The austral biflagellate Chloromonas rubroleosa (Chlorophyceae) is the closest relative of the unusual quadriflagellate genus Chlainomonas, both found in snow.

The quadriflagellate genus Chlainomonas frequently dominates red snow globally. It is unusual in several respects, with two separated pairs of flagella, apparent cell division via extrusion of cytoplasmic threads, and being nested phylogenetically within the biflagellate genus Chloromonas. Here, we showed that the austral species Chloromonas (Cr.) rubroleosa, originally described from Antarctic red snow, is a close biflagellate relative of Chlainomonas, challenging the monophyly of Chlainomonas as currently conceived. Sequences of the 18S rRNA gene robustly linked Cr. rubroleosa with near-identical environmental sequences from Antarctic red snow and Chlainomonas from North America, Japan, and Europe. Furthermore, the 18S rRNA and rbcL gene sequences of Cr. rubroleosa were almost identical to New Zealand and North American collections of Chlainomonas. Cr. rubroleosa and New Zealand Chlainomonas are separated by only a single-base substitution across the ITS1-5.8S-ITS2 rRNA loci (and according to ITS2, the North American collection is the next closest relative). This again raises the possibility that Chlainomonas is a life-cycle stage of vegetatively biflagellate organisms, although this remains confounded by the scarcity of biflagellates in field populations, the apparent cell division by quadriflagellates, and the absence of Chlainomonas-type cells in cultures of Cr. rubroleosa. The latter species is broadly similar to Chlainomonas, being poor at swimming, with similar pigment, chloroplast arrangement and ultrastructure, and is relatively large. Increased size is a feature of the wider clade of "Group D" snow algae. A synthesis of field and laboratory investigations may be needed to unravel the life cycle and correct the systematics of this group.

Lindavia intermedia (Bacillariophyceae) and Nuisance lake Snow in New Zealand: Chitin Content and Quantitative PCR Methods to Estimate Cell Concentrations and Expression of Chitin Synthase1.

Lake snow, caused by the freshwater centric diatom Lindavia intermedia, has become problematic in several large, oligotrophic New Zealand lakes over the past decade. Macroaggregates produced by L. intermedia foul fishing lines, intake screens, and water filters, and have a negative impact on recreational values. It was confirmed that the fibers constituting lake snow are composed of chitin, two chitin synthase genes (chs1 and 2) from L. intermedia were characterized, new qPCR-based tools to quantify the abundance of the species and measure expression of chs2 relative to the reference gene act1 (the product of which has cytoskeletal functions) were developed. The strong heterogeneity and mucilaginous nature of lake snow samples create particular difficulties for calibrations of gene or transcript copy numbers with cell densities and obtaining high yields of mRNA. However, data collected from four lakes during November 2018 and February and May 2019 show that abundance of L. intermedia is always high when lake snow is also abundant, but that a full range of L. intermedia abundance can occur when lake snow is absent, suggesting that chitin production is not obligate in L. intermedia. This result is consistent with the available data for chs2 expression, which suggest higher transcription when lake snow is abundant. Lake snow production by L. intermedia therefore requires an as yet undetermined stimulus independent of cell abundance.

Effect of culturing parameters on the vegetative growth of Haematococcus alpinus (strain lcr-cc-261f) and modeling of its growth kinetics.

The present study investigated the effect of different culture conditions on the vegetative growth of a new species, Haematococcus alpinus (strain LCR-CC-261f) using airlift photobioreactors. The influence of culture medium, aeration rates, CO2 concentration in air-gas mixture, temperature, light intensities, and wavelengths were investigated to achieve sustainable high cell density cultures. Growth parameters were determined by fitting the data to a form of the logistic equation that included a lag phase. The shear-sensitive vegetative cells favored lower aeration rates in the photobioreactors. MLA medium increased to 40 mM nitrate produced high density cultures. Temperatures between 12°C and 18°C, 3% (v/v) CO2 concentration and a narrow photon flux density ranging between 37 and 48 µmol photons · m-2  · s-1 were best suited for growth. The wavelength of the light source also impacted growth and a high cell density of 9.6 × 105 cells · mL-1 was achieved using a mixture of red and blue compared to warm white, red, or blue LEDs.

Improving phylogenetic inference of core Chlorophyta using chloroplast sequences with strong phylogenetic signals and heterogeneous models.

