Phil M. Novis

MOLECULAR PHYLOGENY OF ANTARCTIC PRASIOLA (PRASIOLALES, TREBOUXIOPHYCEAE) REVEALS EXTENSIVE CRYPTIC DIVERSITY1

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).

Two genetic and ecological groups of Nostoc commune in Victoria Land, Antarctica, revealed by AFLP analysis

Microscopy, DNA sequencing, and amplified fragment length polymorphism (AFLP) were used to examine variation within Nostoc commune from collections between 72 and 78°S in Victoria Land, Antarctica. Although there is considerable bias of collected material towards southern latitudes, and this material varies greatly in age (collected between 1984 and 2004), an important new phylogeographic pattern was found. DNA sequencing of the tRNAleu(UAA) region, used recently to define form species N. commune, revealed little variation between collections. AFLP analysis, however, split the collected material according to habitat (irrigated soil communities versus ponds), rather than latitude. These results suggest that environmental factors linked to latitude are not the greatest drivers of genetic variation in Victoria Land. These may operate at a lower level but would require intensive sampling within narrowly defined habitat types at a range of latitudes to uncover. We advocate extensive sampling across local environmental gradients based on water availability, comparative culturing, and development of sequence characterised amplified regions (SCARs) across a range of latitudes in future seasons of the Latitudinal Gradient Project.

Parallela Flint: its phylogenetic position in the Chlorophyceae and the polyphyly of Radiofilum Schmidle

Novis P.M., Lorenz M., Broady P.A. and Flint E.A. 2010. Parallela Flint: its phylogenetic position in the Chlorophyceae and the polyphyly of Radiofilum Schmidle. Phycologia 49: 373–383. DOI: 10.2216/09-65.1 Analysis of chloroplast protein sequences from the genes atpB, rbcL and psaB demonstrates that the ribbon-forming freshwater alga Parallela novae-zelandiae is a member of the Chlorophyceae. We reject earlier classifications of Parallela as a member of the Tetrasporales or Gloeotilales and also confirm the splitting of the latter order. Molecular analysis shows that it is closely related to Radiofilum transversale but not R. conjunctivum, the latter associating with green algae having counterclockwise-displaced flagellar apparatuses. Since R. conjunctivum is the type species of the genus, we refer R. transversale to Parallela transversale. The two species of Parallela share a ribbon-forming mucilaginous habit and an absence of pyrenoids and layered mucilage. Parallela novae-zelandiae differs from P. transversale in lacking bipartite cell walls and has consistently multiseriate filaments, which are only sporadically present in P. transversale and are entirely absent in R. conjunctivum and other described species of Radiofilum. Radiofilum conjunctivum also contains pyrenoids. The bipartite wall condition has apparently evolved more than once during the evolution of the Chlorophyta.

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.

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 Gd3+ 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.

Microbiology of Eutrophic (Ornithogenic and Hydrocarbon-Contaminated) Soil

Antarctic soils are typically low in carbon, nitrogen and phosphorus. Ornithogenic and hydrocarbon-contaminated soils, however, could be considered eutrophic. In this chapter, we review the microbial composition of ornithogenic and hydrocarbon-contaminated soils. Ornithogenic soils form in soils under bird nesting sites. These include those that form under penguin colonies of coastal soils and under bird nests in coastal soils and on nunataks. The soils currently occupied by birds have high levels of C, N and P and a low C:N ratio. The diversity and abundance of microbes in the soils depends on whether they are currently occupied by birds have been abandoned or are adjacent to the colony. Bacteria dominate occupied soils with Firmicutes reported to be prevalent, but in abandoned soils, Proteobacteria are dominant. Among the nematodes Panagrolaimus is most commonly reported from ornithogenic soils. Extensive areas of growth of visible photosynthetic microbes occupy soils adjacent to ornithogenic soils. Prasiola spp and Phormidium spp. are the dominant algae and cyanobacteria, respectively, that are reported. Most investigations of hydrocarbon-contaminated Antarctic soils have focused on heterotrophic bacteria, with a few reports of fungi. Hydrocarbon spills on soils typically occur next to research stations and result in an increase in soil C and a high C:N ratio. The result is a shift in microbial communities towards hydrocarbon-degrading species, predominantly from the Proteobacteria phylum. Among the hydrocarbon-degrading bacteria isolated from Antarctic soils are members of the genera Pseudomonas, Sphingomonas and Rhodococcus. These genera have been observed widely in contaminated temperate soils and have the capability to degrade hydrocarbons. Filamentous fungi from the Ascomycota phylum commonly isolated from contaminated soils include those from the Cadophora, Trichoderma and Mortierella genera, but their ability to degrade hydrocarbons is not always known. There is limited knowledge on the effect of hydrocarbons on Archaea, invertebrates or photosynthetic microbes in hydrocarbon-contaminated Antarctic soil. Our knowledge of eutrophic soils of Antarctica is sparse.

The diatom Lindavia intermedia identified as the producer of nuisance pelagic mucilage in lakes

ABSTRACT Populations of a centric diatom that produces copious extracellular polymeric substance (EPS), known as ‘lake snow’, have developed in several large microtrophic lakes in New Zealand over the past 10 years. The EPS coats fishing lines and blocks water filters. The phenomenon was first noticed in Lake Wanaka in the early 2000s and has recently been reported in Lakes Coleridge and Wakatipu, with single, isolated historical events occurring in Lakes Waikaremoana (confirmed) and Benmore (presumed). The species has been reported from a handful of other lakes in New Zealand, all except one of which are microtrophic-to-oligotrophic. Light and ultrastructural microscopic studies of New Zealand populations, DNA sequencing and comparison with published descriptions identify the causative species as Lindavia intermedia, part of the ‘bodanicoid’ complex. These species are best known from the Northern Hemisphere where they are regarded as confined to nutrient-poor habitats, frequently having disappeared from European lakes as the lakes underwent eutrophication. Lake snow is known from a small number of other lakes in the Northern Hemisphere, but no evidence has been reported linking L. intermedia to the production of lake snow in these lakes. The expected growth characteristics (slow and at depth) of L. intermedia pose difficulties for any prospective containment campaign.

Aquatic nitrogen-fixing cyanobacteria associated with blooms of Didymosphenia geminata: insights from a field study

The nuisance diatom Didymosphenia geminata has recently increased in abundance worldwide and has spread through oligotrophic rivers in the South Island of New Zealand. However, it remains absent from the North Island. Its proliferation in oligotrophic environments has prompted work on phosphorus acquisition, but potential nitrogen acquisition pathways have received little attention. We partially sequenced the nifD gene, encoding a component of nitrogenase, from South Island samples containing D. geminata and compared its diversity with that of North Island sites independently ranked as high, medium and low risk for infestation. Godleyacean cyanobacteria were present in all four South Island provinces investigated, and also in three North Island sites rated medium or high risk. Most high-risk sites were dominated by Nostoc sequences, as were some infested South Island sites. Monte Carlo simulations and the weighted UniFrac metric showed that nifD diversity in low-risk North Island sites differed significantly from sites rated medium and high risk, and from infested South Island sites; however, random resampling showed that the data were insufficient to separate didymo-infested and high-risk sites from medium-risk sites. The relationships we discovered suggest that success of D. geminata in New Zealand could be mediated by indigenous nitrogen-fixing cyanobacteria.

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.