Marvin W. Fawley

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.

Molecular diversity among communities of freshwater microchlorophytes

Current hypotheses on the distribution of freshwater microchlorophytes lead to predictions of low diversity and wide environmental tolerances. Thus, the same few species should be found worldwide in many different habitats. However, these hypotheses are based on a morphospecies concept, which precludes the possibility of numerous cryptic species among these organisms. In this study, we examined the diversity of coccoid green microalgae and chlamydomonads (Chlorophyta) isolated from sites in Minnesota and North Dakota (USA) using techniques of 18S rDNA sequence analysis. Of 93 distinct 18S rDNA sequences identified from among 273 isolates examined by molecular techniques, all but four are new to science. The spatial distribution of organisms represented by these 18S rDNA sequences was not uniform, because some lakes and ponds yielded distinct 18S rDNA types not found at other sites. In addition, organisms generally considered to be cosmopolitan, such as Chlamydomonas reinhardtii and Chlorella vulgaris, were not found. These results challenge predictions of low species number and wide environmental tolerances among these eukaryotic microorganisms.

A SIMPLE AND RAPID TECHNIQUE FOR THE ISOLATION OF DNA FROM MICROALGAE

A simple method for the purification of PCR‐ quality DNA from microalgae is presented. This method uses the detergent dodecyltrimethylammonium bromide coupled with cell breakage by agitation in the presence of glass beads and chloroform. A final purification step involves a commercial cartridge system. The procedure requires only about 1–2 mL of algal culture and can be completed in about 20 min. DNA suitable for PCR has been obtained from several algal lineages using this method, including numerous green algae and stramenopiles.

Phylogenetic analyses of 18s rdna sequences reveal a new coccoid lineage of the prasinophyceae (Chlorophyta)

Phylogenetic analyses of 18S rDNA sequences from 25 prasinophytes, including 10 coccoid isolates, reveals that coccoid organisms are found in at least three prasinophyte lineages. The coccoid Ostreococcus tauri is included in the Mamiellales lineage and P ycnococcus provasolii is allied with the flagellate P seudoscourfieldia marina. A previously undescribed prasinophyte lineage is comprised of the coccoid Prasinococcus cf. capsulatus (CCMP 1407) and other isolates tentatively identified as Prasinococcus sp. (CCMP 1202, CCMP 1614, and CCMP 1194), as well as three unnamed coccoids (CCMP 1193, CCMP 1413, and CCMP 1220). No flagellate organisms are known from this lineage. Organisms of this new lineage share some characteristics of both the Pycnococcaceae and the Mamiellales, although relationships among these separate lineages were not supported by bootstrap analyses. An additional unnamed coccoid isolate (CCMP 1205) is separate from all major prasinophyte lineages. The analyses did not resolve the relationships among the major prasinophyte lineages, although they support previous conclusions that the Prasinophyceae are not monophyletic.

Diversity and ecology of small coccoid green algae from Lake Itasca, Minnesota, USA, including Meyerella planktonica, gen. et sp. nov

M.W. Fawley, K.P. Fawley and H.A. Owen. 2005. Diversity and ecology of small coccoid green algae from Lake Itasca, Minnesota, USA, including Meyerella planktonica, gen. et sp. nov. Phycologia 44: 35–48. We characterized 81 isolates of picoplankton and other small planktonic coccoid green algae from Lake Itasca, Itasca State Park, MN, USA. Sequence analysis and examination by light microscopy revealed three common algae: Pseudodictyosphaerium/Korshpalmella spp. (Chlorophyceae), Choricystis spp. (Trebouxiophyceae) and a previously undescribed autosporic coccoid. The Pseudodictyosphaerium and Korshpalmella species that were evaluated all possessed identical 18S ribosomal DNA (rDNA) and rbcL sequences, but this 18S rDNA sequence differed from the published 18S rDNA sequence for P. jurisii isolated from Europe. These chlorophycean algae were isolated from samples collected from Lake Itasca during all seasons. The 18S rDNA sequence of some of the Itasca isolates of Choricystis was nearly identical to the published sequence for C. minor; however, six different rbcL sequences were detected among the Choricystis isolates and none of these rbcL sequences matched published sequences for isolates from several lakes in Europe. The different rbcL sequence types of Choricystis had distinct seasonal distributions in Lake Itasca. These results extend our knowledge of the distribution of Pseudodictyosphaerium and Choricystis and suggest that Choricystis is even more diverse than previously thought. In addition, the distinct sequences of marker genes for Lake Itasca isolates indicate that these organisms have diverged from the European isolates. The previously undescribed alga isolated from Lake Itasca is a sister taxon to the trebouxiophyte lineage that includes Chlorella vulgaris, but differs from true Chlorella species in that it lacks a pyrenoid. Based upon this difference and molecular analyses, these isolates are described as Meyerella planktonica, gen. et sp. nov. (Trebouxiophyceae, Chlorophyta). All M. planktonica isolates examined possessed identical 18S rDNA, rbcL and ribosomal internal transcribed spacer sequences. Meyerella planktonica was isolated almost exclusively from winter and spring samples.

Changes in the intraisolate genetic structure of Beet necrotic yellow vein virus populations associated with plant resistance breakdown.

The causal agent of rhizomania disease, Beet necrotic yellow vein virus (BNYVV), typically produces asymptomatic root-limited infections in sugar beets (Beta vulgaris) carrying the Rz1-allele. Unfortunately, this dominant resistance has been recently overcome. Multiple cDNA clones of the viral pathogenic determinant p25, derived from populations infecting susceptible or resistant plants, were sequenced to identify host effects on the viral population structure. Populations isolated from compatible plant-virus interactions (susceptible plant-wild type virus and resistant plant-resistant breaking variants) were large and relatively homogeneous, whereas those from the incompatible interaction (resistant plant-avirulent type virus) were small and highly heterogeneous. All populations from susceptible plants had the same dominant haplotype, whereas those from resistant cultivars had a different haplotype surrounded by a spectrum of mutants. Selection and diversification analyses suggest an evolutionary trajectory of BNYVV with positive selection for changes required to overcome resistance, followed by elimination of hitchhiking mutations through purifying selection.

