Julie A. Buchheim

PHYLOGENY OF THE CHLOROPHYCEAE WITH SPECIAL REFERENCE TO THE SPHAEROPLEALES: A STUDY OF 18S AND 26S rDNA DATA

Ultrastructural analyses of the flagellar apparatus suggested that Sphaeroplea, Atractomorpha, the Hydrodictyaceae, and the Neochloridaceae, all of which produce biflagellate motile cells with directly opposed (DO) basal bodies, are allied in an order Sphaeropleales. Recent studies of 18S rDNA sequence data supported an alliance of the DO group, but no data from Sphaeroplea and its allies were included. This investigation presented a test of the phylogenetic hypothesis suggested by the flagellar apparatus evidence using sequence data from the nuclear‐encoded small‐subunit rDNA (18S) and large subunit rDNA (26S) genes, combined with additional taxon sampling. Results from phylogenetic analyses weakly supported monophyly of biflagellate DO taxa and indicated that pyrenoids with cytoplasmic invaginations are present in numerous distinct lineages. Analysis of both molecular data sets supported a class Chlorophyceae comprised of at least six major groups that generally correspond to currently recognized orders or families: Chaetophorales, Chae‐ topeltidales, Chlamydomonadales, Sphaeropleales, Sphaeropleaceae, and Oedogoniales. In addition, Cylindrocapsa, Elakatothrix, Treubaria, and Trochiscia formed a seventh chlorophycean clade that is new to science. This investigation demonstrated that the 26S rDNA gene provides more phylogenetic signal, per unit sequence, than the 18S rDNA gene and that combined analysis yields topologies with more robust support than independent analysis of either data set.

Halotolerant Aerobic Heterotrophic Bacteria from the Great Salt Plains of Oklahoma

The Salt Plains National Wildlife Refuge (SPNWR) near Cherokee, Oklahoma, contains a barren salt flat where Permian brine rises to the surface and evaporates under dry conditions to leave a crust of white salt. Rainfall events dissolve the salt crust and create ephemeral streams and ponds. The rapidly changing salinity and high surface temperatures, salinity, and UV exposure make this an extreme environment. The Salt Plains Microbial Observatory (SPMO) examined the soil microbial community of this habitat using classic enrichment and isolation techniques and phylogenetic rDNA studies. Rich growth media have been emphasized that differ in total salt concentration and composition. Aerobic heterotrophic enrichments were performed under a variety of conditions. Heterotrophic enrichments and dilution plates have generated 105 bacterial isolates, representing 46 phylotypes. The bacterial isolates have been characterized phenotypically and subjected to rDNA sequencing and phylogenetic analyses. Fast-growing isolates obtained from enrichments with 10% salt are predominantly from the gamma subgroup of the Proteobacteria and from the low GC Gram-positive cluster. Several different areas on the salt flats have yielded a variety of isolates from the Gram-negative genera Halomonas, Idiomarina, Salinivibrio, and Bacteroidetes. Gram-positive bacteria are well represented in the culture collection including members of the Bacillus, Salibacillus, Oceanobacillus, and Halobacillus.

Phylogenetic analysis of the ‘Nannochloris-like’ algae and diagnoses of Picochlorum oklahomensis gen. et sp. nov. (Trebouxiophyceae, Chlorophyta)

Abstract A broadly halotolerant new isolate of a small asexual coccoid chlorophyte and six new, related freshwater isolates provided the impetus for a phylogenetic analysis of the so-called ‘Nannochloris-like’ algae within the Trebouxiophyceae. Previous taxonomic disagreements concerning this group had not been rigorously tested with molecular phylogenetic analyses. We show with 1 8S ribosomal DNA (rDNA) sequence phylogeny that 19 of 22 isolates previously assigned to either Nannochloris or Nanochlorum fall within a diverse sister clade to a clade including the four ‘true’ Chlorella species sensu lato. In addition, Marvania geminata, Gloeotila contorta, Chlorella sp. Yanaqocha RA1, Koliella spiculiformis, ‘Chlorella minutissima’ C-1. 1.9, and new Koliella, Gloeotila and Marvania isolates were included in the Nannochloris-like clade. Distinct freshwater and marine or saline lineages comprise at least three major subclades, generally corresponding to cell division pattern. Seven of 14 marine or saline isolates are known (and the others presumed) to divide by autosporulation. Eight freshwater isolates divide by binary fission, including two Koliella, two Gloeotila, N. bacillaris, Chlorella sp. Yanaqocha RA1, and two new unassigned isolates. Four freshwater isolates divide by budding or autosporulation (three Marvania, including CCAP 251/1b, previously assigned to N. coccoides). The autosporic taxa N. eucaryotum UTEX 2502 (marine) and C. minutissima C-1.1.9 (freshwater), which have nearly identical 18S rDNA sequences, are deeper-branching than the freshwater and marine or saline lineages. We propose including the 13 marine or saline, autosporic taxa (excluding N. eucaryotum UTEX 2502) in the new genus Picochlorum until distinctive morphological or biochemical characters are identified that would indicate multiple genera corresponding to subclades. Such characters exist in the freshwater lineages, supporting retention of Koliella, Gloeotila, Marvania and Nannochloris as distinct genera, although each is currently represented by few isolates. Nannochloris at this time may be restricted to N. bacillaris and Chlorella sp. Yanaqocha RA1. We also describe halotolerant P. oklahomensis Hironaka sp. nov. Based on 18S rDNA sequence and lack of chlorophyll b, Nannochloris sp. UTEX 2379 should be reassigned to the Eustigmatophyceae.

