Identification of Paramecium bursaria syngens through molecular markers--comparative analysis of three loci in the nuclear and mitochondrial DNA

This is the first attempt to resolve the phylogenetic relationship between different syngens of Paramecium bursaria and to investigate at a molecular level the intraspecific differentiation of strains originating from very distant geographical locations. Herein we introduce a new collection of five P. bursaria syngens maintained at St Petersburg State University, as the international collection of syngens was lost in the 1960s. To analyze the degree of speciation within Paramecium bursaria, we examined 26 strains belonging to five different syngens from distant and geographically isolated localities using rDNA (ITS1-5.8S-ITS2-5'LSU) fragments, mitochondrial cytochrome c oxidase subunit I (COI), and H4 gene fragments. It was shown that P. bursaria strains of the same syngens cluster together in all three inferred molecular phylogenies. The genetic diversity among the studied P. bursaria strains based on rDNA sequences was rather low. The COI divergence of Paramecium bursaria was also definitely lower than that observed in the Paramecium aurelia complex. The nucleotide sequences of the H4 gene analyzed in the present study indicate the extent of genetic differences between the syngens of Paramecium bursaria. Our study demonstrates the diagnostic value of molecular markers, which are important tools in the identification of Paramecium bursaria syngens.     

基于rDNA ITS序列探讨部分腐霉种的系统发育与其形态特征

基于对73株计58种腐霉和6种疫霉的核糖体DNA的ITS序列分析,以海生疫霉为外围群,按邻接法构建系统发育树,对腐霉的系统发育关系进行了研究。结果表明:在58种腐霉中,Pythium ostracodes,P.chamaehyphon,P.carbonicum,P.montanum和P.vexans归为同一组,介于其它腐霉和疫霉之间,这5种腐霉的孢子囊均为球形;现已归为疫霉属的P.undulatum 单独为一组,它与腐霉的亲缘关系比疫霉更近;其余52种腐霉聚成一大组,这52种腐霉基本上按孢子囊或菌丝膨大体形态分成Ⅰ、Ⅱ两组:第1组31种腐霉, 其中30种腐霉的孢子囊或菌丝膨大体为球形;第Ⅱ组21种腐霉,其中19种腐霉的孢子囊为丝状、瓣状或裂片状。基于ITS序列分析,腐霉属的其它性状如藏卵器壁是否光滑、卵孢子是否满器、雄器的着生方式和数量、异宗配合等呈多元演化。     

一株产阿魏酸酯酶青霉菌株的筛选、鉴定及生长特征

从腐烂的木质纤维中筛选了一株产阿魏酸酯酶的菌株HDFE1,根据其形态特征、rDNAITS1-5.8S-ITS2序列及系统发育分析,鉴定菌株HDFE1为青霉属的橘青霉(Penicillium citrinum Thom)。菌株HDFE1最适生长温度为30°C,最适生长pH为6.0。该菌株在30°C、pH6.0、200r/min培养60h时,阿魏酸酯酶酶活力为最高,达20.75U/L。     

Ostreopsis cf. siamensis and Ostreopsis cf. ovata from the Atlantic Iberian Peninsula: Morphological and phylogenetic characterization

Individuals of Ostreopsis, a genus containing potentially toxic species which affects human health, were collected during summer-autumn 2010 and 2011 from 17 sites located along the Atlantic coast of the Iberian Peninsula, a temperate area which during summer presents contrasting seawater temperatures. Ostreopsis cells were obtained by shaking macroalgae collected from rocky-shore areas bordering accessible beaches. Isolated strains and field samples were analyzed for morphological and phylogenetic characterization where sequences of the ITS1-5.8S-ITS2 region of the rDNA delineated two different species fitting Ostreopsis cf. ovata and Ostreopsis cf. siamensis. By means of calcofluor staining and scanning electron microscopy, it was observed that field samples of both species exhibited a wide and overlapping range of dorsoventral as well as width values. Those cells presented 11–18 pores/100 μm² and were also similar concerning plates shape and size. The main differential feature between the two species was the presence of two sizes of thecal pores (0.07–0.13 μm and 0.15–0.39 μm) in Ostreopsis cf. siamensis and one size (0.24–0.56 μm) in Ostreopsis cf. ovata. A comparison of field vs. cultured cells indicated that field isolates presented larger cells than in culture.     

