Phylogenetic analysis of the SSU rRNA from members of the Chrysophyceae

The nucleotide sequence for the nuclear-encoded small subunit ribosomal RNA gene (SSU rRNA) was determined for 24 species of the Chrysophyceae sensu stricto. These sequences were aligned, using primary and secondary structure, with nine previously published sequences for the Chrysophyceae, 14 for the Synurophyceae, and five for the Eustigmatophyceae (outgroup). Data analyses were the substitution rate calibration distance method using neighbor-joining (TREECON), Kimura 2-parameter neighbor-joining method (PAUP) and the maximum parsimony method (PAUP, PHYLIP). Trees from the analyses were largely congruent, but bootstrap support was weak at many nodes. The analyses recovered clades of uniflagellate and biflagellate organisms associated with current higher level taxonomy (e.g., subclass, order). The genus Ochromonas was polyphyletic, and O. tuberculata in particular was distantly related to the other Ochromonas species in the analysis. The family Paraphysomonadaceae occupied a basal position in three of four analyses. The class Synurophyceae appeared to be embedded within the Chrysophyceae, but bootstrap support was weak (< 50%) in all analyses except the PHYLIP parsimony analysis (= 81%). It was considered premature to place the Synurophyceae back into the Chrysophyceae based upon the analysis of one gene, especially given the ultrastructural and pigment differences between the two groups, but the relationship of these two groups deserves further study.     

39 Phylogeny of aulacoseira (bacillariophyta)

Ochromonas sensu lato is the largest genus in the Chrysophyceae, containing over 100 names. Ochromonas species are biflagellate, naked, plastid-bearing single cells, distinguished from loricate, scaled, colonial and colorless genera. Most, if not all, species of Ochromonas are mixotrophic, i.e., they photosynthesize but they also engulf bacteria and other small prey. Preliminary evidence from SSU rRNA sequences show that Ochromonas is a polyphyletic genus. Ochromonas tuberculata is the most distinct from all other Ochromonas species. The other Ochromonas species (examined thus far) are scattered in three clades. For example, O. danica and O. sphaerocystis are sister to Poterioochromonas stipitata and P. malhamensis . Four additional species (identified by light microscopy as O. elegans Doflein, O. globosa Skuja, O. ovalis Dolfein, O. sociabilis Pringheim ) have SSU rRNA sequences identical to P. malhamensis . Of these, only O. sociabilis has been transferred to Poterioochromonas . Thus, at least some species may be synonymous with others. Two clades of marine species are also known, one containing coastal species and the other containing open ocean species. A number of genera (some also polyphyletic) are interspersed amongst the Ochromonas species (e.g., Chrysolepidomonas, Chrysonephele, Chrysoxys, Cyclonexis, Dinobryon, Epipyxis, Uroglena, Uroglenopsis ). The goal of this research (just beginning) is to establish a monophyletic Ochromonas , probably by assigning some species to other genera (existing or new). One major problem is that the type species, O. triangulata Vysotskii , hasn't been observed in over 100 years, and it is unclear which of several clades of Ochromonas contains the type. Results will be discussed.     

Differential thermal adaptation of clonal strains of a protist morphospecies originating from different climatic zones

