Phylogenetic analysis of <Emphasis Type="Italic">Metarhizium</Emphasis> spp. isolated from soil in Japan

As a result of analyzing the internal transcribed spacer (ITS) and 5′ end of the EF-1α sequence of 145 isolates of Metarhizium spp. isolated from soil in Japan using selective agar medium, eight species were identified. ITS sequence analysis divided the isolates into three clades. Two were identified as M. flavoviride var. pemphigi and M. lepidiotae, respectively. EF-1α sequence analysis identified the other clades as six species: M. anisopliae, M. brunneum, M. guizhouense, M. majus, M. pingshaense and M. robertisii. The distribution of M. flavoviride var. pemphigi was restricted to forest or wood soil, and conidial sizes of M. guizhouense and M. majus were incongruent with the phylogeny based on the sequence of the 5′ end of EF-1α.     

Selection of a DNA barcode for Nectriaceae from fungal whole-genomes

A DNA barcode is a short segment of sequence that is able to distinguish species. A barcode must ideally contain enough variation to distinguish every individual species and be easily obtained. Fungi of Nectriaceae are economically important and show high species diversity. To establish a standard DNA barcode for this group of fungi, the genomes of Neurospora crassa and 30 other filamentous fungi were compared. The expect value was treated as a criterion to recognize homologous sequences. Four candidate markers, Hsp90, AAC, CDC48, and EF3, were tested for their feasibility as barcodes in the identification of 34 well-established species belonging to 13 genera of Nectriaceae. Two hundred and fifteen sequences were analyzed. Intra- and inter-specific variations and the success rate of PCR amplification and sequencing were considered as important criteria for estimation of the candidate markers. Ultimately, the partial EF3 gene met the requirements for a good DNA barcode: No overlap was found between the intra- and inter-specific pairwise distances. The smallest inter-specific distance of EF3 gene was 3.19%, while the largest intra-specific distance was 1.79%. In addition, there was a high success rate in PCR and sequencing for this gene (96.3%). CDC48 showed sufficiently high sequence variation among species, but the PCR and sequencing success rate was 84% using a single pair of primers. Although the Hsp90 and AAC genes had higher PCR and sequencing success rates (96.3% and 97.5%, respectively), overlapping occurred between the intra- and inter-specific variations, which could lead to misidentification. Therefore, we propose the EF3 gene as a possible DNA barcode for the nectriaceous fungi.     

Molecular phylogenetic studies of Lachnum and its allies based on the Japanese material

Molecular phylogenetic studies were carried out based on ITS-5.8S rDNA, the D1–D2 region of the large subunit rRNA gene, RPB2, and combined data of D1–D2 and RPB2 as well as these three genes on 36 species among 7 genera for Lachnum and allied genera in the family Hyaloscyphaceae. In the combined data of all three regions, seven strongly supported clades were obtained. The same clades were also recognized in most of the trees based on each gene, and the combined data of D1–D2 and RPB2, although some of them were not strongly supported. Four clades represented Albotricha, Brunnipila, Incrucipulum, and Lachnellula, respectively, whereas Lachnum was distributed to the remaining three clades. The molecular phylogenies strongly supported a group of species with granulate hairs, and we suggest the concept of Lachnaceae should be restricted to these species. Based on the molecular phylogenetic analysis, three new combinations—Incrucipulum longispineum, I. radiatum, and Lachnellula pulverulentum from Lachnum—are proposed.     

The phylogenetic position of an <Emphasis Type="Italic">Armillaria</Emphasis> species from Amami-Oshima,a subtropical island of Japan,based on elongation factor and ITS sequences

An undetermined Armillaria species was collected on Amami-Oshima, a subtropical island of Japan. The phylogenetic position of the Armillaria sp. was determined using sequences of the elongation factor-1α (EF-1α) gene and the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). The phylogenetic analyses based on EF-1α and ITS sequences showed that this species differs from known Japanese taxa of Armillaria. The sequences of this species and A. novae-zelandiae from Southeast Asia were contained in a strongly supported clade, which was adjacent to a well-supported sister clade containing A. novae-zelandiae from Australia and New Zealand.     

Divergence of the IGS rDNA in Fusarium proliferatum and Fusarium globosum reveals two strain specific non-orthologous types

A phylogenic analysis of Fusarium proliferatum and closely related species was performed using the most variable part within the intergenic spacer of the nuclear ribosomal DNA (IGS) and compared with a previously reported phylogeny performed in the same group of samples with a partial region of the nuclear single copy gene encoding the elongation factor 1α (EF-1α). The phylogenies from both genomic sequences were not concordant and revealed the presence of two non-orthologous IGS types, named types I and II, in F. proliferatum and Fusarium globosum.     

