Identification of species in the genus Pichia by restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal DNA gene

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database (). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.     

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) .     

A Molecular Phylogenetic Investigation of Bakuella,Anteholosticha, and Caudiholosticha (Protista,Ciliophora, Hypotrichia) Based on Three‐Gene Sequences

Traditionally classifications of the Urostyloida have been mainly based on morphology and morphogenesis. Recent molecular phylogenetic analyses have been largely based on single‐gene data for a limited number of taxa. Consequently, incongruence has arisen between the morphological/morphogenetic and the molecular data. In this study, the three phylogenetic markers (SSU rDNA, ITS1‐5.8S‐ITS2 region, and LSU‐rDNA) of three urostyloid genera represented by four species (Bakuella granulifera, Anteholosticha monilata, Caudiholosticha sylvatica, and C. tetracirra) were sequenced to investigate their phylogeny. The results show that: (1) all three genera should be regarded as the members of the order Urostyloida within the subclass Hypotrichia, as indicated by morphological characters; (2) phylogenetic analyses and sequence similarities both indicate that neither Anteholosticha nor Caudiholosticha are monophyletic and the systematic assignment of both genera awaits further evaluation; and (3) Bakuella has a closer relationship with Urostyla than with bakuellids (e.g. Apobakuella and Metaurostylopsis), suggesting Bakuella may belong to the family Urostylidae rather than the family Bakuellidae.     

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.     

Molecular and morphological characterization of a poorly known marine ciliate, Myoschiston duplicatum precht 1935: implications for phylogenetic relationships between three morphologically similar genera -- Zoothamnium, Myoschiston, and Zoothamnopsis (Ciliophora, Peritrichia, Zoothamniidae)

We studied the morphology and molecular phylogeny of Myoschiston duplicatum, a peritrich ciliate that has been recorded as an epibiont of crustaceans, but which we also identified on marine algae from Korea. The important morphological characteristics revealed by silver staining of Myoschiston species have not been described because they are rarely collected. Using morphological methods, we redescribed the type species of the genus, Myoschiston duplicatum, and provided an improved diagnosis of Myoschiston. In addition, the coding regions for nuclear small subunit (SSU) rRNA and internal transcribed spacer 1‐5.8S‐internal transcribed spacer 2 sequences were sequenced. Phylogenetic analyses that included available SSU rDNA sequences of peritrichs from GenBank strongly supported a position of M. duplicatum within the family Zoothamniidae. In addition, phylogenetic analyses were performed with single datasets (ITS1‐5.8S‐ITS2) and combined datasets (SSU rDNA + ITS1‐5.8S‐ITS2) to explore further the phylogenetic relationship in the family Zoothamniidae between the three morphologically similar genera—Zoothamnium, Myoschiston, and Zoothamnopsis.     

Morphology and molecular phylogeny of a new hypotrich ciliate,Anteholosticha songi nov. spec., and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991 (Ciliophora,Hypotrichia)

A new urostylid ciliate, Anteholosticha songi nov. spec., isolated from forest soil in Tibet, and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991, isolated from a freshwater pond in the USA, are investigated in terms of their morphology, ontogenesis, and molecular biology. Anteholosticha songi nov. spec. is characterized by a slender to ellipsoidal body measuring 160–205 × 40–55 μm in vivo; rod-shaped yellowish cortical granules arranged in irregular short rows; four dorsal kineties; adoral zone consisting of 35–40 membranelles; three frontal, one buccal, one parabuccal, two frontoterminal, two pretransverse, and four to six transverse cirri and 14–25 midventral pairs; 12–22 ellipsoidal macronuclear nodules longitudinally arranged in pairs left of cell mid-line. Supplemental information on morphogenesis in Holosticha pullaster is also presented. The phylogenetic relationship of Anteholosticha and Holosticha inferred from SSU rDNA sequence data are concordant with previous studies and showing that Holosticha is monophyletic whereas Anteholosticha is polyphyletic and should be split into two or more genera.     