Eco-physiological variation and local adaptation are key issues in microbial ecology. Here, we investigated the thermal adaptation of 19 strains of the same Spumella morphospecies (Chrysophyceae, Heterokonta). In order to test for local adaptation and the existence of specific ecotypes we analysed growth rates of these strains, which originated from different climate regions. We applied temperature-adaptation as an eco-physiological marker and analysed growth rates of the different Spumella strains at temperatures between 0 degrees C and 35 degrees C. The temperatures allowing for maximal growth of strains from temperate and warm climatic zones ranged between 19.9 degrees C and 33.4 degrees C. Phylogenetically, most of these 'warm'-adapted strains fall into two different previously defined 18S rDNA Spumella clusters, one of them consisting of mostly soil organisms and the other one being a freshwater cluster. As a rule, the 'warm'-adapted strains of the soil cluster grew slower than the 'warm'-adapted isolates within the freshwater cluster. This difference most probably reflect different strategies, i.e. the formation of cysts at the expense of lower growth rates in soil organisms. In contrast, as expected, all isolates from Antarctica were cold-adapted and grew already around melting point of freshwater. Surprisingly, optimum temperature for these strains was between 11.8 degrees C and 17.7 degrees C and maximum temperature tolerated was between 14.6 degrees C and 23.5 degrees C. Our data indicate that despite the relatively high optimal temperature of most Antarctic strains, they may have a relative advantage below 5-10 degrees C only. Based on the thermal adaptation of the flagellate strains the Antarctic strains were clearly separated from the other investigated strains. This may indicate a limited dispersal of flagellates to and from Antarctica. Even if the latter assumption needs support from more data, we argue that the high levels of eco-physiological and molecular microdiversity indicate that the current species concepts do not sufficiently reflect protist eco-physiological differentiation.     

Particle handling during interception feeding by four species of heterotrophic nanoflagellates

High resolution video-microscopy was used to observe grazing patterns of the heterotrophic nanoflagellates Cafeteria roenbergensis, Bodo saltans, Spumella sp., and Ochromonas sp. Spumella and Ochromonas enclose food particles with pseudopodia while Cafeteria and Bodo engulf particles by invagination of the cell surface. The following parameters of the feeding process were quantified: frequency of flagellar beating, speed of particles in different positions of the feeding current, food size selection, feeding rate, and the time budget for the handling of particles. The mean handling times differed between 94 s for Cafeteria and 4 s for Ochromonas for ingested particles. Handling times for ingested particles were significantly longer than for non-captured particles. Long handling times were calculated to be disadvantageous only for flagellates which propel a high water volume per hour (esp. Ochromonas) or live in a bacteria-rich environment. Our model calculations may provide a reasonable theoretical explanation for a concentration-dependent behavioural variability of the feeding strategy of different heterotrophic nanoflagellates (HNF) species.     

Confusing Selective Feeding with Differential Digestion in Bacterivorous Nanoflagellates

Food selectivity and the mechanisms of food selection were analyzed by video microscopy for three species (Spumella, Ochromonas, Cafeteria) of interception-feeding heterotrophic nanoflagellates. The fate of individual prey particles, either live bacteria and/or inert particles, was recorded during the different stages of the particle-flagellate-interaction, which included capture, ingestion, digestion, and egestion. The experiments revealed species-specific differences and new insights into the underlying mechanisms of particle selection by bacterivorous flagellates. When beads and bacteria were offered simultaneously, both particles were ingested unselectively at similar rates. However, the chrysomonads Spumella and Ochromonas egested the inert beads after a vacuole passage time of only 2-3 min, which resulted in an increasing proportion of bacteria in the food vacuoles. Vacuole passage time for starved flagellates was significantly longer compared to that of exponential-phase flagellates for Spumella and Ochromonas. The bicosoecid Cafeteria stored all ingested particles, beads as well as bacteria, in food vacuoles for more then 30 min. Therefore "selective digestion" is one main mechanism responsible for differential processing of prey particles. This selection mechanism may explain some discrepancies of former experiments using inert particles as bacterial surrogates for measuring bacterivory.     

A rapid method for differentiating Saccharomyces sensu stricto strains from other yeast species in an enological environment

During programs for the selection of enological yeasts, several hundred natural isolates are usually screened. The scope of these operations is to isolate strains possessing good fermentative properties without necessarily arriving at a precise species designation: in other words, to detect strains belonging to the Saccharomyces sensu stricto complex. In the present study, a pair of primers, designed within the variable D1/D2 region of the 26S subunit of ribosomal yeast RNA, have been constructed. These generate an amplification fragment of 471 bp that is specific for the seven Saccharomyces sensu stricto species, while no signal was obtained for Saccharomyces sensu lato strains (17 species) or for another 18 selected species commonly found in enological environments. A second pair of primers was also constructed, within the 18S rRNA gene, composed of perfectly conserved sequences common for all 42 yeast species examined, which generate a 900 bp (c.) band for all strains. This was used as a positive experimental control in multiplex PCR analysis using all four primers.     

Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history.     

The Influence of Preculture Conditions and Food Quality on the Ingestion and Digestion Process of Three Species of Heterotrophic Nanoflagellates

The influence of prey characteristics such as motility and size as well as of predator characteristics such as satiation and preculturing diet on the feeding process of interception feeding heterotrophic nanoflagellates was investigated. Three species of gram-negative bacteria, one species of gram-positive bacteria, two species of cyanobacteria (Synechococcus) and inert latex particles were fed as prey particles for three species of heterotrophic nanoflagellates (Spumella, Ochromonas, Cafeteria). Ingestion rates depended on the satiation of the flagellates and especially on the filling status of the food vacuoles. In addition, the ingestion rates depended on the characteristics of the food particle and were modified by pre-culturing the flagellates on either Pseudomonas putida or Bacillus subtilis. Digestion was found to be particle-specific. Cyanobacteria were excreted a few minutes after ingestion whereas heterotrophic bacteria were stored and digested in the food vacuoles. The spectrum of ingested particles is not identical to that of digested particles and thus neither the diet of the flagellates nor their impact on bacterial communities can be calculated simply from food vacuole content. "Selective digestion" could be shown to be an important selection mechanism concerning natural food particles. The digestion strategies of Cafeteria on the one hand and Spumella and Ochromonas on the other hand may be an important factor to explain protozoan species composition and succession in the field. In addition to bacterial abundance and grazing pressure by metazooplankton, the bacterial speciescomposition as well as biochemical variations within bacterial species may influence protozoan species composition and abundance.     

Phylogenetic diversity and whole-cell hybridization of oxymonad flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes

SSU rRNA genes of oxymonad protists from the hindgut of the wood-feeding termite Reticulitermes flavipes were PCR-amplified using a newly designed oxymonad-specific forward primer and a newly designed reverse primer specific for termite gut flagellates. After cloning, the clone library was sorted into four groups by RFLP analysis and nearly full-length SSU rRNA gene sequences were obtained for representative clones from each group. Phylogenetic analysis revealed that sequences of all four groups formed a monophyletic cluster with the only other existing SSU rRNA gene sequence of oxymonads. Using whole-cell hybridization with clone-specific fluorescently labeled probes, each of the four clone groups could be assigned to a specific morphotype, which were identified as Dinenympha gracilis, Dinenympha fimbriata, and so-far undescribed species of Pyrsonympha and Dinenympha. Our results demonstrate that the morphological variety of oxymonads is not caused by the presence of different developmental stages of the same organism, but that the various morphotypes represent different species.     

Novel eukaryotic lineages inferred from small-subunit rRNA analyses of oxygen-depleted marine environments

Microeukaryotes in oxygen-depleted environments are among the most diverse, as well as the least studied, organisms. We conducted a cultivation-independent, small-subunit (SSU) rRNA-based survey of microeukaryotes in suboxic waters and anoxic sediments in the great Sippewisset salt marsh, Cape Cod, Mass. We generated two clone libraries and analyzed approximately 300 clones, which contained a large diversity of microeukaryotic SSU rRNA signatures. Only a few of these signatures were closely related (sequence similarity of >97%) to the sequences reported earlier. The bulk of our sequences represented deep novel branches within green algae, fungi, cercozoa, stramenopiles, alveolates, euglenozoa and unclassified flagellates. In addition, a significant number of detected rRNA sequences exhibited no affiliation to known organisms and sequences and thus represent novel lineages of the highest taxonomical order, most of them branching off the base of the global phylogenetic tree. This suggests that oxygen-depleted environments harbor diverse communities of novel organisms, which may provide an interesting window into the early evolution of eukaryotes.     