Molecular phylogeny of Pseudokeronopsis (Protozoa, Ciliophora, Urostylida), with reconsideration of three closely related species at inter- and intra-specific levels inferred from the small subunit ribosomal RNA gene and the ITS1-5.8S-ITS2 region sequences

In order to address the confused taxonomy of morphotypes within the genus Pseudokeronopsis , the small subunit ribosomal RNA (SS rRNA) gene and the internal transcribed spacer (ITS1)-5.8S-ITS2 region for 13 populations/clones of morphologically similar Pseudokeronopsis species, Pseudokeronopsis flava , Pseudokeronopsis rubra and Pseudokeronopsis carnea , were sequenced and compared at inter- and intra-specific levels. Different geographical/time interval isolates belonging to the same species were slightly divergent, while clones from the same strain had identical SS rRNA gene sequences. Compared with the SS rRNA gene sequence, the sequence of the ITS1-5.8S-ITS2 region of Pseudokeronopsis revealed higher diversity, with nucleotide dissimilarities of 3.9–4.9% at the intra-specific level and 8.0–10.5% at the inter-specific level. There were several minor differences among ITS2 secondary structures of three Pseudokeronopsis species, while three Pseudok. carnea populations were identical. Phylogenetic analyses using multiple algorithms and different species selections confirm that Pseudok. flava , Pseudok. rubra and Pseudok. carnea form a monophyletic group within the urostylids. The result also supports the closest relationship of Pseudokeronopsis and Pseudourostyla that forms the so-called Acaudalia Berger (2006) .     

Molecular phylogeny of Nothoholosticha (Protozoa,Ciliophora, Urostylida) and systematic relationships of the Holosticha-complex

The marine ciliate Nothoholosticha is characterized by having a Holosticha-like ciliature pattern but without migratory frontoterminal cirri. However, its systematic position and its relationship to other members of the Holosticha-complex have not yet been resolved. In order to gain deeper insights into these relationships, the small subunit rRNA (SSrRNA) gene and the rRNA internal transcribed spacer and 5.8SrRNA (ITS1–5.8S-ITS2) region of two marine Anteholosticha species, as well as the ITS1–5.8S-ITS2 region of N. fasciola, were sequenced, and molecular trees (BI, ML, NJ and MP trees) were constructed. Although our analyses failed to conclusively resolve the phylogeny of this assemblage, certain conclusions could be drawn. Firstly, Nothoholosticha is a valid genus that is more closely related to Pseudokeronopsis than to other Holosticha-complex genera. Secondly, sequence analyses and phylogenetic trees of several Anteholosticha species revealed a high molecular diversity, which does not support the monophyly of this genus. Thirdly, the current assignment of certain well-known genera, e.g. Holosticha, Anteholosticha, Apokeronopsis, Parabirojimia, Psammomitra, Diaxonella, Metaurostylopsis and Thigmokeronopsis, to the families Bakuellidae (sensu Berger 2006), Urostylidae (sensu Berger 2006) or Holostichidae (sensu Berger 2006) is challenged by the molecular data presented here. And fourthly, the families Holostichidae and Pseudokeronopsidae (sensu Lynn 2008) are probably paraphyletic, and their systematic assignments await further evaluation.     

Studies on keratinophilic fungi. XI. Chrysosporium undulatum sp. nov.

Chrysosporium undulatum sp. nov., showing intermediate characteristics between Chrysosporium and Malbranchea, is described and illustrated. This taxon is characterised by long and curved to loosely coiled hyphal appendages, deflected to wavy fertile hyphae forming dense tufts and small, subglobose, frequently intercalary, verrucose conidia. Restriction enzyme analysis and comparison of sequences of the ITS1-5.8S-ITS2 ribosomal DNA region of the most similar species strongly support this proposal.     

Polymorphism of Paramecium pentaurelia (Ciliophora, Oligohymenophorea) strains revealed by rDNA and mtDNA sequences

Paramecium pentaurelia is one of 15 known sibling species of the Paramecium aurelia complex. It is recognized as a species showing no intra-specific differentiation on the basis of molecular fingerprint analyses, whereas the majority of other species are polymorphic. This study aimed at assessing genetic polymorphism within P. pentaurelia including new strains recently found in Poland (originating from two water bodies, different years, seasons, and clones of one strain) as well as strains collected from distant habitats (USA, Europe, Asia), and strains representing other species of the complex. We compared two DNA fragments: partial sequences (349 bp) of the LSU rDNA and partial sequences (618 bp) of cytochrome B gene. A correlation between the geographical origin of the strains and the genetic characteristics of their genotypes was not observed. Different genotypes were found in Kraków in two types of water bodies (Opatkowice—natural pond; Jordan's Park—artificial pond). Haplotype diversity within a single water body was not recorded. Likewise, seasonal haplotype differences between the strains within the artificial water body, as well as differences between clones originating from one strain, were not detected. The clustering of some strains belonging to different species was observed in the phylogenies.     

