MOLECULAR PHYLOGENY AND PHENOTYPIC VARIATION IN THE HETEROTROPHIC GREEN ALGAL GENUS PROTOTHECA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

Species of the heterotrophic green microalgal genus Prototheca and related taxa were phylogenetically analyzed based on the nuclear small subunit (SSU) and the 5′ end of the large subunit (LSU) rRNA gene (rDNA) sequences. We propose restricting the genus Prototheca to the four species: P. moriformis Krüger, P. stagnora (Cooke) Pore, P. ulmea Pore, and P. zopfii Krüger. The main diagnostic feature of these taxa is the absence of growth on trehalose.Of these, it was suggested that P. moriformis should be merged into P. zopfii; P. moriformis and three varieties of P. zopfii constituted a paraphyletic assemblage with estimated short evolutionary distances. The trehalose‐assimilating strains (Prototheca wickerhamii Tubaki et Soneda strains and Auxenochlorella protothecoides (Krüger) Kalina et Pun?ochá?ová SAG 211‐7a), together with an invertebrate pathogen Helicosporidium sp., diverged before the radiation of the four species of Prototheca in the SSU rDNA and composite (SSU rDNA plus LSU rDNA) analyses. Comparison between the results from physiological data in this work (fermentative pattern) and those described earlier (growth requirements) lead us to propose a hypothesis that the phenotypic variation, which did not represent diagnostic characters for species delimitation, may reflect the history of genetic diversification within the genus Prototheca as inferred from rDNA sequence characters.     

A further phylogenetic study of the heterotrophic dinoflagellate genus, Protoperidinium (Dinophyceae) based on small and large subunit ribosomal RNA gene sequences

The heterotrophic marine dinoflagellate genus Protoperidinium is the largest genus in the Dinophyceae. Previously, we reported on the intrageneric and intergeneric phylogenetic relationships of 10 species of Protoperidinium, from four sections, based on small subunit (SSU) rDNA sequences. The present paper reports on the impact of data from an additional 5 species and, therefore, an additional two sections, using the SSU rDNA data, but now also incorporating sequence data from the large subunit (LSU) rDNA. These sequences, in isolation and in combination, were used to reconstruct the evolutionary history of the genus. The LSU rDNA trees support a monophyletic genus, but the phylogenetic position within the Dinophyceae remains ambiguous. The SSU, LSU and SSU + LSU rDNA phylogenies support monophyly in the sections Avellana, Divergentia, Oceanica and Protoperidinium, but the section Conica is paraphyletic. Therefore, the concept of discrete taxonomic sections based on the shape of 1′ plate and 2a plate is upheld by molecular phylogeny. Furthermore, the section Oceanica is indicated as having an early divergence from other groups within the genus. The sections Avellana and Excentrica and a clade combining the sections Divergentia/Protoperidinium derived from Conica‐type dinoflagellates independently. Analysis of the LSU rDNA data resulted in the same phylogeny as that obtained using SSU rDNA data and, with increased taxon sampling, including members of new sections, a clearer idea of the evolution of morphological features within the genus Protoperidinium was obtained. Intraspecific variation was found in Protoperidinium conicum (Gran) Balech, Protoperidinium excentricum (Paulsen) Balech and Protoperidinium pellucidum Bergh based on SSU rDNA data and also in Protoperidinium claudicans (Paulsen) Balech, P. conicum and Protoperidinium denticulatum (Gran et Braarud) Balech based on LSU rDNA sequences. The common occurrence of base pair substitutions in P. conicum is indicative of the presence of cryptic species.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

Isolation, characterization, and fermentative pattern of a novel thermotolerant Prototheca zopfii var. hydrocarbonea strain producing ethanol and CO2 from glucose at 40 °C