PHYLOGENY OF THE HYDRODICTYACEAE (CHLOROPHYCEAE): INFERENCES FROM rDNA DATA1

The hydrodictyacean green algal lineage has been the focus of much research due to the fossil record of at least some members, their ornamented cell walls, and their distinctive reproductive strategies. The phylogeny of the family was, until recently, exclusively morphology based. This investigation examines hydrodictyacean isolates from several culture collections, focusing on sequences from ribosomal data: 18S rDNA, 26S rDNA (partial), and internal transcribed spacer (ITS)‐2 data. Results from phylogenetic analyses of independent and combined data matrices support the Hydrodictyaceae as a monophyletic lineage that includes isolates of Chlorotetraedron, Hydrodictyon, Pediastrum, Sorastrum, and Tetraedron. Phylogenetic analyses of rDNA data indicate that the three‐dimensional coenobium of Hydrodictyon is evolutionarily distinct from the three‐dimensional coenobium of Sorastrum. The more robust aspects of the ITS‐2 data corroborate the 18S+26S rDNA topology and provide a structural autapomorphy for the Hydrodictyaceae and Neochloridaceae, that is, an abridgment of helix IV in the secondary structure. The rDNA data do not support monophyly of Pediastrum but rather suggest the existence of four additional hydrodictyacean genera: Monactinus, Parapediastrum, Pseudopediastrum, and Stauridium.

DNA-Repair Potential of Halomonas spp. from the Salt Plains Microbial Observatory of Oklahoma

The Great Salt Plains (GSP), an unvegetated, barren salt flat that is part of the Salt Plains National Wildlife Refuge near Cherokee, Oklahoma, is the site of the Salt Plains Microbial Observatory. At the GSP the briny remains of an ancient sea rise to the surface, evaporate under dry conditions, and leave crusts of white salt. Adaptation to this environment requires development of coping mechanisms providing tolerance to desiccating conditions due to the high salinity, extreme temperatures, alkaline pH, unrelenting exposure to solar UV radiation, and prevailing winds. Several lines of evidence suggest that the same DNA repair mechanisms that are usually associated with UV light or chemically induced DNA damage are also important in protecting microbes from desiccation. Because little is known about the DNA repair capacity of microorganisms from hypersaline terrestrial environments, we explored the DNA repair capacity of microbial isolates from the GSP. We used survival following exposure to UV light as a convenient tool to assess DNA repair capacity. Two species of Halomonas (H. salina and H. venusta) that have been isolated repeatedly from the GSP were chosen for analysis. The survival profiles were compared to those of Escherichia coli, Pseudomonas aeruginosa, and Halomonas spp. from aquatic saline environments. Survival of GSP organisms exceeded that of the freshwater organism P. aeruginosa, although they survived no better than E. coli. The GSP isolates were much more resistance to killing by UV than were the aquatic species of Halomonas reported in the literature [Martin et al. (2000) Can J Microbiol 46:180−187]. Unlike E. coli, the GSP isolates did not appear to have an inducible, error-prone repair mechanism. However, they demonstrated high levels of spontaneous mutation.

Phylogeny of chloromonas (chlorophyceae): A study of 18S ribosomal RNA gene sequences

The unicellular, biflagellate genus Chloromonas differs from its ally, Chlamydomonas, primarily by the absence of pyrenoids in the vegetative stage of the former. As with most green flagellate genera, little is known about phylogenetic affinities within and among Chloromonas species. Phylogenetic analyses of nuclear‐encoded small‐subunit ribosomal RNA gene sequences demonstrate that a sampling of five Chloromonas taxa, obtained from major culture collections, do not form a monophyletic group. However, only three of these isolates, Chloromonas clathrata, Chloromonas serbinowi, and Chloromonas rosae, are diagnosable morphologically as Chloromonas species by the absence of a pyrenoid in the vegetative stage. The three diagnosable Chloromonas taxa form an alliance with two pyrenoid‐bearing chlamydomonads, Chlamydomonas augustae and Chlamydomonas macrostellata. With the exception of Chloromonas serbinowi, which represents the basal lineage within the clade, each of the diagnosable Chloromonas taxa and their pyrenoidbearing Chlamydomonas allies were isolated originally from mountain soils, snow, or cold peat. These observations suggest that habitat, independent of pyrenoid status, may be most closely linked to the natural history of this clade of chlamydomonad flagellates.