Phylogenetic relationships within the green algal phylum Chlorophyta have proven difficult to resolve. The core Chlorophyta include Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Pedinophyceae and Chlorodendrophyceae, but the relationships among these classes remain unresolved and the monophyly of Ulvophyceae and Trebouxiophyceae are highly controversial. We analyzed a dataset of 101 green algal species and 73 protein-coding genes sampled from complete and partial chloroplast genomes, including six newly sequenced ulvophyte genomes (Blidingia minima NIES-1837, Ulothrix zonata, Halochlorococcum sp. NIES-1838, Scotinosphaera sp. NIES-154, Caulerpa brownii and Cephaleuros sp. HZ-2017). We applied the Tree Certainty (TC) score to quantify the level of incongruence between phylogenetic trees in chloroplast genomic datasets, and show that the conflicting phylogenetic trees of core Chlorophyta stem from the most GC-heterogeneous sites. With removing the most GC-heterogeneous sites, our chloroplast phylogenomic analyses using heterogeneous models consistently support monophyly of the Chlorophyceae and of the Trebouxiophyceae, but the Ulvophyceae was resolved as polyphyletic. Our analytical framework provides an efficient approach to reconstruct the optimal phylogenetic relationships by minimizing conflicting signals.

A new Eocene fossil of the genus Phycopeltis (Ulvophyceae, Chlorophyta).

A fossil of the aerophytic green algal genus Phycopeltis (Trentepohliaes, Ulvophyceae) dated to 35 Ma, is reported from the Pikopiko Fossil Forest, Southland, New Zealand. Previous reports of fossilized Phycopeltis have been subsequently synonymized with fungi by other authors; however, our specimen is not vulnerable to their criticisms. Inflated cells present in two approximately concentric rings are interpreted as gametangia, with irregular structures resembling the gametangial pores of modern material; sporophytic material is absent. The fossil resembles the modern disc-forming species P. novae-zelandiae, P. expansa, and P. arundinacea. The limited material available prevents the assignation of a specific epithet, but the habit and dimensions of the fossil clearly fall within those of modern representatives of the genus. Its single cell thickness throughout, absence of distinct melanization, and larger size demonstrate that it is not a fungal shield. The specimen constitutes arguably the most convincing fossil belonging to Trentepohliales, and the first unambiguously for the genus Phycopeltis. It is consistent in age with other known fossils of the order that, when combined with molecular evidence, suggests a terrestrial radiation far more recent than that of land plants.

Inclusion of chloroplast genes that have undergone expansion misleads phylogenetic reconstruction in the Chlorophyta.

PREMISE OF THE STUDY: Chlorophytes comprise a substantial proportion of green plant diversity. However, sister-group relationships and circumscription of the classes Chlorophyceae, Trebouxiophyceae, and Ulvophyceae have been problematic to resolve. Some analyses support a sister relationship between the trebouxiophycean Leptosira and chlorophyceans, potentially altering the circumscription of two classes, also supported by a shared fragmentation in the chloroplast gene rpoB. We sought to determine whether the latter is a synapomorphy or whether the supporting analyses are vulnerable to systematic bias. METHODS: We sequenced a portion of rpoB spanning the fragmented region in strains for which it had not previously been sampled: four Chlorophyceae, six counterclockwise (CCW) group (ulvophyceans and trebouxiophyceans) and one streptophyte. We then explored the effect of subsampling proteins and taxa on phylogenetic reconstruction from a data set of 41 chloroplast proteins. KEY RESULTS: None of the CCW or streptophyte strains possessed the split in rpoB, including inferred near relatives of Leptosira, but it was found in all chlorophycean strains. We reconstructed alternative phylogenies (Leptosira + Chlorophyceae and Leptosira + Chlorellales) using two different protein groups (Rpo and Rps), both subject to coding-region expansion. A conserved region of RpoB remained suitable for analysis of more recent divergences. CONCLUSIONS: The Rps sequences can explain earlier findings linking Leptosira with the Chlorophyceae and should be excluded from phylogenetic analyses attempting to resolve deep nodes because their expansion violates the assumptions of substitution models. We reaffirm that Leptosira is a trebouxiophycean and that fragmentation of rpoB has occurred at least twice in chlorophyte evolution.