Impact of cultivation on characterisation of species composition of soil bacterial communities

The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates. The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples. Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium. In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively. Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group. However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria. In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus. In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones. A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively. All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample. Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences. This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated during direct analysis of the same soil and from a wide range of other sample types studied elsewhere. The study demonstrates the value of fine-scale molecular analysis for identification of laboratory isolates and indicates the culturability of approximately 1% of the total population but under a restricted range of media and cultivation conditions.     

Antigenic, phenotypic and molecular characterization confirms Babesia odocoilei isolated from three cervids

Babesia isolates from an elk (Cervus elaphus canadensis) and a caribou (Rangifer tarandus caribou) with fatal infections were compared to Babesia odocoilei (Engeling isolate) from white-tailed deer (Odocoileus virginianus) by experimental infection, serologic, and small subunit ribosomal RNA (SSU rRNA) gene sequence analysis studies. Both the indirect fluorescent antibody test and immunoprecipitation assays demonstrated antigenic variation among the isolates. Experimental infection studies showed no clinical differences among the isolates. Nucleotide sequence analysis showed that the elk and caribou Babesia sp. isolates possessed SSU rRNA genes with identical sequences to that of B. odocoilei. A phylogenetic tree constructed from SSU rRNA gene sequences shows that B. odocoilei is most closely related to Babesia divergens, both of which branch together in the true babesia clade.     

Phylogeny of lobose amoebae based on actin and small-subunit ribosomal RNA genes

Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyze actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the "Gymnamoebia sensu stricto" and the Archamoebae (pelobionts + entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.     

Unexpected effects of prey dimensions and morphologies on the size selective feeding by two bacterivorous flagellates (Ochromonas sp. and Spumella sp.)

Current models on protistan size-selective feeding assume that contact probability is the factor that largely explains observed food preferences. Contact probability is generally expected to be positively correlated with prey size and therefore to explain observed food selection for larger prey items. We critically tested these basic assumptions on size-selective feeding using the interception-feeding chrysomonad nanoflagellates Ochromonas sp. and Spumella sp. Mechanisms of differential feeding were studied during distinct stages of the selection process (i.e. contact probability, capture efficiency, ingestion efficiency, and differential digestion) by means of high-resolution video microscopy. Food selection was investigated using a mixture of microspheres ranging from 0.3-2.2 microm in diam., as well as a mixed bacterial community. In contrast to current model assumptions, the contact probability was highest for microspheres of intermediate size (0.9-1.2 microm), but was not generally positively correlated with prey size over the whole prey size range. Capture and ingestion also proved to be involved in size selection: these patterns were also independent of the food concentration (p = 0.968 for Ochromonas, p = 0.971 for Spumella). Even though the capture rate was significantly higher for attached flagellates than for swimming flagellates (p < 0.001), size selectivity was not affected (p > 0.05). Our results indicate that: (i) size selection is not actively regulated by these flagellates, but is a passive process; (ii) contact probability is not generally positively correlated with prey size, but shows a maximum for intermediate-sized prey in the prey size spectrum of 0.3-2.2 microm; and (iii) selection steps other than contact probability are crucial for size selection and should be integrated in models on size selection.     

The structure of extrusive organelles in alveolate and other heterotrophic flagellates

The structure of ejective organelles (extrusomes) in 12 species of heterotrophic flagellates belonging to 8 taxonomic groups is considered. Trichocysts of various structures have been found in colpodellids, Spumella and Metromonas. Kinetocysts of amoeboid flagellates, thaumatomonads, contain a cylindrical element. These kinetocysts are attached to bacteria after discharge. When being discharged, trichocysts of colpodellids produce filaments which have transversal striation. Discobolocysts have been found in excavate flagellate Reclinomonas, and ejectisomes--in Goniomonas. Toxicysts have been marked in carnivorous alveolate flagellate Colponema. The most of studied extrusomes lie inside the cytoplasm, while exstrusomes in chrysomonad Spumella are also found inside the nucleus. Trichocysts of colpodellids and Metromonas as well as ejectisomes of Goniomonas are located near the branches of Golgi apparatus. The comparison of the data obtained confirms the hypothesis about the correlation of taxonomic position of flagellates and the structure of their extrusomes that allows in some cases using these features as phylogenetic markers.     