A multigene phylogeny of theGibberella fujikuroi species complex: Detection of additional phylogenetically distinct species

Phylogenetic relationships within theGibberella fujikuroi species complex were extended to newly discovered strains using nucleotide characters obtained by sequencing polymerase chain reaction (PCR) amplified DNA from 4 loci used in a previous study [nuclear large subunit 28S rDNA, nuclear ribosomal internal transcribed spacer (ITS) region, mitochondriaal small subunit (mtSSU) ribosomal DNA, and β-tubulin] together with two newly sampled protein-encoding nuclear genes, translation elongation factor EF-1α and calmodulin. Sequences from the ribosomal ITS region were analyzed separately and found to contain of two highly divergent, nonorthologous ITS2 types. Phylogenetic analysis of the individual and combined datasets identified 10 new phylogenetically distinct species distributed among the following three areas: 2 within Asia and 4 within both Africa and South America. Hypotheses of the monophyly ofFusarium subglutinans and its two formae speciales, f. sp.pini and f. sp.ananas, were strongly rejected by a likelihood analysis. Maximum parsimony results further indicate that the protein-encoding nuclear genes provide considerably more phylogenetic signal that the ribosomal genes sequenced. Relative apparent synapomorphy analysis was used to detect long-branch attraction taxa and to obtain a statistical measure of phylogenetic signal in the individual and combined datasets.     

Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China

The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear‐cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH‐psbA and trnL‐F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra‐ and inter‐specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH‐psbA (100%), trnL‐F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH‐psbA and trnL‐F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers.     

Development of chromosome 6D-specific markers for α-gliadin genes and their use in assessing dynamic changes at the Gli-2 loci

To develop chromosome 6D-specific point mutation (PM) markers for α-gliadin genes, 79 α-gliadin sequences cloned from Aegilops tauschii and another 40 α-gliadin genes with known chromosome locations were used in multi-sequence alignment and phylogenic analysis. Additional multiple alignment adjustments were performed manually to facilitate discovery of putative chromosome 6D-specific point mutations. A total of 85 PM primers were designed to detect 68 candidate chromosome 6D-specific point mutations. Experimental tests revealed 11 chromosome 6D-specific PM markers by using genomic DNA from homoeologous group 6 nullisomic–tetrasomic lines of Chinese Spring and putative diploid and tetraploid ancestors of hexaploid wheat as PCR templates. Detection of PM markers in one synthetic hexaploid wheat and its parental lines indicated that some α-gliadin genes were lost from Gli-2 loci during the formation of hexaploid wheat by amphidiploidization of the genomes of Triticum turgidum and Ae. tauschii. Detection of these PM markers in Ae. tauschii, T. aestivum and its four subspecies indicated that at least two genetically distinct sources of Ae. tauschii contributed germplasm to the D genome of T. aestivum.     

Higher efficiency of ISSR markers over plastid psbA‐trnH region in resolving taxonomical status of genus Ocimum L.

High level of morphological as well as chemical variability exists within the genus Ocimum, and its taxonomy and phylogenetic relationships are still doubtful. For evaluating interspecific genetic relationships among the Ocimum species, genotyping with intersimple sequence repeat (ISSR) markers and sequence analyses of noncoding psbA‐trnH intergenic region belonging to chloroplast DNA were carried out. Although ISSR markers are highly efficient and reproducible, they have not been used extensively in phylogenetic studies. The use of the plastidial barcode candidate was expected to provide more variable and informative insight into evolutionary rates, and was thus employed as a phylogenetic marker to assess interspecific relationships. This study revealed that the ISSR markers were more efficient than psbA‐trnH sequences in resolving the current status of Ocimum L. genus. Distance‐ and character‐based methodological approaches applied on the molecular data with biparental and maternal inheritance were used for deducing the phylogenetic relationships among Ocimum species. Average polymorphic information content (0.344) and resolving power (6.285) depicted through ISSR markers proved to be efficient in discriminating the studied species of Ocimum. The primers used in this study revealed 99.585% polymorphism across the species demonstrating the polymorphic nature of ISSR markers.     