A novel thermotolerant strain of the achlorophyllous micro-alga Prototheca was isolated from a hot spring. The isolate was found to produce an appreciable amount of ethanol and CO2 from glucose under anoxic conditions at both 25 and 40 degrees C; this type of alcohol fermentation has not yet been reported in the genus Prototheca. Moreover, it also evolved gas from sucrose after a time lag at 40 degrees C. Its taxonomic characteristics coincided with those of Prototheca zopfii var. hydrocarbonea, and phylogenetic analysis, based on a small-subunit (SSU) rDNA sequence, also revealed a close relationship between the two strains. D-lactic acid, ethanol, CO2 and a trace of acetic acid were produced from glucose, but L-lactic acid, formic acid, and H2 were not. At 25 degrees C, D-lactic acid and ethanol were produced in approximately equimolar amounts under N2/H2/CO2, whereas ethanol production was predominant under N2. More ethanol was produced at 40 degrees C than at 25 degrees C irrespective of the gas composition in the atmosphere. This is the first report on gas production from glucose and on the changes in the fermentative patterns as a function of temperature for the genus Prototheca.     

In vitro acitivity of ribostamycin against Prototheca sp.

The in vitro activity of ribostamycin against algae of the genus Prototheca was evaluated by minimum inhibitory concentration (MIC) studies on solid media. Concentrations of 4 mcg/ml were required to inhibit 100% of the P. zopfii strains; 16 mcg/ml inhibited 100% of the P. stagnora strains and 95% of the P. wickerhamii strains. These values are inferior to plasma concentrations obtained after injection of ribostamycin. It is likely that this antibiotic could be effective in the treatment of protothecosis in man.     

淡水刚毛藻目一新组合种及其系统发育分析

作者于2012-2014年先后在湖北省采集到4株刚毛藻目的丝状绿藻。经鉴定,其形态特征和莫拉瓦刚毛藻Cladophora moravica Gardavsk(1986)非常相似;但相较于刚毛藻属各个种的形态,4株藻体更接近黑孢藻科的分类特征。基于SSU和LSU rDNA序列构建的系统发育树显示,4株刚毛藻目的丝状绿藻均属于黑孢藻科拟湖球藻属(Aegagropilopsis)。据此,作者建议将该种名称作为一个新组合处理,即莫拉瓦拟湖球藻Aegagropilopsis moravica(Dvoˇrák)Zhao et Liu com.nov。拟湖球藻属仅含有3个种:莫拉瓦拟湖球藻、硬枝拟湖球藻和棒状拟湖球藻,其形态特征与湖球藻相近且易混淆,故采用核基因组序列分析方法区分二者是非常有效的。     

Appearance of Colonies of Prototheca on CHROMagar Candida medium

The microorganisms capable of producing opportunist infections include the yeast-like organisms of the genus Candida, and the unicellular algae of the genus Prototheca, which share common features and can, therefore, lead to confusion. Their colonies are almost identical and they grow in the same culture media used routinely in mycology. CHROMagar Candida is a new chromogenic differential isolation medium that facilitates the presumptive differentiation of some of the most clinically important yeast-like organisms. To our knowledge, the use of CHROMagar Candida with Prototheca spp. has not been reported in the literature. This report describes the growth of 151 strains of Prototheca on CHROMagar Candida compared to the growth of a total of 326 well-characterized yeast organisms of the genera Candida, Cryptococcus, Trichosporon, Geotrichum, and Saccharomyces. It is clinically relevant to note that algae of the genus Prototheca (P. wickerhamii, P. zopfii, and P. stagnora) and of the genus Candida parapsilosis produced similar cream-colored colonies on CHROMagar Candida medium. Based on their growth on CHROMagar, a new species of Candida is described, C. zeylanoides, which has blue-green colonies. The colonies of two species of Trichosporon are also differentiated: the blue-green colonies of T. beigelii and the pink colonies of T. capitatum. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cryptic Speciation in the Genus Cryptoglena (Euglenaceae) Revealed by Nuclear and Plastid SSU and LSU rRNA Gene