Mesostigmatophyceae, a new class of streptophyte green algae revealed by SSU rRNA sequence comparisons

Complete nuclear-encoded SSU rRNA sequences have been obtained from three taxa of streptophyte green algae (Klebsormidium nitens, Nitella capillaris, Chaetosphaeridium globosum) and two strains of the scaly green flagellate Mesostigma viride. Phylogenetic analyses of 70 taxa of Viridiplantae (Chlorophyta and Streptophyta) and 57 taxa of streptophyte green algae and embryophyte plants using distance, parsimony and likelihood methods revealed a novel monophyletic lineage among the Streptophyta comprising the genera Mesostigma and Chaetosphaeridium. This lineage is described here as the Mesostigmatophyceae classis nova. Our analyses demonstrate that (1) scaly green flagellates (prasinophytes) are polyphyletic, (2) a scaly green flagellate is a member of the Streptophyta and forms a clade with the oogamous, filamentous Chaetosphaeridium to the exclusion of all other known streptophyte green algae, (3) a previously published SSU rRNA sequence of Chaetosphaeridium (AF113506) is chimeric and contains part of a fungal SSU rRNA, and (4) the phylogenetic relationships between the Mesostigmatophyceae and other streptophyte green algae remain unresolved by SSU rRNA sequence comparisons.     

Ultrastructure and taxonomy of Paraphysomonas (Chrysophyceae) and related genera 3

New observations are presented on the internal ultrastructure of the scale–bearing chrysophycean genera Chromophysomonas, Chrysosphaerella , the new genus Polylepidomonas and 15 species of Paraphysomonas. These data show that the pigmented genera Chromophysomonas, Chrysosphaerella and Polylepidomonas have a generally similar internal structure and that their taxonomic separation is based only on differences in scale structure. The structure of Paraphysomonas resembles that of these genera but the cells always possess a leucoplast rather than a chloroplast. In cell structure, the pigmented genera resemble the naked genus Ochromonas while Paraphysomonas resembles Spumella , the colourless counterpart of Ochromonas. Evaluation of the differences between these genera and the scale–bearing genera Mallomonas and Synura has led to the conclusion that Chromophysomonas, Chrysosphaerella, Polylepidomonas and Paraphysomonas should no longer be classified within the family Mallomonadaceae. The new family Paraphysomonadaceae is established to include Chrysophyceae with an Ochromonas type of cell structure but which also produce silica scales.     

Polyphasic taxonomy of Aspergillus fumigatus and related species

The variability within Aspergillus fumigatus Fresenius and related species was examined using macro-, micro-morphology, growth temperature regimes and extrolite patterns. In addition, DNA analyses including partial beta-tubulin, calmodulin and actin gene sequences were used. Detailed examination of strains, considered as A. fumigatus earlier, showed that they could be divided into four groups including A. fumigatus sensu stricto, A. lentulus and two new species. The intraspecific genetic variability within A. fumigatus sensu stricto was low, the sequence differences among 23 strains of the species was at most two bases in each partial beta-tubulin and calmodulin gene. However, intraspecific morphological diversity within the species was high and delineation of the species was equivocal. Therefore, beta-tubulin and calmodulin gene sequences could be critical determinants for the delineation of the A. fumigatus sensu stricto species. A. lentulus including isolates from clinical origin, Korean soil and from a dolphin clustered into an isolated group based on beta-tubulin, calmodulin and actin gene sequences, differing from A. fumigatus by morphological characters, growth temperature and extrolite profile. A. lentulus produces the extrolites auranthine, cyclopiazonic acid, a dimeric indole of unknown structure, neosartorin, some pyripyropens, terrein and some tryptoquivalins and tryptoquivalons. Two pair of isolates (CBS 117194, 117186 and 117520, 117519) clustered into separate groups from A. fumigatus and the other Aspergillus section Fumigati species, including the teleomorph Neosartorya, are proposed as two new species. A. fumigatiaffinis spec. nov. produces the extrolites auranthine, cycloechinulin, helvolic acid, neosartorin, palitantin, pyripyropens, tryptoquivalins and tryptoquivalons, and A. novofumigatus spec. nov. produces the extrolites cycloechinuline, helvolic acid, neosartorin, palitantin and terrein.     