Multigene‐based phylogeny of Urostylida (Ciliophora,Hypotrichia), with establishment of a novel family

The Urostylida is a major taxon of hypotrichs with many unresolved evolutionary relationships. Due to incomplete or inaccurate character states and a paucity of morphogenetic data, the phylogeny of several taxa within urostylids is unresolved. Molecular phylogeny studies based on single gene (SSU rDNA) data may lead to conflict between morphological classification and SSU rDNA tree. In this work, 20 new sequences (SSU rDNA, ITS1‐5.8S‐ITS2 and LSU rDNA) of five genera of urostylids are provided to further investigate the phylogenetic relationships of this group. The main findings are as follows: (i) the establishment of Hemicycliostylidae, a novel family presently including Hemicycliostyla and Australothrix, is supported by both single gene and concatenated phylogenies; (ii) all molecular data support the exclusion of Eschaneustyla from the family Epiclintidae; (iii) Australothrix, Bergeriella and Thigmokeronopsis are distinctly separated in all gene trees although they share the character that each posterior streak generates the ventral row together with the midventral pair; (iv) compared with closely related genera in all trees, that is Metaurostylopsis and Apourostylopsis, Neourostylopsis is characterized by having more than three frontal cirri arranged in distinct or indistinct corona rather than the length of the midventral complex; (v) Hemicycliostyla and Pseudourostyla, two morphologically similar genera, do not form a monophyletic group in all molecular trees, suggesting that the bicorona, multiple marginal cirral rows and high numbers of dorsal kineties may result from convergent evolution; (vi) species of Bakuella fall into three separate clades in all trees suggesting that this genus needs to be split.     

Isolation and identification of endophytic fungus SX01, a red pigment producer from <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L

Fungal pigments are a potential resource as natural food colorant. Endophytic fungus SX01, which is able to produce abundant soluble red pigments, was isolated from the twigs of Ginkgo biloba L. For further research and utilization of this strain and its secondary metabolites, morphological and molecular characteristics of SX01 was identified. Morphological identification which employed light microscope and scanning electron microscope showed that SX01 was a species of genera Penicillium. ITS1-5.8S-ITS2 region and 18S rRNA gene were then cloned, and the molecular phylogeny of these two sequences proved that strain SX01 was Penicillium purpurogenum.     

Morphology,morphogenesis, and molecular phylogeny of a new marine urostylid ciliate (Ciliophora,Stichotrichia) from the South China Sea,and a brief overview of the convergent evolution of the midventral pattern within the Spirotrichea

The morphology, infraciliature, morphogenetic events, and small subunit ribosomal RNA (SSU rRNA) gene‐based phylogeny of a stichotrich ciliate, Bergeriella ovata gen. et sp. nov. , found in coastal waters off Daya Bay, Guangdong, South China, were investigated. The new taxon has an atypical midventral pattern, and is characterized by several rather unusual morphological features: frontal cirri forming an irregular tricorona; one nonmigratory row located right of the midventral rows, which is morphogenetically derived from the posteriormost fronto‐ventral‐transverse (FVT) streak; conspicuously enlarged postoral ventral cirri, whereas the left ventral cirri are delicate and arranged in oblique rows. Based on both the morphological and morphogenetical data, a new family, Bergeriellidae fam. nov. , is proposed. In the SSU rRNA phylogenetic trees, B. ovata gen. et sp. nov. groups with the classic urostylids (urostylids s.s.) with high nodal support. Our phylogenetic analyses have revealed at least seven separate clades for midventral pattern‐bearing taxa (e.g. Pattersoniella, Neokeronopsis, Rigidothrix, urostylids s.s., Uroleptidae, Holostichidae + Psammomitridae, and Pseudoamphisiellidae), suggesting that the order Urostylida Jankowski, 1979 sensu Lynn, 2008 is a polyphyletic assemblage, and that the convergent evolution of the urostylid midventral pattern might have occurred more times among the spirotrichs than has previously been recognized. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 697–710.     

Atypical non-lipid-dependent strains of <Emphasis Type="Italic">Malassezia furfur</Emphasis>

Members of the yeast genus Malassezia, including atypical strains, are lipophilic except for Malassezia pachydermatis. New physiological features that characterize atypical Malassezia strains are mainly associated with alteration in Tween assimilation pattern — such isolates still require lipids for growth. We isolated three non-lipid-dependent strains of Malassezia from patients with diagnosed atopic dermatitis (AD). These isolates could not be identified to the species level via their physiological properties. Phylogenetic trees, based on the D1/D2 regions of the 26S rDNA gene sequences and nucleotide sequences of the ITS1-5.8S-ITS2 rRNA region, showed the isolates to belong to Malassezia furfur. Three non-lipid dependent isolates from AD skin were conspecific, and sequences analysis proved them to be M. furfur.     