Reconsideration of systematic relationships within the order Euplotida (Protista, Ciliophora) using new sequences of the gene coding for small-subunit rRNA and testing the use of combined data sets to construct phylogenies of the Diophrys-complex

Comprehensive molecular analyses of phylogenetic relationships within euplotid ciliates are relatively rare, and the relationships among some families remain questionable. We performed phylogenetic analyses of the order Euplotida based on new sequences of the gene coding for small-subunit RNA (SSrRNA) from a variety of taxa across the entire order as well as sequences from some of these taxa of other genes (ITS1-5.8S-ITS2 region and histone H4) that have not been included in previous analyses. Phylogenetic trees based on SSrRNA gene sequences constructed with four different methods had a consistent branching pattern that included the following features: (1) the “typical” euplotids comprised a paraphyletic assemblage composed of two divergent clades (family Uronychiidae and families Euplotidae–Certesiidae–Aspidiscidae–Gastrocirrhidae), (2) in the family Uronychiidae, the genera Uronychia and Paradiophrys formed a clearly outlined, well-supported clade that seemed to be rather divergent from Diophrys and Diophryopsis, suggesting that the Diophrys-complex may have had a longer and more separate evolutionary history than previously supposed, (3) inclusion of 12 new SSrRNA sequences in analyses of Euplotidae revealed two new clades of species within the family and cast additional doubt on the present classification of genera within the family, and (4) the intraspecific divergence among five species of Aspidisca was far greater than those of closely related genera. The ITS1-5.8S-ITS2 coding regions and partial histone H4 genes of six morphospecies in the Diophrys-complex were sequenced along with their SSrRNA genes and used to compare phylogenies constructed from single data sets to those constructed from combined sets. Results indicated that combined analyses could be used to construct more reliable, less ambiguous phylogenies of complex groups like the order Euplotida, because they provide a greater amount and diversity of information.     

Development of an oligonucleotide microarray for the detection and monitoring of marine dinoflagellates

Harmful Algal Blooms (HABs), mainly caused by dinoflagellates and diatoms, have great economic and sanitary implications. An important contribution for the comprehension of HAB phenomena and for the identification of risks related to toxic algal species is given by the monitoring programs. In the microscopy-based monitoring methods, harmful species are distinguished through their morphological characteristics. This can be time consuming and requires great taxonomic expertise due to the existence of morphologically close-related species. The high throughput, automation possibility and specificity of microarray-based detection assay, makes this technology very promising for qualitative detection of HAB species. In this study, an oligonucleotide microarray targeted to the ITS1-5.8S-ITS2 rDNA region of nine toxic dinoflagellate species/clades was designed and evaluated. Two probes (45-47 nucleotides in length) were designed for each species/clade to reduce the potential for false positives. The specificity and sensitivity of the probes were evaluated with ITS1-5.8S-ITS2 PCR amplicons obtained from 20 dinoflagellates cultured strains. Cross hybridization experiments confirmed the probe specificity; moreover, the assay showed a good sensitivity, allowing the detection of up to 2 ng of labeled PCR product. The applicability of the assay with field samples was demonstrated using net concentrated seawater samples, un-spiked or spiked with known amounts of cultured cells. Despite the general application of microarray technology for harmful algae detection is not new, a peculiar group of target species/clades has been included in this new-format assay. Moreover, novelties regarding mainly the probes and the target rDNA region have allowed sensitivity improvements in comparison to previously published studies.     