PHYLOGENY OF LOBOCHARACIUM (CHLOROPHYCEAE) AND ALLIES: A STUDY OF 18S AND 26S rDNA DATA1

The phylogenetic affinities of Lobocharacium coloradoense were investigated by analysis of combined 18S and 26S rDNA data. Results from both parsimony and likelihood methods supported a close alliance among Lobocharacium, Characiosiphon, and Characiochloris. These three taxa formed a clade near the base of the “Dunaliella” group within the chlamydomonad lineage. Protosiphon, which exhibits a siphonous habit similar to Characiosiphon and Lobocharacium, was not resolved as a close ally of the latter two taxa. The Lobocharacium alliance was characterized by the presence of an attachment pad associated with the nonmotile vegetative stage and pyrenoids that possess cytoplasmic invaginations. The pyrenoid feature is an ultrastructural trait that has now been observed in five different chlorophycean lineages. The Lobocharacium–Characiosiphon–Characiochloris clade is not predicted by any classifications of green algae. Additional taxon and data sampling need to be completed to resolve inconsistencies between the molecular phylogenetic evidence and at least some of the current family‐level taxa.

The blood alga: phylogeny of Haematococcus (Chlorophyceae) inferred from ribosomal RNA gene sequence data

The status of the green algal genera Haematococcus and Stephanosphaera has been a source of debate among algal systematists. A phylogenetic alliance between Haematococcus (sensu lato) and the colonial Stephanosphaera was affirmed by earlier molecular phylogenetic investigations. Although the data suggested that the genus Haematococcus may not be a monophyletic group, taxon sampling limited the scope of any potential taxonomic revision. Results from new molecular phylogenetic analyses of data from the 18S and 26S rRNA genes support the establishment of a separate genus, Balticola, as originally proposed by Droop in 1956. Haematococcus remains as a valid genus, with H. pluvialis as its only member. The monotypic status of H. pluvialis is supported both by molecular phylogenetic analyses of the ribosomal RNA genes and assessments of molecular evolution in the ITS2 sequences of H. pluvialis strains. The near-complete absence of compensatory base changes in a sequence-structure analysis of the highly variable ITS2 gene from more than 40 geographically diverse isolates of H. pluvialis corroborates the unity of the species inferred from molecular phylogenetic analyses of 18S and 26S rRNA gene sequence data.

HYPERSALINE SOIL SUPPORTS A DIVERSE COMMUNITY OF DUNALIELLA (CHLOROPHYCEAE)1

Numerous isolates of the green halophile Dunaliella were studied as part of a survey of microbial diversity at the Great Salt Plains (GSP) in Oklahoma, USA. The GSP is a large (∼65 km2) salt flat with extreme temporal and spatial fluctuations in salinity and temperature. Although the flagellate halophile Dunaliella is common worldwide, nearly all cultured isolates are from saline habitats that are primarily aquatic rather than primarily terrestrial. The diverse GSP Dunaliella strains exhibit three morphotypes: a predominantly motile form, a motile form with a prominent palmelloid phase (nonmotile, mucilage rich), and a palmelloid form with a weakly motile phase. All had broad salinity optima well below typical in situ salinities at the GSP, and two of the palmelloid isolates grew as well in freshwater as in highly saline media. Molecular phylogenetic and evolutionary analyses revealed that Dunaliella from the GSP (and two similar habitats in the Great Basin, USA) are allied with D. viridis Teodor. but possess phylogenetic diversity in excess of existing global isolates from aquatic habitats. In addition, isolates from primarily terrestrial habitats exhibit statistically higher rates of nucleotide substitution than the phylogenetically homogeneous set of primarily aquatic Dunaliella taxa. We hypothesize that dynamically extreme saline soil habitats may select for different and more diverse Dunaliella lineages than more stable saline aquatic habitats. We also propose Dunaliella as a tractable microbial model for in situ testing of evolutionary and phylogeographic hypotheses.

AN UNRECOGNIZED ANCIENT LINEAGE OF GREEN PLANTS PERSISTS IN DEEP MARINE WATERS 1

We provide molecular phylogenetic evidence that the obscure genera Palmophyllum Kütz. and Verdigellas D. L. Ballant. et J. N. Norris form a distinct and early diverging lineage of green algae. These palmelloid seaweeds generally persist in deep waters, where grazing pressure and competition for space are reduced. Their distinctness warrants recognition as a new order, the Palmophyllales. Although phylogenetic analyses of both the 18S rRNA gene and two chloroplast genes (atpB and rbcL) are in agreement with a deep‐branching Palmophyllales, the genes are in conflict about its exact phylogenetic placement. Analysis of the nuclear ribosomal DNA allies the Palmophyllales with the prasinophyte genera Prasinococcus and Prasinoderma (Prasinococcales), while the plastid gene phylogeny placed Palmophyllum and Verdigellas as sister clade to all other Chlorophyta.