Phyletic and evolutionary relationships ofBrachyscome lineariloba (Compositae)

A comparison of karyotypes ofBrachyscome breviscapis (2n = 8),B. lineariloba cytodemes E (2n = 10), B (2n = 12) and C (2n = 16) suggests that these species have a homoelogous basic set of four chromosome pairs, two large pairs and two small, and that theB. lineariloba cytodemes E, B and C are related toB. breviscapis by successive additions of small chromosomes. A pronounced asynchrony of chromosome condensation between these large and small chromosomes has been observed. In the artificial hybrids betweenB. dichromosomatica (2n = 4) ×B. breviscapis, and theB. lineariloba cytodemes, theB. dichromosomatica chromosomes are similar in size and condensation behaviour to the small chromosomes ofB. breviscapis and ofB. lineariloba cytodemes E, B and C. Meiotic pairing in these hybrids also demonstrates the strong affinities between these chromosomes. It is suggested thatB. breviscapis may be of amphidiploid origin between a species with two large early condensing chromosome pairs and another,B. dichromosomatica-like species with two small late condensing pairs. It seems most likely that the additional small and late condensing chromosomes inB. lineariloba cytodemes E, B and C are derived from theB. dichromosomatica-like parent, and that each addition increases vigour, fecundity and drought tolerance, allowing these cytodemes to colonize more open and arid environments. Transmission of the univalents in the quasidiploidB. lineariloba cytodeme E was verified as being via the pollen, and not via the embryo sacs.The cytology ofBrachyscome lineariloba (Compositae, Asteroidae), 10.     

A monophyletic origin of the B chromosomes ofBrachycome dichromosomatica (Asteraceae)

The A and B chromosomes of different karyotype variants (cytodemes A1, A2, A3 and A4) ofBrachycome dichromosomatica were analysed by computer-aided chromosome image analysis and fluorescencein situ hybridisation (FISH). Ribosomal DNA and the B chromosome-specific sequence Bd49 were detected on all B chromosomes. In addition to minor size variation of the Bs, polymorphism of the rDNA and Bd49 position and copy number revealed two major types of B chromosomes. The B chromosomes of all the cytodemes were indistinguishable from each other in length, but that of A3 showed evidence of rearrangements consistent with its long-term geographic isolation. The results presented suggest a monophyletic origin of the B chromosomes ofB. dichromosomatica.     

Organisation and origin of a B chromosome centromeric sequence from Brachycome dichromosomatica

Brachycome dichromosomatica is an Australian native daisy that has two pairs of A chromosomes and up to three B chromosomes in some populations. A putative B-specific tandem repeat DNA sequence (Bd49) was isolated previously. Here we describe further characterisation of this sequence and investigate its possible origin. Southern analysis showed that all individual B chromosomes examined have highly methylated tandem repeats of Bd49 but differences in banding pattern for distinct B isolates suggested that the sequence is in a state of flux. Using in situ hybridisation, the sequence was shown to be located at the centromeric region of the B chromosome. Southern analysis of genomic DNA with Bd49 demonstrated that multiple copies of the sequence exist in the genomes of B. eriogona, B. ciliaris, B. segmentosa and B. multifida (none of which have B chromosomes) whereas other species tested (including 0B plants of B. dichromosomatica and 0B B. curvicarpa and B. dentata) have few or no copies. Genomic clones and Bd49-like sequences derived by the polymerase chain reaction (PCR) were obtained from five species but determination of phylogenetic relationships within the genus and inference as to the possible origin of the B chromosome were problematic because of extensive intragenomic heterogeneity of the sequences.     

Length variation in the internal transcribed spacers of ribosomal DNA in Picea abies and related species

The structure and variation of nuclear ribosomal DNA (rDNA) units of Picea abies, (L.) Karst. was studied by restriction mapping and Southern hybridization. Conspicuous length variation was found in the internal transcribed spacer (ITS) region of P. abies, although the length of this region is highly conserved both within and among most of the plant species. Two types of ITS variants (A and B), displaying a size difference of 0.5 kb in the ITS2 region, were present within individuals of P. abies from Sweden, Central Europe and Siberia. A preliminary survey of 14 additional Eurasian and North American species of Picea suggested that length variation in the ITS region is widespread in this genus. Alltogether three length variants (A, B and C) were identified. Within individuals of eight Picea species, two length variants were present within the genome (combinations of A and B variants in P. glehnii, P. maximowiczii, P. omorika, P. polita and P. sitchensis and variants B and C in P. jezoensis, P. likiangensis and P. spinulosa). Within individuals from five species, however only one rDNA variant was present in their genome (variant A in P. aurantiaca, P. engelmannii, P. glauca, P. koraiensis and P. koyamai; variant B in P. bicolor). The ITS length variation will be useful as a molecular marker in evolutionary studies of the Picea species complex, whose phylogeny is controversial. The presence of intraindividual variation in, and shared polymorphism of the, ITS length variants raises questions about the occurrence of interspecific hybridization during the evolutionary history of Picea.     