Protist genetic diversity in the acidic hydrothermal environments of Lassen Volcanic National Park, USA

We examined eukaryote genetic diversity in the hydrothermal environments of Lassen Volcanic National Park (LVNP), Northern California. We sampled hydrothermal areas of the Bumpass Hell, Sulfur Works, Devil's Kitchen, and Boiling Springs Lake sites, all of which included diverse acidic pools, mud pots, and streams with visible algal mats and biofilms. Temperatures varied from 15 to 85 degrees C and pH from 1.7 to 5.8. DNA extraction methods compared by denaturing gradient gel electrophoresis fingerprinting exhibited similar patterns, and showed limited diversity of eukaryotic small subunit (SSU) rRNA genes compared with prokaryotes. We successfully amplified eukaryotic SSU rRNA genes from most environments up to 68 degrees C. Cloned rDNA sequences reveal acidophilic protists dominate eukaryotes in LVNP hydrothermal environments. Most sites showed phototrophic assemblages dominated by chlorophytes and stramenopiles (diatoms and chrysophytes). Heterotrophic taxa, though less abundant, included diverse alveolates (ciliates), amoebae, and flagellates. Fungi were also found at most sites, and metazoans (hexapods, nematodes, platyhelminths) were sometimes detected in less acidic environments, especially in algal mats. While many cloned rDNA sequences showed 95%-99% identity to known acidophilic isolates or environmental clones from other acidic sites (Rio Tinto), sequence diversity generally declined both with decreasing pH and increasing temperature, and both were controlling physical variables on the abundance and distribution of organisms at our sites. However, a pool at 68 degrees C with pH 1.7 yielded the greatest number of distinct sequences. While some were likely contaminants from nearby cooler sites, we suggest that Lassen's acidic hydrothermal features may harbor novel protists.     

Comparative morphology and phylogenetic placement of two microsclerotial black fungi from Sphagnum

Capnobotryella renispora and Scleroconidioma sphagnicola form black, irregularly shaped microsclerotia that are indistinguishable in gross morphology on leaves of Sphagnum fuscum. In culture, microsclerotia of these fungi were similar, in that mature component cells possessed thick, highly melanized cell walls, poorly defined organelles, large lipid bodies and simple septa. They were different in morphogenesis, in the way their component cells were organized and in disseminative propagules. Microsclerotia of S. sphagnicola formed phialidic conidiogenous cells on their surface, whereas in C. renispora, adjacent cells in mature microsclerotia often separated from each other by septum schizolysis and formed chlamydospores. The identification of C. renispora from Sphagnum is provisional despite a 100% ITS sequence match with data for a culture derived from the type strain. No holoblastic, reniform conidia typical of the species were formed in nature or in culture, and the SSU sequence for a separately preserved culture of the ex-type strain was markedly divergent. Parsimony analyses of nuclear ribosomal DNA sequences showed that these two fungi were related to separate orders of Dothideomycetes. Both SSU and ITS data supported a close relationship for S. sphagnicola to the Dothideales sensu stricto, while the closest ITS match was to Rhizosphaera spp. In the SSU analyses, C. renispora was nested within the Capnodiales.