A PCR based SNPs marker for specific characterization of English walnut (<Emphasis Type="Italic">Juglans regia</Emphasis> L.) cultivars

English walnut (Juglans regia L.) is the most economically important species from all the 21 species belonging to the genus Juglans and is an important and healthy food as well as base material for timber industry. The aim of this study was to develop a simple technique for specific characterization of English walnut using DNA method. The first and second internal transcribed spacers (ITS1 and ITS2) as well as the intervening 5.8S coding region of the rRNA gene for 18 cultivars of J. regia L. isolated from different geographic origins were characterized. The size of the spacers sequences ranged from 257 to 263 bases for ITS1 and from 217 to 219 bases for ITS2. Variation of GC contents has also been observed and scored as 55–56.7 and 57.1–58.9% for ITS1 and ITS2, respectively. This data exhibited the presence of polymorphism among cultivars. Alignment of the ITS1-5.8S-ITS2 sequences from 18 walnut cultivars showed that there were 244 single nucleotide polymorphisms (SNPs) and 1 short insertion–deletion (indel) at 5′ end ITS1. Amplification refractory mutation system strategy was successfully applied to the SNP markers of the ITS1 and ITS2 sequences for the fingerprinting analysis of 17 on 18 walnut cultivars. The prediction of ITS1 and ITS2 RNA secondary structure from each cultivar was improved by detecting key functional elements shared by all sequences in the alignments. Phylogenetic analysis of the ITS1-5.8S-ITS2 region clearly separated the isolated sequences into two clusters. The results showed that ITS1 and ITS2 region could be used to discriminate these walnut cultivars.     

Yeast communities associated with artisanal mezcal fermentations from <Emphasis Type="Italic">Agave salmiana</Emphasis>

The aims of this work were to characterize the fermentation process of mezcal from San Luis Potosi, México and identify the yeasts present in the fermentation using molecular culture-dependent methods (RFLP of the 5.8S-ITS and sequencing of the D1/D2 domain) and also by using a culture-independent method (DGGE). The alcoholic fermentations of two separate musts obtained from Agave salmiana were analyzed. Sugar, ethanol and major volatile compounds concentrations were higher in the first fermentation, which shows the importance of having a quality standard for raw materials, particularly in the concentration of fructans, in order to produce fermented Agave salmiana must with similar characteristics. One hundred ninety-two (192) different yeast colonies were identified, from those present on WL agar plates, by RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA gene, with restriction endonucleases, HhaI, HaeIII and HinfI. The identified yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identifications were confirmed by sequencing the D1-D2 region of the 26S rRNA gene. With the PCR-DGGE method, bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii were clearly detected, confirming the results obtained with classic techniques.     

Co-transformation of <Emphasis Type="Italic">Panax</Emphasis> major ginsenosides Rb<Subscript>1</Subscript> and Rg<Subscript>1</Subscript> to minor ginsenosides C–K and F<Subscript>1</Subscript> by <Emphasis Type="Italic">Cladosporium cladosporioides</Emphasis>

Rb₁ and Rg₁ are the major ginsenosides in protopanaxadiol and protopanaxatriol. Their content in ginsenosides was 23.8 and 17.6%, respectively. A total of 22 isolates of β-glucosidase producing microorganisms were isolated from the soil of a ginseng field using Esculin-R2A agar. Among these isolates, the strain GH21 showed the strongest activities to convert ginsenoside Rb₁ and Rg₁ to minor ginsenosides compound-K and F₁, respectively. Ginsenosides Rb₁ and Rg₁ bioconversion rates were 74.2 and 89.3%, respectively. Meanwhile, the results demonstrated that the ginsenoside Rg₁ could change the biotransformation pathway of ginsenoside Rb₁ by inhibiting the formation of the intermediate metabolite gypenoside-XVII. GH21 was identified as a Cladosporium cladosporioides species based on the internal transcribed spacers (ITS) ITS1-5.8S-ITS2 rRNA gene sequences constructed phylogenetic trees.     

Phylogenetic relationships among Myrceugenia, Blepharocalyx, and Luma (Myrtaceae) based on paired-sites models and the secondary structures of ITS and ETS sequences

To establish relationships among the genera Blepharocalyx, Luma, and Myrceugenia, the phylogeny of tribe Myrteae was reconstructed based on secondary structures of the sequences of ITS (ITS1-5.8S-ITS2) and ETS regions from 93 taxa belonging to 29 genera. The DNA sequences were aligned according to their secondary structure and analysed with Bayesian inference (BI) using base substitution models for RNA, which can differentiate between the regions consisting of base pairs (helices) and unpaired bases (loops). The best-fit models were RNA7C for ITS and RNA7B for ETS, which differ in how they predict double substitutions and symmetry of the frequency of base pairs. The phylogenetic hypothesis was compared with results obtained using classical 4-state models, the results of maximum parsimony, and using sequence alignment without considering the secondary structure. Analyses show low support along the backbone of the consensus trees, those using substitution models of paired sites showing more highly supported derived clades than substitution models of independent sites. All tests were consistent and differed in the positioning of certain groups, but they also showed that Blepharocalyx, Luma, and Myrceugenia arise from three independent lineages that are not closely related. The substitution model for RNA shows that Luma is a monophyletic group and sister to the other genera of Myrteae, except for Myrtus communis. Myrceugenia appears as a monophyletic group, except for M. fernandeziana, which is related to Blepharocalyx, a genus that appears to be biphyletic.     