Karyological stability and microevolution of a cell suspension culture ofBrachycome dichromosomatica (Asteraceae)

A long-term suspension culture ofBrachycome dichromosomatica (2n = 4) was induced from a cotyledon-derived callus. Subcultures were obtained every week up to three years. The bulk of the cultures displayed a stable diploid karyotype, while one cell line evolved with 2n = 5 chromosomes in the 86th reinoculation. No further chromosomal change occurred also in that cell line. It is assumed that the fifth chromosome is the expression of a trisomy 2.The chromatin ultrastructure was of the species-specific chromomeric type in the wild-type line, while the trisomic line displayed more condensed chromatin, what probably indicates a rather inactive state of the extra-chromosome.Brachycome dichromosomatica is suggested to represent an ideal species to follow-up karyotype stability and/or variation in cell culture.As a former student W. N. dedicates this paper in gratitude and admiration to Prof. DrElisabeth Tschermak-Woess on the occasion of her 70th birthday. Prof.Woess with her scientific work has stimulated in an unique manner the study of nuclear structures in plants, of endopolyploidy and polytene chromosomes, and has thus established the basis for the rapidly increasing research in these fields.     

A repetitive DNA sequence common to the different B chromosomes of the genus Brachycome

Dot-like micro B chromosomes of Brachycome dichromosomatica were analysed for their sequence composition. Southern hybridization patterns of a total micro B probe to genomic DNA from plants with and without micro Bs demonstrated that the micro Bs shared sequences with the A chromosomes. In addition to telomere, rDNA and common A and B chromosome sequences, a new B-specific, highly methylated tandem repeat (Bdm29) was detected. After in situ hybridization with Bdm29 the entire micro B chromosome was labelled and clustering of the condensed micro Bs could be observed at interphase. A high number of Bdm29-like sequences were also found in the larger B chromosomes of B. dichromosomatica and in other Bs within the genus Brachycome. Received: 30 May 1997; in revised form: 20 August 1997 / Accepted: 20 August 1997     

Cytogenetics of protoplast cultures of Brachycome dichromosomatica and Crepis capillaris and regeneration of plants

Summary Callus derived protoplasts of Brachycome dichromosomatica (2n=2x=4) and Crepis capillaris (2n=2x=6) have been regenerated into karyologically normal plants, i.e. plants without visible alterations of the diploid chromosome set. However, metaphase analysis of protoplast cultures derived from both callus as well as mesophyll cells showed karyological changes in the overwhelming majority of cells in both species leading to multinucleated, polyploid and aneuploid cells. Furthermore, callus derived protoplasts sometimes exhibited changes at the chromosome level as indicated by translocations. The vast majority of aberrant karyotypes arose from failures during mitosis and cytokinesis, pointing to inadequate microtubules as a possible underlying cause. Karyological events of the kind described herein greatly affect the plating efficiency of isolated protoplasts and the viability of protoplast derived calli. Plant regeneration, although demonstrated in this study for the first time in both species, seems to be limited to rarely occurring, protoplast-derived colonies with a relatively stable genome. Our experiments, performed with chromosomal model species, emphasize the need for controlled, non-mutagenic culture conditions.     

Lengths and nucleotide sequences of the internal spacers of nuclear ribosomal DNA in gymnosperms and pteridophytes

The nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA were analyzed in species belonging to gymnosperms and pteridophytes. The lengths of the ITSs of sixteen species of gymnosperms and seven species of pteridophytes were estimated. The gymnosperms have ITS1 regions larger than those observed in the pteridophytes and angiosperms (ca. 610–3100 bp versus 159–360 bp). On the other hand, the ITS2 regions appear to be of a conserved length (182–370 bp). We have determined the complete nucleotide sequences of ITS regions from four gymnosperm species and five pteridophyte species by cloning the PCR products. Sequence analysis showed the presence of three short tandem arranged subrepeats of about 70 bp in the 1112 bp ITS1 ofEphedra fragilis. Pyrimidine rich (about 90%) DNA segments of 40–50 bp were observed in the ITS1 ofGinkgo biloba. A highly conserved 16 bp long sequence known to be present in the ITS1 of the angiosperm species has been also found in the ITS1 ofCycas revoluta, Taxus baccata andEphedra fragilis. Dedicated to Prof.Emilio Battaglia.     