Phylogenetic Position of the Mitochondrion-Lacking Protozoan Trichomonas tenax,Based on Amino Acid Sequences of Elongation Factors 1α and 2

Major parts of amino-acid-coding regions of elongation factor (EF)-1α and EF-2 in Trichomonas tenax were amplified by PCR from total genomic DNA and the products were cloned into a plasmid vector, pGEM-T. The three clones from each of the products of the EF-1α and EF-2 were isolated and sequenced. The insert DNAs of the clones containing EF-1α coding regions were each 1,185 bp long with the same nucleotide sequence and contained 53.1% of G + C nucleotides. Those of the clones containing EF-2 coding regions had two different sequences; one was 2,283 bp long and the other was 2,286 bp long, and their G + C contents were 52.5 and 52.9%, respectively. The copy numbers of the EF-1α and EF-2 gene per chromosome were estimated as four and two, respectively. The deduced amino acid sequences obtained by the conceptual translation were 395 residues from EF-1α and 761 and 762 residues from the EF-2s. The sequences were aligned with the other eukaryotic and archaebacterial EF-1αs and EF-2s, respectively. The phylogenetic position of T. tenax was inferred by the maximum likelihood (ML) method using the EF-1α and EF-2 data sets. The EF-1α analysis suggested that three mitochondrion-lacking protozoa, Glugea plecoglossi, Giardia lamblia, and T. tenax, respectively, diverge in this order in the very early phase of eukaryotic evolution. The EF-2 analysis also supported the divergence of T. tenax to be immediately next to G. lamblia. Received: 15 February 1996 / Accepted: 28 June 1996     

Gain and loss of elongation factor genes in green algae

Background  

Two key genes of the translational apparatus, elongation factor-1 alpha (EF-1α) and elongation factor-like (EFL) have an almost mutually exclusive distribution in eukaryotes. In the green plant lineage, the Chlorophyta encode EFL except Acetabularia where EF-1α is found, and the Streptophyta possess EF-1α except Mesostigma, which has EFL. These results raise questions about evolutionary patterns of gain and loss of EF-1α and EFL. A previous study launched the hypothesis that EF-1α was the primitive state and that EFL was gained once in the ancestor of the green plants, followed by differential loss of EF-1α or EFL in the principal clades of the Viridiplantae. In order to gain more insight in the distribution of EF-1α and EFL in green plants and test this hypothesis we screened the presence of the genes in a large sample of green algae and analyzed their gain-loss dynamics in a maximum likelihood framework using continuous-time Markov models.     

Phylogenetic Relationships Within the Genus Equus and the Evolution of α and θ Globin Genes

Sequences of the α1, α2 and θ globin genes from six equid species have been determined to investigate relationships within the genus Equus. Analyses using standard phylogenetic methods, or an approach designed to account for the effects of gene conversion between the α genes, gave broadly similar results and show that the horses diverged from the zebra/ass ancestor ∼2.4 million years ago and that the zebra and ass species arose in a rapid radiation ∼0.9 million years ago. These results from the α genes are corroborated by θ gene data and are in contrast to mitochondrial DNA studies of the phylogeny of this genus, which suggest a more gradual set of speciation events. Received: 22 April 1997 / Accepted: 20 July 1998     

Plant DNA‐barcode library and community phylogeny for a semi‐arid East African savanna

Applications of DNA barcoding include identifying species, inferring ecological and evolutionary relationships between species, and DNA metabarcoding. These applications require reference libraries that are not yet available for many taxa and geographic regions. We collected, identified, and vouchered plant specimens from Mpala Research Center in Laikipia, Kenya, to develop an extensive DNA‐barcode library for a savanna ecosystem in equatorial East Africa. We amassed up to five DNA barcode markers (rbcL, matK, trnL‐F, trnH–psbA, and ITS) for 1,781 specimens representing up to 460 species (~92% of the known flora), increasing the number of plant DNA barcode records for Africa by ~9%. We evaluated the ability of these markers, singly and in combination, to delimit species by calculating intra‐ and interspecific genetic distances. We further estimated a plant community phylogeny and demonstrated its utility by testing if evolutionary relatedness could predict the tendency of members of the Mpala plant community to have or lack “barcode gaps”, defined as disparities between the maximum intra‐ and minimum interspecific genetic distances. We found barcode gaps for 72%–89% of taxa depending on the marker or markers used. With the exception of the markers rbcL and ITS, we found that evolutionary relatedness was an important predictor of barcode‐gap presence or absence for all of the markers in combination and for matK, trnL‐F, and trnH–psbA individually. This plant DNA barcode library and community phylogeny will be a valuable resource for future investigations.     

Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress

Real-time RT-PCR is a powerful technique for the measurement of gene expression, but its accuracy depends on the stability of the internal reference gene(s) used for data normalization. Tobacco (Nicotiana tabacum) is an important model in studies of plant gene expression, but stable reference genes have not been well-studied in the tobacco system. We address this problem by analysing the expression stability of eight potential tobacco reference genes. Primers targeting each gene (18S rRNA, EF-1α, Ntubc2, α- and β-tubulin, PP2A, L25 and actin) were developed and optimized. The expression of each gene was then measured by real-time PCR in a diverse set of 22 tobacco cDNA samples derived from developmentally distinct tissues and from plants exposed to several abiotic stresses. L25 and EF-1α demonstrated the highest expression stability, followed by Ntubc2. Measurement of L25 and EF-1α was sufficient for accurate normalization in either the developmental or stress-treated samples, but Ntubc2 was also required when considering the entire sample set. Analysis of a tobacco circadian gene (NTCP-23) verified these reference genes in an additional context, and all techniques were optimized to enable a high-throughput approach. These results provide a foundation for the more accurate and widespread use of real-time RT-PCR in tobacco.     