Species boundaries in tintinnid ciliates: a case study--morphometric variability, molecular characterization, and temporal distribution of Helicostomella species (Ciliophora, Tintinnina)

Tintinnids are a large group of planktonic ciliates with diverse morphologies. The range of variability in lorica shapes and sizes can be very high even within a single species depending on life stages and environmental conditions, which makes the delimitation of different species based on morphological criteria alone very difficult. Accordingly, comparisons of morphological and molecular variability in tintinnids are necessary to provide a pragmatic approach for establishing species boundaries within this diverse and poorly understood group. We investigated the temporal distribution of species of the hyaline tintinnid Helicostomella (Ciliophora, Tintinnina), which were collected daily from September 2008 to August 2009 in Jangmok Bay of Geoje Island on the south coast of Korea. Two forms - a long form and a short form, were discovered. The long form was found in cold waters in February and March whereas the short form occurred in warm waters from June to October. Thus, these two forms were seasonally isolated. However, all the morphological characteristics for these two forms overlap to some degree. A comparison of the small subunit (SSU) rDNA, ITS1-5.8S-ITS2, and ITS2 sequences from two forms revealed 0.5%, 3.8%, and 5.6% divergences, respectively. Morever, one compensatory base change (CBC) and three hemi-CBCs were identified from ITS2 secondary structures of these two forms. All these data suggest that these two forms represent two distinct species despite their highly similar lorica morphology. The phylogenetic position of the genus Helicostomella was also examined using SSU rDNA sequences.     

Taxonomic characterization of two marine peritrichous ciliates, Pseudovorticella clampi n. sp. and Zoothamnium pararbuscula n. sp. (Ciliophora: Peritrichia), from North China

The morphology, infraciliature, and silverline system of two new peritrichous ciliates, Pseudovorticella clampi n. sp. and Zoothamnium pararbuscula n. sp., have been investigated based on both living and silver-impregnated specimens. Partial sequence of 18S-ITS1-5.8S rDNA of Z. pararbuscula is also determined in order to compare it with the closely related congener, Zoothamnium arbuscula. Zoothamnium pararbuscula can be distinguished from its close form Z. arbuscula by the different habitats, the appearance of the main stalk, the position of the contractile vacuole, and the information derived from 18S-ITS1-5.8S rDNA sequence analysis. Pseudovorticella clampi n. sp. is distinguished from its congeners by its body shape and size, pellicle granules, habitat, and number of transverse silverlines.     

Molecular characterisation of the species of the genus Zygosaccharomyces

The restriction fragments polymorphisms of the mitochondrial DNA and the PCR fragment that comprised the internal transcribes spacers and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 40 strains from the 10 species of the genus Zygosaccharomyces, including the new species Z. lentus were examined. The RFLP's of the ITS-5.8S region showed a specific restriction pattern for each species, including the new species Z. lentus. The only exception were the species Z. cidri and Z. fermentati that produced identical restriction profiles. The electrophoretic chromosome patterns confirmed the differences between the species of this genus, including the phylogenetic closest species Z. cidri and Z. fermentati. They present few chromosomes ranging from 3 bands (4 or 5 chromosomes) for Z. florentinus to 7 bands (8 to 10 chromosomes) for Z. cidri and Z. fermentati. The strain level resolution power of RFLP's of mtDNA of this genus enabled the characterisation of strains from the same species, even where they are isolated from the same substrate. However, in the cases of Z. bailii and Z. lentus, electrophoretic karyotyping there was considerable variation.     

Multiple origins of the symbioses in Paramecium bursaria

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.     

Morphological and molecular phylogenetic analysis of two Saprolegnia sp. (Oomycetes) isolated from silver crucian carp and zebra fish

Two Saprolegnia isolates, JY isolated from silver crucian carp (Carassius auratus gibelio Bloch) and BMY isolated from zebra fish (Brachydanio rerio Hamilton) came from infections occurring concurrently in different locations in China. To confirm whether the two isolates were from the same Saprolegnia clone, comparative studies have been carried out based on their morphological, physiological and molecular characteristics. Observations showed that morphologically (both asexual and sexual organs) the two isolates were broadly similar and both isolates underwent repeated zoospore emergence. Comparing 704 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found isolates JY and BMY shared an identical ITS sequence with a minor variation (99.6 % similarity). Forty available sequences for representatives Saprolegnia spp. belonged to four phylogenetically separate clades. The two studied isolates fell within clade I that comprised a group of isolates which showed almost an identical ITS sequence but had been identified as a number of different morphological species. Our findings suggest that isolates JY and BMY appear to belong to the S. ferax clade and this clade (I) contains a number of closely related phylogenetic species. This is distinct from the more common fish pathogenic isolates, which belong to the S. parasitica clade (III) and are characterized by having cysts decorated by bundles of long hooked hairs and two further clades (II and IV) containing largely saprotrophic or soil born species.     