The photosynthetic euglenoid genus Cryptoglena is differentiated from other euglenoid genera by having a longitudinal sulcus, one chloroplast, two large trough‐shaped paramylon plates positioned between the chloroplast and pellicle, and lack of metaboly. The genus contains only two species. To understand genetic diversity and taxonomy of Cryptoglena species, we analyzed molecular and morphological data from 25 strains. A combined data set of nuclear SSU and LSU and plastid SSU and LSU rRNA genes was analyzed using Bayesian, maximum likelihood, maximum parsimony, and distance (neighbor joining) methods. Although morphological data of all strains showed no significant species‐specific pattern, molecular data segregated the taxa into five clades, two of which represented previously known species: C. skujae and C. pigra, and three of which were designated as the new species, C. soropigra, C. similis, and C. longisulca. Each species had unique molecular signatures that could be found in the plastid SSU rRNA Helix P23_1 and LSU rRNA H2 domain. The genetic similarity of intraspecies based on nr SSU rDNA ranged from 97.8% to 100% and interspecies ranged from 95.3% to 98.9%. Therefore, we propose three new species based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

Detection of Helicosporidium spp. in metagenomic DNA

Distinct isolates of the invertebrate pathogenic alga Helicosporidium sp., collected from different insect hosts and different geographic locations, were processed to sequence the 18S rDNA and β-tubulin genes. The sequences were analyzed to assess genetic variation within the genus Helicosporidium and to design Helicosporidium-specific 18S rDNA primers. The specificity of these primers was demonstrated by testing not only on the Helicosporidium sp. isolates, but also on two trebouxiophyte algae known to be close Helicosporidium relatives, Prototheca wickerhamii and Prototheca zopfii. The genus-specific primers were used to develop a culture-independent assay aimed at detecting the presence of Helicosporidium spp. in environmental waters. The assay was based on the PCR amplification of 18SrDNA gene fragments from metagenomic DNA preparations, and it resulted in the amplification of detectable products for all sampled sites. Phylogenetic analyses that included the environmental sequences demonstrated that all amplification products clustered in a strongly supported, monophyletic Helicosporidium clade, thereby validating the metagenomic approach and the taxonomic origin of the produced environmental sequences. In addition, the phylogenetic analyses established that Helicosporidium spp. isolated from coleopteran hosts are more closely related to each other than they are to the isolate collected from a dipteran host. Finally, the phylogenetic trees depicted intergeneric relationships that supported a Helicosporidium-Prototheca cluster but did not support a Helicosporidium-Coccomyxa grouping, suggesting that pathogenicity to invertebrates evolved at least twice independently within the trebouxiophyte green algae.     

Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.     

Identification of the Prototheca Species by Immunofluorescence

Studies were carried out to develop fluorescent antibody reagents for the identification of the Prototheca species and for their differentiation from morphologically similar fungi of various genera in formalin-fixed tissues. Antisera against representative isolates of P. filamenta, P. moriformis, P. stagnora, P. wickerhamii, and P. zopfii were produced in rabbits. Antiglobulins, labeled with fluorescein-isothiocyanate that intensely stained most cells of the homologous species, were selected for use as potential diagnostic reagents. By adsorbing the conjugates with selected heterologous cross-staining protothecae, reagents that were both sensitive and specific were obtained. Evaluation of the adsorbed conjugates with sections of tissue infected with protothecae, sections of tissue infected with morphologically similar fungi, and cultures of protothecae showed that these reagents are useful for the rapid and reliable identification of the Prototheca species.     

