple origins of colonial green flagellates from unicells: evidence from molecular and organismal characters.

Phylogenetic hypotheses generated from cladistic analysis of organismal and molecular data are shown to be generally congruent and/or complementary for comparisons of unicellular and colonial green algae in the Chlorophyceae. Cladistic analysis of organismal character data corroborates the alliance of colonial Stephanosphaera with unicellular Haematococcus (Haematococcaceae sensu Smith), inferred from previous studies of nuclear-encoded rRNA sequence data. The organismal data also support monophyly of the colonial Volvocaceae (sensu Smith). Alliances of other unicellular taxa, including those ascribed to the "Euchlamydomonas" Hauptgruppe (sensu Ettl), are not resolved by organismal characters principally because the structure of the data is skewed to shared ancestral characters (symplesiomorphies) and unique characters (autapomorphies) which define individual taxa only. Reanalysis of rRNA sequence data, with additional sequence data for critical taxa, does not support monophyly of the colonial Volvocaceae (sensu Smith). However, these data are weak in the support of the alternate hypothesis of nonmonophyly. In contrast, relationships among most unicellular flagellates are unambiguously resolved by the molecular data. Although the failure of the sequence data to resolve relationships among colonial flagellates appears to be due to a sampling of conservative sequences, an ancient, rapid radiation event or taxon sampling bias may also be contributing to the ambiguity problem. Results from analysis of a combined data set (organismal and molecular) are generally consistent with the inferences of the organismal character data regarding the colonial flagellates and are also consistent with the inferences of the sequence data regarding the unicellular taxa.

Molecular phylogeny of the volvocine flagellates.

Phylogenetic studies of approximately 2,000 bases of sequence from the large and small nuclear-encoded ribosomal RNAs are used to investigate the origins of the genus Volvox. The colonial and multicellular genera currently placed in the family Volvocaceae form a monophyletic group that is significantly closer phylogenetically to Chlamydomonas reinhardtii than it is to the other unicellular green flagellates that were tested, including Chlamydomonas eugametos, Chlorella pyrenoidosa, and Haematococcus lacustris. Statistical analysis of 251 phylogenetically informative nucleotide positions rejects the "volvocine lineage" hypothesis, which postulates a monophyletic evolutionary progression from unicellular organisms (such as Chlamydomonas), through colonial organisms (e.g., Gonium, Pandorina, Eudorina, and Pleodorina) demonstrating increasing size, cell number, and tendency toward cellular differentiation, to multicellular organisms having fully differentiated somatic and reproductive cells (in the genus Volvox). The genus Volvox appears not to be monophyletic. Volvox capensis falls outside a lineage containing other representatives of Volvox (V. aureus, V. carteri, and V. obversus), and both of these Volvox lineages are more closely related to certain colonial genera than they are to each other. This implies either a diphyletic origin of Volvox from different colonial volvocacean ancestors, a phylogenetic derivation of some of the colonial genera from a multicellular (i.e., Volvox) ancestor, or both. Considered together with previously published observations, these results suggest that the different levels of organizational and developmental complexity found in the Volvocaceae represent alternative stable states, among which evolutionary transitions have occurred several times during the phylogenetic history of this group.