Keratinophilic Fungi Recovered from Muddy Soil in Cairo Vicinities,Egypt

One hundred samples of muddy soil were collected from seven areas in the vicinity of Cairo and screened for the presence of keratinophilic fungi by using hair baiting isolation technique. Forty isolates of keratinophilic fungi were recovered and identified by recognition of their cultures, macro- and micromorphological features. Their physiological and molecular characteristics were studied by determination of their ubiquinone (Coenzyme Q) composition and DNA sequences of (ITS1-5.8S-ITS2) and 18S rRNA region sequences. The Keratinophilic isolates were identified as Chrysosporium carmichaelii, C. queenslandicum, C. zonatum, C. indicum, Aphanoascus mephitalis, and Uncinocarpus reesii. Chrysosporium zonatum was the most prevalent species and represented 42.5% of the total number of isolates. Each of C. carmichaelii and C. queenslandicum were equal in their prevalence and represented 15%. C. indicum comes next constituting 12.5%; followed by Uncinocarpus reesii which represented 10%. The least prevalent species in our study was Aphanoascus mephitalis, which was represented only 5% of the total keratinophilic isolates.     

Ribosomal DNA Comparisons of Globodera from Two Continents

Ribosomal DNA (rDNA) sequence data were compared for five species of Globodera, including G. rostochiensis, G. pallida, G. virginiae, and two undescribed Globodera isolates from Mexico collected from weed species and maintained on Solanum dulcamara. The rDNA comparisons included both internal transcribed spacers (ITS1 and ITS2), the 5.8S rRNA gene, and small portions of the 3'' end of the 18S gene and the 5'' end of the 28S gene. Phylogenetic analysis of the rDNA sequence data indicated that the two potato cyst nematodes, G. pallida and especially G. rostochiensis, are closely related to the Mexican isolates, whereas G. virginiae is relatively dissimilar to the others and more distantly related. The data are consistent with the thesis that Mexico is the center of origin for the potato cyst nematodes.     

MORPHOLOGY AND MOLECULAR PHYLOGENY OF AUREOPHYCUS ALEUTICUS GEN. ET SP. NOV. (LAMINARIALES,PHAEOPHYCEAE) FROM THE ALEUTIAN ISLANDS1

A previously unknown species of kelp was collected on Kagamil Island, Aleutian Islands. The species can be easily distinguished from any known laminarialean alga: the erect sporophytic thallus is composed of a thin lanceolate blade attaining ～2 m in height and ～0.50 m in width, without midrib, and the edge of the blade at the transition zone is thickened to form a V‐shape; the stipe is solid and flattened, slightly translucent, attaining ～1 m in length; the holdfast is semidiscoidal and up to 0.15 m in diameter. Anatomically, the blade has the typical trumpet‐shaped hyphae characteristic of the Chordaceae and derived foliose laminarialean species (i.e., Alariaceae/Laminariaceae/Lessoniaceae). No hair pits or mucilaginous structures were observed on the blade or stipe. No fertile sporophytes were collected, but abundant juvenile sporophytes were observed in the field. In the molecular phylogenetic analyses using chloroplast rbcL gene, nuclear ITS1‐5.8S‐ITS2 rDNA, and mitochondria nad6 DNA sequences, the new species (Aureophycus aleuticus gen. et sp. nov.) showed a closer relationship with Alariaceae of conventional taxonomy, or the “Group 1” clade of Lane et al. (2006) including Alaria and related taxa than with other groups, although the species was not clearly included in the group. Aureophycus may be a key species in elucidating the evolution of the Alariaceae within the Laminariales. Because of the lack of information on reproductive organs and insufficient resolution of the molecular analyses, we refrain from assigning the new species to a family, but we place the new species in a new genus in the Laminariales.     

Comparisons of Isolates of Heterodera avenae using 2-D PAGE Protein Patterns and Ribosomal DNA

Six geographic isolates of Heterodera avenae, including two isolates each from Sweden, Australia, and the United States, were compared on the basis of 2-D PAGE protein patterns and the complete DNA sequence for the two internal transcribed ribosomal DNA spacers (rDNA ITS1 and ITS2) and the 5.8S rRNA gene. The protein pattern data and rDNA ITS sequence data both indicated that the Swedish Gotland strain of H. avenae differed markedly from the rest of the isolates. Protein patterns for the Australia isolates differed more from a Swedish strict H. avenae isolate and isolates from Oregon and Idaho, than the two U.S. isolates and the Swedish strict H. avenae isolate differed from each other. Except for the Gotland strain isolate, the rDNA ITS sequences were highly conserved among all of the H. avenae isolates, just as we earlier found them to be conserved among species of the schachtii group of Heterodera.