Polar steroids from <Emphasis Type="Italic">Solaster endeca</Emphasis> starfish and the physiological activity of polar steroids from three starfish species

Four polyhydroxylated steroids, new (20R)-5α-cholestan-3β,6α,8,15α,24,26-hexaol (I) and known (20R,25S)-5α-cholestan-3β,6α,8,15β,16β,26-hexaol, (20R,25S)-5α-cholestan-3β,6α,15β,16β,26-pentaol, and marthasterone sulfate were isolated from the Solaster endeca starfish inhabiting the Sea of Okhotsk and characterized. Steroid (I) contains a 24,26-dihydroxylated side chain, which is unique for starfish polyols. The isolated steroids and related metabolites from two starfish species of the Evasterias genus (in total, 15 compounds) were weakly cytotoxic in a human HeLa cell culture and some of them were inhibitors of non-specific esterase from mouse Ehrlich carcinoma. The effects of these compounds on the p53 protein activity were studied in a yeast two-hybrid test system and both inhibitors and stimulators of this activity were found among them.     

Mycobiota of the Takamatsuzuka and Kitora Tumuli in Japan,focusing on the molecular phylogenetic diversity of <Emphasis Type="Italic">Fusarium</Emphasis> and <Emphasis Type="Italic">Trichoderma</Emphasis>

In an effort to clarify the cause of the deterioration of the colorfully painted murals that adorn the inner walls of the small stone chambers in the Takamatsuzuka and Kitora Tumuli in Japan, we enumerated the fungi that were isolated from moldy spots on the plaster walls collected between May 2004 and April 2005. The 262 fungal isolates from 79 samples of both tumuli were identified as approximately 100 species based on their phenotypic characters. Fusarium, Trichoderma, and Penicillium species were the predominant colonizers in the stone chamber interior and adjacent areas of both tumuli. In addition to the 28S phylogeny, neighbor-joining and Bayesian phylogenies of partial EF-1-alpha gene sequences revealed 24 genetically diverse fusaria in the Takamatsuzuka and Kitora Tumuli. Most of the fusaria were nested in clade 3 of the Fusarium solani species complex (FSSC); however, a few isolates were members of the F. oxysporum species complex (FOSC) clade or the F. avenaceum/F. tricinctum species complex clade. The FSSC isolates were compared with those detected in the Lascaux cave in France. In addition, a partial EF-1α gene phylogeny indicated that 13 Trichoderma isolates clustered in the Harzianum-Virens clade and 5 isolates in the Viride clade or Trichoderma sect. Longibrachiatum. Our analyses suggest that most of the fungi recovered from both tumuli are typically soil dwellers. First two authors contributed equally to this work     

Evolutionary analysis of the APA genes in the <Emphasis Type="Italic">Phaseolus</Emphasis> genus: wild and cultivated bean species as sources of lectin-related resistance factors?

The APA (Arcelin/Phytohemagglutinin/α-Amylase inhibitor) gene family is composed of various members, present in Phaseolus species and coding for lectin and lectin-related seed proteins having the double role of storage and defense proteins. Here members of the APA family have been identified by immunological, functional, and molecular analyses and representative genes were sequenced in nine wild species of Phaseolus. All taxa possessed at least one member of the true lectin gene. No arcelin type sequences have been isolated from the species examined. Among the wild species studied, only P. costaricensis contained an α-amylase inhibitor (α-AI). In addition P. augusti, P. maculatus, P. microcarpus, and P. oligospermus showed the presence of the lectin-related α-amylase inhibitor-like (AIL) genes and α-AI activity. Data from Southern blot analysis indicated the presence of only one lectin gene in P. parvulus and P. filiformis, while an extensive gene duplication of the APA locus was found in the other Phaseolus species. Phylogenetic analysis carried out on the nucleotide sequences showed the existence of two main clusters and clearly indicated that lectin-related genes originated from a paralogous duplication event preceding the development of the ancestor to the Phaseolus genus. The finding of detectable α-AI activity in species containing AIL genes suggests that exploiting APA genes variability in the Phaseolus genus may represent a valuable tool to find new members that may have acquired insecticidal activities. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.