Molecular phylogeny of the genus Stereocaulon (Stereocaulaceae, lichenized ascomycetes)

In the present study phylogenetic relationships of the genus Stereocaulon (lichenized ascomycetes) were examined using DNA sequences from the ITS1–5.8 S–ITS2 rDNA gene cluster and from the protein-coding β-tubulin gene. In addition to the fruticose species traditionally classified in Stereocaulon, representatives of the crustose species that have recently been transferred to the genus were included. Muhria, a monotypic genus that is morphologically similar to Stereocaulon, differing only in apothecia ontogeny, was also incorporated. The analyses included 101 specimens from the ingroup representing 49 taxa. Sequences from both DNA regions were analysed simultaneously using direct optimization under the parsimony optimality criterion. The results support the inclusion of the crustose species and Muhria in Stereocaulon, while the current infrageneric classification is not supported. As Muhria is securely nested within Stereocaulon the new combination Stereocaulon urceolatum comb. nov. (syn. Muhria urceolata) is made. Further, species concepts need to be re-examined, as some species do not appear as monophyletic entities in the phylogeny.     

Fungal Infection of Mantis Shrimp (<Emphasis Type="Italic">Oratosquilla oratoria</Emphasis>) Caused by Two Anamorphic Fungi Found in Japan

Two fungal pathogens of the mantis shrimp (Oratosquilla oratoria) in Yamaguchi and Aichi Prefectures, Japan are described as the new species Plectosporium oratosquillae and Acremonium sp. (a member of the Emericellopsis marine clade). Both fungi infect the gills of the mantis shrimp, which become brown or black due to melanization. The former species is characterized by its slow growth on artificial seawater yeast extract peptone glucose (PYGS) agar, pale yellow to pale orange or grayish yellow colonies, short cylindrical solitary phialides with a wavy tip, and one-celled ellipsoidal conidia. Although lacking the two-celled conidia demonstrated by the type species Plectosporium tabacinum, the taxonomic placement of the new species was confirmed by DNA sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA (ITS1, 5.8S rDNA and ITS2). Acremonium sp., the other causal pathogen, differs from P. oratosquillae by its fast growth on PYGS agar, pale orange to salmon-colored colonies, long, slender conidiophores consisting of solitary phialides with tips lacking an undulate outline, and typically cylindrical conidia. Analysis of ITS and β-tubulin gene sequences placed this fungus within the phylogenetically distinct Emericellopsis (anam. Acremonium) marine clade. Various physiological characteristics of both pathogens were also investigated. This is the first report of a fungal infection found on the mantis shrimp in Japan.     

A molecular phylogeny of the genus Gagea (Liliaceae) in Germany inferred from non-coding chloroplast and nuclear DNA sequences

The systematics of the mainly yellow flowered Gagea species complex (Liliaceae) has long been considered difficult because only a few phenotypic features within this genus and as a result of hypothesized interspecific hybridisation. A molecular phylogenetic study of seven Gagea species (G. bohemica, G. lutea, G. minima, G. pomeranica, G. pratenis, G. spathacea and G. villosa) from Germany has been undertaken, based on plastid DNA sequences (trnL(UAA)-trnF(GAA), psbA-trnH) and on the nuclear ribosomal internal transcribed spacer (ITS). Sequence divergence within the Gagea species ranges up to 15.5% for psbA-trnH, 22.0% for trnL-trnF and 23.7% for ITS (ITS1 + 5.8S rRNA + ITS2). Two subspecies of Gagea bohemica: G. bohemica subsp. saxatilis and G. bohemica subsp. bohemica are characterized by trnL-trnF data and morphological features. Analysis of the ITS region shows that G. pomeranica represents a hybrid of G. pratensis and G. lutea. Lloydia serotina was initially used as an outgroup species, but it was placed within the investigated Gagea species in the psbA-trnH and the trnL-trnF phylogenetic tree.     