An estuarine species of the alga Vaucheria (Xanthophyceae) displays an increased capacity for turgor regulation when compared to a freshwater species.

Turgor regulation is the process by which walled organisms alter their internal osmotic potential to adapt to osmotic changes in the environment. Apart from a few studies on freshwater oomycetes, the ability of stramenopiles to turgor regulate has not been investigated. In this study, turgor regulation and growth were compared in two species of the stramenopile alga Vaucheria, Vaucheria erythrospora isolated from an estuarine habitat, and Vaucheria repens isolated from a freshwater habitat. Species were identified using their rbcL sequences and respective morphologies. Using a single cell pressure probe to directly measure turgor in Vaucheria after hyperosmotic shock, V. erythrospora was found to recover turgor after a larger shock than V. repens. Threshold shock values for this ability were >0.5 MPa for V. erythrospora and <0.5 MPa for V. repens. Recovery was more rapid in V. erythrospora than V. repens after comparable shocks. Turgor recovery in V. erythrospora was inhibited by Gd(3+) and TEA, suggesting a role for mechanosensitive channels, nonselective cation channels, and K(+) channels in the process. Growth studies showed that V. erythrospora was able to grow over a wider range of NaCl concentrations. These responses may underlie the ability of V. erythrospora to survive in an estuarine habitat and restrict V. repens to freshwater. The fact that both species can turgor regulate may indicate a fundamental difference between members of the Stramenopila, as research to date on oomycetes suggests they are unable to turgor regulate.

MOLECULAR PHYLOGENY OF ANTARCTIC PRASIOLA (PRASIOLALES, TREBOUXIOPHYCEAE) REVEALS EXTENSIVE CRYPTIC DIVERSITY(1).

Trebouxiophytes of the genus Prasiola are well known in Antarctica, where they are among the most important primary producers. Although many aspects of their biology have been thoroughly investigated, the scarcity of molecular data has so far prevented an accurate assessment of their taxonomy and phylogenetic position. Using sequences of the chloroplast genes rbcL and psaB, we demonstrate the existence of three cryptic species that were previously confused under Prasiola crispa (Lightfoot) Kützing. Genuine P. crispa occurs in Antarctica; its presence was confirmed by comparison with the rbcL sequence of the type specimen (from the Isle of Skye, Scotland). Prasiola antarctica Kützing is resurrected as an independent species to designate algae with gross morphology identical to P. crispa but robustly placed in a separate lineage. The third species is represented by specimens identified as P. calophylla (Carmichael ex Greville) Kützing in previous studies, but clearly separated from European P. calophylla (type locality: Argyll, Scotland); this alga is described as P. glacialis sp. nov. The molecular data demonstrated the presence of P. crispa in Maritime and Continental Antarctica. P. antarctica was recorded from the Antarctic Peninsula and Shetland Islands, and P. glacialis from the Southern Ocean islands and coast. Such unexpected cryptic diversity highlights the need for a taxonomic reassessment of many published Antarctic records of P. crispa. The results also indicate that marine species of Prasiola form a well-supported monophyletic group, whereas the phylogenetic diversity of freshwater species is higher than previously suspected (at least three separate lineages within the genus include species living in this type of environments).

Identification and characterization of freshwater algae from a pollution gradient using rbcL sequencing and toxicity testing.

One approach in toxicity testing using microalgae is to assess the modulation of light energy absorbed as a result of exposure to contaminants. In this study, four strains of microalgae were isolated to obtain a variety of taxa for testing from sites receiving various levels of environmental stressors around Christchurch, New Zealand. The strains were characterized by partially sequencing rbcL, a routinely used gene in plant phylogenetics with a large existing database of strains. Based on morphological observation and gene sequences, the strains were identified as Chlorella sp., Neochloris sp., and Choricystis minor. The isolates were exposed to the herbicide glyphosate and the metal zinc, and their responses were measured using the ToxY-PAM system. Chlorella sp. was the most sensitive. Two strains of Choricystis minor were isolated from different ponds in an effluent gradient at a sewage treatment plant. Analysis of variance indicated that the isolate from the least contaminated pond was more sensitive to zinc (although regression analysis did not show this result). This suggests that the selective pressure exerted on algal strains by a contamination gradient over short a distance is detectable by both genetic and physiological methods, with implications for sourcing appropriate indicator organisms from the environment.