EVALUATING THE MORPHOSPECIES CONCEPT IN THE SELENASTRACEAE (CHLOROPHYCEAE, CHLOROPHYTA)1

Isolates of the genera Monoraphidium Kom.‐Legn., Ankistrodesmus Corda and Raphidocelis Hindák emend. Marvan et al. were cultured from two areas in Minnesota and North Dakota, USA. These isolates were identified to species level (when possible), using light microscopy and standard monographs and then characterized by 18S rDNA sequence analysis. Phylogenetic analyses indicated that in some cases, 18S rDNA sequences from these isolates were very similar, but not identical to the sequences of other isolates of the same morphospecies from different parts of the world. However, some isolates that were identified as the same species actually belong to different lineages within the Selenastraceae, whereas other isolates with identical or nearly identical 18S rDNA sequences possessed rather different morphologies. Overall, our data suggest that the application of a broad morphospecies concept to the Selenastraceae has resulted in an underestimation of the species diversity of this family and probably erroneous conclusions about the distribution of species.

Winter phytoplankton community structure in three shallow temperate lakes during ice cover

The general model of seasonal phytoplankton succession in temperate lakes suggests that winter phytoplankton growth is minimal under ice-cover. However, some studies have found diverse phytoplankton communities during winter. The primary objectives of this study were to determine the species composition and the changes in the winter phytoplankton community structure under the ice. For 2 consecutive winters, phytoplankton samples were collected under ice-cover at 4 sites on 3 lakes in Arrowwood National Wildlife Refuge (ANWR), near Pingree, North Dakota. Ninety taxa were identified and enumerated. Densities of several of these taxa frequently exceeded 106 cells l−1. The winter phytoplankton communities in these lakes were dominated by flagellates, principally cryptomonads, a synurophyte (Synura uvella), small chrysophytes (Chrysococcus spp., Kephyrion spp.) and a dinoflagellate (Peridinium aciculiferum), as well as non-flagellate microchlorophytes (Monoraphidium spp., Ankistrodesmus spp., and Pseudodictyosphaerium sp.), a cyanobacterium (Gloeocapsa aeruginosa) and centric diatoms (Stephanodiscus minutulus, S. parvus and Cyclotella meneghiniana).

PHOTOSYNTHETIC PIGMENTS OF PSEUDOSCOURFIELDIA MARINA AND SELECT GREEN FLAGELLATES AND COCCOID ULTRAPHYTOPLANKTON: IMPLICATIONS FOR THE SYSTEMATICS OF THE MICROMONADOPHYCEAE (CHLOROPHYTA)1

Photosynthetic pigments of the green flagellate Pseudoscourfieldia marina (Throndsen) Manton (Micromonadophyceae) are similar to those of the coccoid Pycnococcus provasolii Guillard; prasinoxanthin is the predominant carotenoid. Other organisms that possess prasinoxanthin also possess additional pigments not found in either P. marina or P. provasolii. Uriolide, a xanthophyll previously described from the coccoid done URI 266G, was also found in Mantoniella squamata (Manton et Parke) Desikachary, Micromonas pusilla Manton et Parke and Mamiella gilva (Parks et Rayns) Moestrup, all flagellate members of the Mamiellales, and the coccoid clone IV E5G. Other unidentified carotenoids were also present in M. squamata, M. pusilla, and M. gilva. These results suggest that P. marina and the coccoid organisms URI 266G and IV E5G may be related to the Mamiellales, and that P. provasolii may be more closely related to P. marina than to M. squamata, M. pusilla, and M. gilva.

The diversity of Scenedesmus and Desmodesmus (Chlorophyceae) in Itasca State Park, Minnesota, USA

J.L. Johnson, M.W. Fawley and K.P. Fawley. 2007. The diversity of Scenedesmus and Desmodesmus (Chlorophyceae) in Itasca State Park, Minnesota, USA. Phycologia 46: 214–229. DOI: 10.2216/05-69.1 Species of Scenedesmus and Desmodesmus are ubiquitous in freshwater ecosystems. However, phenotypic plasticity coupled with the minute variations between named species make the identification of taxa by light microscopy extremely difficult, especially from field samples. As a result, the actual level of diversity and distributions of these genera are not known. In this study, the diversity of Scenedesmus and Desmodesmus from Itasca State Park (ISP), Minnesota, USA was studied using culture techniques followed by light microscopy and internal transcribed spacer (ITS-2) rDNA sequence analysis. From 100 characterized isolates of Scenedesmus, Desmodesmus and allied isolates, 34 different sequence types were detected, 24 of which are different enough from each other to correspond to distinct taxa. This diversity greatly exceeds that described for these genera in previous studies of ISP algae using light microscopy alone. The sequences of five ISP isolates identically matched those of previously described species or isolates, and sequences of six ISP isolates differed by eight bases or less from those of previously described species. The isolation of Desmodesmus species from Itasca State Park with sequences identical to those from isolates from highly disjunct locations around the world suggests that these species are widely distributed. Results from this study also indicate that isolates with very similar (< 8 substitutions) ITS-2 sequences tend to have only very slight morphological variation. We conclude that culturing and ITS-2 sequence analysis is an effective technique for investigating species level diversity in Scenedesmus and Desmodesmus.