Marked Intragenomic Heterogeneity and Geographical Differentiation of nrDNA ITS in <Emphasis Type="BoldItalic">Larix</Emphasis><Emphasis Type="BoldItalic">potaninii</Emphasis> (Pinaceae)

Nuclear ribosomal DNA (nrDNA) of gymnosperms, especially Pinaceae, is characterized by slow concerted evolution and exhibits substantial ITS-region length variation (975–3663 bp), in sharp contrast to the narrow range (565–700 bp) in angiosperms. Here we examined intra- and intergenomic heterogeneity of the nrDNA internal transcribed spacer (ITS) region in four varieties of Larix potaninii, a species from the mountainous areas of western China. Two clones with more than a 100-bp deletion in ITS1 were detected in L. potaninii var. chinensis and L. potaninii var. australis, respectively. The deletion resulted in the loss of most part, including the motif sequence, of subrepeat 1 (SR1). Sequence divergence and phylogenetic analyses showed that some clones would be pseudogenes given their low GC content, high substitution rates, unique positions in the phylogenetic trees, or significant length variation. These clones might represent orphons or paralogues at minor loci resulting from large-scale gene or chromosome reorganization. Some recombinants characterized by chimeric structure and discordant phylogenetic positions in partitioned sequence analyses indicate that unequal crossing-over plays an important role in the process of nrDNA concerted evolution. In addition, some varieties of L. potaninii might have experienced an nrDNA founder effect parallel to their geographical isolation.     

Phylogenetic analysis of <Emphasis Type="Italic">Antrodia</Emphasis> species and <Emphasis Type="Italic">Antrodia camphorata</Emphasis> inferred from internal transcribed spacer region

The species of Antrodia are one of the difficult-to-classify and obscure groups of poroid Aphyllophorales based on morphological appearance. However, it is becoming increasingly important to reliably identify the entire suite of Antrodia camphorata strains and Antrodia species due to the potential pharmaceutical value of their biologically active ingredients. In this study, the internal transcribed spacer (ITS) region of the ribosomal RNA gene (rDNA) was sequenced and phylogenetically analyzed in a number of Antrodia fungal species and strains. ITS amplicons from the Antrodia species tested ranged in size from 543 to 610 bp; the size of the ITS of A. camphorata strains ranged from 592 to 596 bp. The overall sizes of ITS2 and 5.8S ribosomal RNA gene of all A. camphorata strains tested in this study were shown to be 217 and 158 bp, respectively. A phylogenetic analysis of ITS data generated, which included sequences of 11 A. camphorata strains and nine other Antrodia species, showed three clearly distinct groups. Group 1 includes A. camphorata, Antrodia salmonea, and Antrodia carbinca strains. Within Group 2, Antrodia sinuosa and Antrodia xantha were clustered together. Group 3 contained Antrodia albida, A. heteromorpha, A. serialis, and A. malicola. The observed sequence diversity among ITS alleles provided an effective tool for differentiating strains of A. camphorata, A. salmonea, A. xantha, A. sinuosa, or A. serialis. Polymorphisms arising within the ITS1-5.8S-ITS2 region can provide practical markers for establishing a foundation for the further expansion of an ITS sequence database of medically important fungi.     

Genetic divergence and phylogenetic analysis of genus <Emphasis Type="Italic">Jatropha</Emphasis> based on nuclear ribosomal DNA ITS sequence

The genus Jatropha belongs to the family Euphorbiaceae having significant economic importance. The present investigation was undertaken with an aim to understand phylogenetic relationships among seven species (J. curcas, J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica, and J. tanjorensis.) which are widely distributed in India, using nuclear ribosomal DNA ITS sequence (nrDNA ITS) and to compare the results with multilocus marker analysis systems reported earlier for the same genus. The size variation obtained among sequenced nrDNA ITS regions was narrow and ranged from 647 to 654 bp. The overall mean genetic distance (GD) of genus Jatropha was found to be 0.385. Highest interspecific GD (0.419) was found between J. glandulifera and J. multifida. The least interspecific GD (0.085) was found between J. gossypifolia and J. tanjorensis. The highest intraspecific GD was observed in J. podagrica (0.011) and least in J. gossypifolia (0.002). The phylogram obtained using nrDNA ITS sequence showed congruence with the phylograms obtained using multilocus markers system reported earlier with minor variations. The present study also strongly supports high phylogentic closeness of J. curcas and J. integerrima. The only exception found was J. podagrica which clustered with J. multifida in earlier based on multilocus marker analysis, was clustered with J. curcas in the present analysis. The sequence data generated in the present investigation will help for further studies in intraspecies population, and their phylogentic analysis, biogeographical, molecular evolution studies and also pave way for future phylogetic and/or evolution studies among the other groups belongs to the family Euphorbiaceae.     