基于核内核糖体小亚基序列的蝗总科系统发育关系分析

用核糖体SSURdna全序列对蝗总科（Acridoidea）进行了分子系统学研究。依据测定的8种蝗虫的SSU Rdna全序列 (平均 1.844 bp),并从GenBank中选取了6种内群种类和2种外群种类的SSU Rdna同源序列,进行序列分析。利用Clustal、MEGA 和 PHYLIP 软件构建分子系统树（距离邻接法Neighbor-Joining,NJ;最小进化法 Minimum Evolution）。结果显示： (1) 蝗总科是一个单系类群;(2) 锥头蝗科（Chrotogonidae）和瘤锥蝗科（Pyrgomorphidea）亲缘关系较近,为蝗总科最原始的类群;(3) 网翅蝗科（Arcypteridae）和槌角蝗科（Gomphoceridae）有较近的亲缘关系; (4) 斑翅蝗科 （Oedipodidae）为最进化的类群; (5) SSU Rdna序列保守性强,转换transition）取代的速率大于或接近颠换（transversion）取代的速率;(6) 在系统树中,总科首先分离,大多数同科不同属的类群以高置信度聚合在一起,说明SSU Rdna序列适合用于蝗总科的系统发育关系分析。     

一株淡水多甲藻的形态观察和多甲藻属系统发育分析

通过形态学方法将中国淡水藻种库一株编号为FACHB-329的甲藻鉴定为楯形多甲藻不等变种Peridinium umbonatum var.inaequale Lemmermann,并且描述了此甲藻所产生的孢囊形态。还对该藻的SSU和LSU rDNA序列进行测序,系统发育分析的结果也支持形态鉴定。系统发育研究结果表明多甲藻属是多系起源的,本研究所用的多甲藻属类群可以形成两个大的分枝——Peridinium umbonatum类群形成的分枝和狭义多甲藻属类群（Peridinium sensu stricto）形成的分枝。并且形态学和分子数据都显示了P.umbonatum类群与狭义多甲藻属类群之间的差异。P.umbonatum类群形成的淡水单系类群虽然没有高度的自展支持但获得了不同的系统发育分析的支持,而狭义多甲藻属类群形成的单系类群获得了高度的支持。淡水多甲藻P.aciculiferum和P.wierzejskii则与海洋斯氏藻属关系密切。     

Phylogenetic relationships among Phialocephala species and other ascomycetes

Phialocephala was established for species in the Leptographium complex that produce conidia from phialides at the apices of dark mononematous conidiophores. Some species previously included in Phialocephala were re-allocated to Sporendocladia because they resembled Thielaviopsis in having ring-wall-building conidial development and conidia with two attachment points that emerge in false chains. Despite this significant realignment of the genus, a great deal of morphological heterogeneity remains in Phialocephala. The objective of this study was to consider the heterogeneity among Phialocephala spp. based on comparisons of sequence data derived from the large and small subunits (LSU and SSU) of the rRNA operon of species in Phialocephala. Phialocephala dimorphospora, the type species of the genus, and P. fortinii grouped with genera of the Helotiales in phylogenetic trees generated based on the LSU and SSU datasets. Phialocephala xalapensis and P. fusca clearly are unrelated to Phialocephala sensu stricto and should represent a new genus in the Ophiostomatales. Phialocephala compacta resides with representatives of the Hypocreales, and we believe that it represents a distinct genus. Phialocephala scopiformis and P. repens are not closely related to the other Phialocephala species and group within the Dothideales. The morphological heterogeneity among species of Phialocephala clearly is reflected by phylogenetic analysis of sequence data from two conserved rRNA gene regions. Appropriate genera now need to be found to accommodate these fungi.     

Plagiodinium ballux sp. nov. (Dinophyceae), a deep (36 m) sand dwelling dinoflagellate from subtropical Japan

A new species of marine sand‐dwelling dinoflagellate, Plagiodinium ballux N. Yamada, Dawut, R. Terada & T. Horiguchi is described from a deep (36 m) seafloor off Takeshima Island, Kagoshima Prefecture, Japan in the subtropical region of the northwest Pacific. The species is thecate and superficially resembles species of Prorocentrum, but possesses an extremely small epitheca. The cell varies from ovoid to a rounded square, and is small (15.0–22.5 μm in length) and laterally compressed. The thecal plates are smooth and the thecal plate arrangement (Po, 1′, 0a, 5″, 5C, 2S, 5?, 0p, 1″″) is similar to that of Plagiodinium belizeanum, the type species of the genus. Molecular phylogenetic analyses based on SSU rDNA and partial LSU rDNA reveal that the dinoflagellate is closely related to P. belizeanum, but it can be clearly distinguished by its size and cell shape. This suite of morphological and molecular differences leads to the conclusion that this deep benthic dinoflagellate represents a new species of the genus Plagiodinium.     