Detection of yeast species also occurring in substrates associated with animals and identification of a novel dimorphic species in Verbascum flowers from Georgia

The molecular taxonomic analysis of yeasts isolated from Verbascum flowers collected in central Georgia identified strains that could be assigned to the species Cryptococcus adeliensis, Cryptococcus magnus and Moniliella megachiliensis detected previously also in substrates associated with insects and other animals and a hitherto undescribed species for which the name Candida verbasci is proposed. The new species forms slightly pink colonies, propagates by mostly unipolar budding, forms invasive pseudomycelium, and the sequences of its D1/D2 LSU rRNA genes and ITS1-5.8S-ITS2 regions indicate close phylogenetic relationship with a group of species that form a cluster basal to the Candida albicans/Lodderomyces elongisporus clade. The type strain is 11-1055^T. It has been deposited in Centralbureau voor Schimmelcultures (Utrecht, the Netherlands) as CBS 12699^T, the National Collection of Agricultural and Industrial Microorganisms (Budapest, Hungary) as NCAIM Y.02048^T and the Culture Collection of Yeasts (Bratislava, Slovakia) as CCY 29-185-1^T. The GenBank accession numbers for nucleotide sequences of the C. verbasci type strain are: JX515981 (D1/D2 domain of the 26S rRNA gene) and JX515982 (ITS1-5.8S-ITS2). Mycobank: MB 801391.     

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.     

Inter- and intrasporal nuclear ribosomal gene sequence variation within one isolate of arbuscular mycorrhizal fungus, Diversispora sp.

Most molecular ecological studies of arbuscular mycorrhizal fungi (AMF) have been based on the rRNA gene sequences. However, information about intraspecific nucleotide variation is still limited in these fungi. In this study, we calculated the inter- and intrasporal nucleotide variation of Diversispora sp. EE1 using 78 cloned sequences from four spores within a ca 4960 bp fragment of the nuclear ribosomal operon spanning the near full length small ribosomal subunit (SSU) rRNA gene, the full internal transcribed spacer (ITS: ITS1-5.8S-ITS2) and ca 2740 bp of the large ribosomal subunit (LSU) rRNA gene. Data for each marker region (SSU, ITS and LSU) originated from the very same spores. Sequence variation resulting from point mutations and small indels was recorded in all regions. Highest sequence variation was observed in the ITS region at both the inter- and intrasporal levels. The ITS1 component was more variable than ITS2, whilst the 5.8S gene was the least variable component of the ITS region. Evolutionary divergence of gene copies between spores was intermediate for the LSU and lowest for the SSU. The SSU and the LSU genes had relatively similar evolutionary divergence per spore. Sequence variant richness was not exhaustive for any of the marker regions, indicating that multiple sequences per spore from multiple spores are needed when characterizing a species. This study provides reference sequences for ecological studies, permitting identification of AMF using any of the ribosomal regions or primer systems.     

Phylogenetic relationships among 28 spirotrichous ciliates documented by rDNA

The contiguous sequence of the SSU rDNA, ITS 1, 5.8S, ITS 2, and approximately 1370 bp at the 5(') end of the LSU rDNA was determined in 25 stichotrichs, one oligotrich, and two hypotrichs. Maximum parsimony, neighbor-joining, and quartet-puzzling analyses were used to construct individual phylogenetic trees for SSU rDNA, for LSU rDNA, and ITS 1+5.8S+ITS 2, as well as for all these components combined. All trees were similar, with the greatest resolution obtained with the combined components. Phylogenetic relationships were largely consistent with classical taxonomy, with notable disagreements. DNA sequences indicate that Oxytricha granulifera and Oxytricha longa are rather distantly related. The oligotrich, Halteria grandinella, is placed well within the order Stichotrichida. Uroleptus pisces and Uroleptus gallina probably belong to different genera. Holosticha polystylata (family Holostichidae) and Urostyla grandis (family Urostylidae) are rather closely related. These rDNA sequence analyses imply the need for some modifications of classical taxonomic schemes.     

基于核糖体基因的鳞钙皮菌种内分子关系分析

为探讨不同地域的鳞钙皮菌Didymium squamulosum种内分子亲缘关系,通过PCR扩增鳞钙皮菌子实体及原质团DNA,得到SSU、ITS1-5.8S-ITS2 rRNA基因区域,并以SSU、5.8S rRNA基因片段构建NJ亲缘关系树。