Phylogenetic relationships in the genera<Emphasis Type="Italic"> Zostera</Emphasis> and<Emphasis Type="Italic"> Heterozostera</Emphasis> (Zosteraceae) based on<Emphasis Type="Italic"> matK</Emphasis> sequence data

Phylogenetic analysis of the plastid (chloroplast) DNA matK gene of Zosteraceae species was undertaken. A molecular phylogenetic tree based on matK sequence data showed the monophyly of Heterozostera tasmanica and subgenus Zosterella and did not support the separation of Heterozostera from the genus Zostera. The tree based on matK supported the monophyly of the subgenus Zostera, and showed that Zosteraceae consist of three main groups: Phyllospadix, which is clearly defined by being dioecious; the subgenus Zosterella and Heterozostera; and the subgenus Zostera. Character-state reconstruction of chromosome number and geographic distribution for our molecular phylogenetic tree showed that 2n=12 is a plesiomorphic character for Zostera and Heterozostera, that the chromosome number was doubled or tripled in two lineages, and that the initial speciation of Zostera and Heterozostera occurred in the Northern Hemisphere. The matK tree showed the close affinity of Z. noltii and Z. japonica, which have disjunct distributions. Zostera marina, which is the only widely distributed species in the subgenus Zostera, also occurring in the northern Atlantic, was shown to be embedded within other subgenus Zostera species.     

Sectional relationships in the genus <Emphasis Type="Italic">Musa</Emphasis> L. inferred from the PCR-RFLP of organelle DNA sequences

The objective of this study was to construct a molecular phylogeny of the genus Musa using restriction-site polymorphisms of the chloroplast (cpDNA) and mitochondrial DNA (mtDNA). Six cpDNA and two mtDNA sequences were amplified individually in polymerase chain reaction (PCR) experiments in 13 species representing the four sections of Musa. Ensete ventricosum (W.) Ch. was used as the outgroup. The amplified products were digested with ten restriction endonucleases. A total of 79 restriction-site changes were scored in the sample. Wagner parsimony using the branch and bound option defined two lines of evolution in Musa. One lineage comprised species of the sections Australimusa and Callimusa which have a basic number of x = 10 chromosomes, while most species of sections Eumusa and Rhodochlamys (x = 11) formed the other lineage. Musa laterita Cheesman (Rhodochlamys) had identical organellar genome patterns as some subspecies of the Musa acuminata Colla complex. The progenitors of the cultivated bananas, M. acuminata and Musa balbisiana Colla, were evolutionarily distinct from each other. Musa balbisiana occupied a basal position in the cladogram indicating an evolutionarily primitive status. The close phylogenetic relationship between M. laterita and M. acuminata suggests that species of the section Rhodochlamys may constitute a secondary genepool for the improvement of cultivated bananas.Communicated by H.F. Linskens     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

Phylogenetic relationships among annual and perennial species of the genus <Emphasis Type="Italic">Cicer</Emphasis> as inferred from ITS sequences of nuclear ribosomal DNA

The cladistic analysis of the DNA sequences of the internal transcribed spacers of ribosomal cistrons (ITS1 and ITS2) for 20 species of Cicer L. (among which all the annuals), shows that various sections of the genus are not monophyletic. Annual species do not form a clade: C. arietinum, in fact, is closely related to both C. echinospermum and C. reticulatum, whereas C. bijugum, C. judaicum, and C. pinnatifidum form a separate clade. The annual C. cuneatum is sister group to the perennial C. canariense and both are archaic species within the genus. C. yamashitae is, on the contrary, the only annual species belonging to a group of perennials, within which close relationships are evident between C. graecum and C. montbretii as well as among a group of mainly Asian species.     