Evolutionary dynamics of multiple group I introns in nuclear ribosomal RNA genes of endoparasitic fungi of the genus Cordyceps

A large number of group I introns were discovered in coding regions of small and large subunits of nuclear ribosomal RNA genes (SSU rDNA and LSU rDNA) in ascomycetous fungi of the genus CORDYCEPS: From 28 representatives of the genus, we identified in total 69 group I introns which were inserted at any of four specific sites in SSU rDNA and four specific sites in LSU rDNA. These group I introns reached sizes of up to 510 bp, occurred in up to eight sites in the same organism, and belonged to either subgroup IB3 or subgroup IC1 based on their sequence and structure. Introns inserted at the same site were closely related to each other among Cordyceps fungi, whereas introns inserted at different sites were phylogenetically distinct even in the same species. Mapped on the host phylogeny, the group I introns were generally not restricted to a particular lineage, but, rather, widely and sporadically distributed among distinct lineages. When the phylogenetic relationships of introns inserted at the same site were compared with the phylogeny of their hosts, the topologies were generally significantly congruent to each other. From these results, the evolutionary dynamics of multiple group I introns in Cordyceps fungi was inferred as follows: (1) most of the group I introns were already present at the eight sites in SSU and LSU rDNAs of the ancestor of the genus Cordyceps; (2) the introns have principally been immobile and vertically transmitted throughout speciation and diversification of Cordyceps fungi, which resulted in the phylogenetic congruence between the introns at the same site and their hosts; (3) in the course of vertical transmission, the introns have repeatedly been lost in a number of lineages independently, which has led to the present sporadic phylogenetic distribution of the introns; and (4) a few acquisitions of new introns, presumably through horizontal transmission, were identified in the evolutionary history of the genus Cordyceps, while no transpositions were detected. Losses of group I introns in SSU rDNA have occurred at least 27 times in the evolutionary course of the 28 Cordyceps members.     

INTRAGENOMIC NUCLEOTIDE POLYMORPHISM AMONG SMALL SUBUNIT (18S) RDNA PARALOGS IN THE DIATOM GENUS SKELETONEMA (BACILLARIOPHYTA)1

Morphological features of the siliceous cell wall traditionally have been used to diagnose and classify species of diatoms, though an increasing number of studies distinguish new species, in part, by phylogenetic analysis of rDNA sequences. Intragenomic sequence variation is common among the hundreds to thousands of rDNA cistrons present within a genome, and this variation has strong potential to obscure species boundaries based on rDNA sequences. We screened six Skeletonema culture strains for intragenomic nucleotide polymorphisms in the small subunit (SSU) rDNA gene and found that all strains had polymorphic sites, with proportions ranging from 0.57% to 1.81%. In all cases, transitions accounted for more than 70% of nucleotide differences at polymorphic sites. Polymorphic sites were split nearly evenly in the SSU rRNA molecule between the base‐paired regions of helices (52%) and the unpaired regions of loops and bulges (48%). Phylogenetic analysis showed that SSU rDNA genotypes were monophyletic for two of the six culture strains examined. Genotypes from the other four culture strains either showed little or no phylogenetic structure compared with genotypes of other conspecific culture strains or had phylogenetic structure that was incongruent with existing species boundaries. Moderate to strong support for monophyly was recovered for four of the seven species included in the analysis. Phylogenetic results combined with the low sequence divergence of SSU rDNA genotypes within species suggest that concerted evolution has not proceeded to completion in these species and/or that the rate at which variation is being generated exceeds the rate at which concerted evolution is expunging variation.