Molecular phylogenetic analysis of key <Emphasis Type="Italic">Jatropha</Emphasis> species inferred from nrDNA ITS and chloroplast (<Emphasis Type="Italic">trn</Emphasis>L-F and <Emphasis Type="Italic">rbc</Emphasis>L) sequences

The genus Jatropha (Euphorbiaceae) contains species that are of significant economic and ornamental value. However, Jatropha breeding material is rather limited due to incomplete information regarding phylogenetic relationships among germplasm resources. Phylogenetic analyses were performed based on the internal transcribed spacer of nuclear ribosomal DNA (nrDNA ITS), two chloroplast regions (trnL-F and rbcL), and the combined (ITS+trnL-F+rbcL) dataset among twenty-five specimens representing six key Jatropha species. Phylogenetic relationships of Jatropha were well resolved between subgenus Curcas and subgenus Jatropha, and demonstrated the intermediate position of section Polymorphae among sections of both subgenera. Jatropha curcas and J. integerrima demonstrated a close phylogenetic relationship. The molecular data agreed with the morphological classification that recognized J. multifida and J. podagrica in sec. Peltatae. The distinct intraspecific divergence that occurred in J. curcas could be attributed to restricted gene flow caused by geographical isolation and different ecological conditions. Phylograms produced with trnL-F and rbcL sequence data suggested slow rates of sequence divergence among Jatropha spp., while the ITS gene tree had good resolution suggesting high genetic variation of ITS among Jatropha species.     

Phylogenetic relationships in genus <Emphasis Type="Italic">Arachis</Emphasis> based on ITS and 5.8S rDNA sequences

Background  

The genus Arachis comprises 80 species and it is subdivided into nine taxonomic sections (Arachis, Caulorrhizae, Erectoides, Extranervosae, Heteranthae, Procumbentes, Rhizomatosae, Trierectoides, and Triseminatae). This genus is naturally confined to South America and most of its species are native to Brazil. In order to provide a better understanding of the evolution of the genus, we reconstructed the phylogeny of 45 species using the variation observed on nucleotide sequences in internal transcribed spacer regions (ITS1 and ITS2) and 5.8 S of nuclear ribosomal DNA.     

Phylogenetic relationships in the genus <Emphasis Type="Italic">Primula</Emphasis> L. (Primulaceae) inferred from the ITS region sequences of nuclear rDNA

The nucleotide sequences of the nuclear rDNA ITS regions were determined for 34 species of the genus Primula L. and one species of the genus Cortusa L., family Primulaceae Vent., and used to infer the phylogenetic relationships among these species. In this analysis species of the Russian flora and the flora of adjacent territories were studied for the first time. The results clarified the taxomic structure of the genus Primula and confirmed the entity of some of its sections; but not the subgenera sensu J.Richards. Our data do not support an independent status of the genus Cortusa, placing it as one of the terminal lineages of the section Cortusoides Balf. f. in the genus Primula.     

<Emphasis Type="Italic">Umbelopsis gibberispora</Emphasis> sp. nov. from Japanese leaf litter and a clarification of <Emphasis Type="Italic">Micromucor ramannianus </Emphasis>var. <Emphasis Type="Italic">angulisporus</Emphasis>

 Umbelopsis gibberispora is described as a new species in the genus Umbelopsis, Umbelopsidaceae, Mucorales. The species differs from others in this genus by ellipsoidal sporangiospores with unilaterally thickened walls. Phylogenetic analyses based on nuclear large subunit ribosomal DNA (nLSU rDNA) partial sequences suggest that U. gibberispora, U. swartii, and U. westeae form a clade together with the strains of Umbelopsis ramanniana. The ex-type strain of Micromucor ramannianus var. angulisporus is found to be very close to Umbelopsis vinacea, whereas other isolates identified under the former name in the sense of Linnemann fall in the U. ramanniana subclade. For these isolates, a new species, Umbelopsis angularis, is introduced. Phylogenetic relationships among Umbelopsis species are discussed related to their attributes of the sporangial wall and mature spore shapes. Received: August 27, 2002 / Accepted: March 11, 2003 Acknowledgments We thank Dr. Takashi Ohsono, Graduate School of Agriculture, Kyoto University, Japan, for providing the strain of U. gibberispora (CBS 109328). We also thank Dr. Wieland Meyer, University of Sydney, Australia for access to the phylogenetic tree based on ITS sequence data before publishing, and Dr. Richard C. Summerbell, Centraalbureau von Schimmelcultures, the Netherlands, for linguistic corrections.     

Phylogenetic relationships in genus <Emphasis Type="Italic">Geopora</Emphasis> (Pyronemataceae,Pezizales)

Species delimitation in the genus Geopora (Pyronemataceae) is complicated because of small number of differentiating characters, values of which tend to overlap among the species. Current classification relies mainly on size and shape of ascospores and fruit-bodies, position of the apothecia in the ground and length of excipular hairs. We measured ascospores in ca. 90 Geopora specimens. Sequences of internal transcribed spacer (ITS) rDNA gene were obtained to investigate phylogenetic relationships in the genus. Maximum parsimony (MP) and Bayesian analyses reveal that the well-supported clades detected often do not correspond to the species concepts based on morphological characters. Nine out of the ten lineages include specimens which were initially identified as belonging to different species. The dimensions of ascospores vary to great extent within the lineages. The size and shape of fruit-bodies, length of excipular hair and hymenium colour are mostly homogenous within each clade; however, these characters coincide to a great extent among the lineages and the latter can be assessed only from fresh fruit-bodies. Nuclear DNA content, and accordingly, ploidy level do not provide evidence for species distinction. Geopora arenicola, G. tenuis and G. sepulta were recognized as monophyletic species. Geopora foliacea and G. cervina could not be explicitly delimited and the G. cervina complex comprising three clades was introduced.     

A new <Emphasis Type="Italic">Dactylella</Emphasis> species from <Emphasis Type="Italic">Orbilia alba</Emphasis>

A new Dactylella species, Dactylella alba was isolated from the ascospores of Orbilia alba collected in Wenshan County, Yunnan Province, China. Conidiophores were either not branched or occasionally branched, bearing divergent sterigmata on the tip with single conidium on each. Conidia were elongated ellipsoids, 1–2 septate, mostly 1 septate. By combining the ITS sequence with morphological characteristics, a new anamorphic species is described and illustrated together with its teleomorph.     

Phylogenetic relationships in Iranian <Emphasis Type="Italic">Scutellaria</Emphasis> (Lamiaceae) based on nuclear ribosomal ITS and chloroplast <Emphasis Type="Italic">trn</Emphasis>L-F DNA data

The genus Scutellaria (Lamiaceae, Scutellarioidae) includes taxa with a wide range of pharmacological traits, which make this genus economically important. Previous taxonomical studies on Scutellaria were mainly based on morphological and chromosomal characters. The infrageneric classification of the genus has been the subject of a long controversy among authors, and delimitation of taxa remains problematic. In this study, the phylogeny of the genus was evaluated using nrDNA ITS and trnL-F sequences, with a focus on the 42 Iranian taxa belonging to subgenera Apeltanthus and Scutellaria. In both ITS and trnL-F trees, there were two main clades within the genus corresponding to the two subgenera Scutellaria and Apeltanthus. In the subg. Scutellaria, the sect. Anaspis was monophyletic, but the monophyly of sect. Scutellaria was not supported. In this subgenus, S. galericulata was located away from other species in both trees, supported the isolation of this species from the sect. Scutellaria and inclusion in the sect. Galericularia as was previously considered by some authors. In subg. Apeltanthus, sect. Apeltanthus and sect. Lupulinaria were not monophyletic; furthermore, the monophyly of subsect. Lupulinaria was not supported. Based on the results of this study, it can be suggested that the sectional classification of both subgenera Apeltanthus and Scutellaria should be revised.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the genus <Emphasis Type="Italic">Clivia</Emphasis>

We have developed a protocol for the in vitro propagation of the genus Clivia. Shoots were regenerated when fragments of the peduncle-pedicel junction (PP junction) from young inflorescences were used as explants. The optimal media for PP junction were Murashige and Skoog (MS)-based medium containing 10 M of 6-benzyladenine (BA) and 10 M of 2,4-dichlorophenoxyacetic acid (2,4-D) or MS supplemented with 5 M BA, 10 M -naphthaleneacetic acid (NAA), 250 mg l^-1 glutamine and 500 mg l^–1 casein hydrolysate and their usage depended on the breeding lines. Multiplication from initiations and in vitro seedlings was the best when the explants were cut longitudinally through the meristem and placed on MS plus 44 M BA. Plantlets were transferred on to hormone -free MS medium with charcoal for rooting.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.