Molecular phylogenetic analyses of nuclear and plastid DNA sequences support dysploid and polyploid chromosome number changes and reticulate evolution in the diversification of Melampodium (Millerieae, Asteraceae)

Chromosome evolution (including polyploidy, dysploidy, and structural changes) as well as hybridization and introgression are recognized as important aspects in plant speciation. A suitable group for investigating the evolutionary role of chromosome number changes and reticulation is the medium-sized genus Melampodium (Millerieae, Asteraceae), which contains several chromosome base numbers (x = 9, 10, 11, 12, 14) and a number of polyploid species, including putative allopolyploids. A molecular phylogenetic analysis employing both nuclear (ITS) and plastid (matK) DNA sequences, and including all species of the genus, suggests that chromosome base numbers are predictive of evolutionary lineages within Melampodium. Dysploidy, therefore, has clearly been important during evolution of the group. Reticulate evolution is evident with allopolyploids, which prevail over autopolyploids and several of which are confirmed here for the first time, and also (but less often) on the diploid level. Within sect. Melampodium, the complex pattern of bifurcating phylogenetic structure among diploid taxa overlain by reticulate relationships from allopolyploids has non-trivial implications for intrasectional classification.     

Molecular phylogeny ofSalicaceae and closely relatedFlacourtiaceae: Evidence from 5.8 S, ITS 1 and ITS 2 of the rDNA

A ribosomal DNA region, including the entire 5.8S RNA gene and the internal transcribed spacers ITS 1 and ITS 2, was used for studying the phylogeny ofSalicaceae and the relationship betweenSalicaceae andFlacourtiaceae. The length of the ITS regions withinSalicaceae andFlacourtiaceae was similar to that found in other angiosperms. The GC content of both ITS regions was high, varying 62.7-72.2%. The most parsimonious tree clusters the wind-pollinatedChosenia bracteosa among theSalix species, suggesting that it should be included in the genusSalix. The grouping withinSalix leaves subg.Salix as paraphyletic, for which reason the subgeneric division is questionable.Populus was monophyletic and formed a sister group toSalix. The interspecific variation of the ITS sequences was very small inSalicaceae, which is in contradiction to the age of the group according to the evidence from fossil data.Idesia polycarpa fromFlacourtiaceae shows great sequence similarity withSalicaceae, but the analysis of 5.8S rDNA supports monophyly of the four species ofFlacourtiaceae sampled for this study.     

Chromosomal location of genes controlling seed proteins in species related to wheat

Summary The seed proteins of Chinese Spring wheat stocks which possess single chromosomes from other plant species related to wheat have been separated by gel electrophoresis in the presence of sodium dodecyl sulphate. Marker protein bands have been detected for both arms of barley chromosome 5, chromosome E (= 1R) and B (= 2R) of rye, chromosomes A,B (= 1C^u) and C (= 5C^u) of Aegilops umbellulata and chromosomes I and III of Agropyron elongatum. These studies, and previous findings, indicate that chromosome 5 of barley, chromosome 1R of rye, chromosome I of Ag. elongatum and possibly chromosome 1C^u of Ae. umbellulata are similar to chromosomes 1A, 1B and 1D in hexaploid wheat in that they carry genes controlling prolamins on their short arms and genes controlling high-molecular-weight (apparent molecular weight greater than 86,000) seed protein species on their long arms. These findings support the idea that all these chromosomes are derived from a common ancestral chromosome and that they have maintained their integrity since their derivation from that ancestral chromosome.     

Molecular phylogeny of Phebalium (Rutaceae: Boronieae) and related genera based on the nrDNA regions ITS 1+2

Parsimony analyses of the internal transcribed spacer regions of nuclear ribosomal DNA (ITS 1 & ITS 2) for 38 taxa sampled from the Phebalium group (Rutaceae: Boronieae) and two outgroups confirm that, with the exception of Phebalium sensu stricto and Rhadinothamnus, six of the currently recognised genera within the group are monophyletic. The data indicate that Phebaliums. str. is paraphyletic with respect to Microcybe, and Rhadinothamnus is paraphyletic with respect to Chorilaena. Rhadinothamnus and Chorilaena together are the sister group to Nematolepis. Drummondita, included as an outgroup taxon, clustered within the ingroup as sister to Muiriantha and related to Asterolasia.The phylogeny suggests that the evolution of major clades within a number of these genera (e.g. Phebalium) relates to vicariance events between eastern and south-western Australia. Leionema is an eastern genus, with the most basal taxon being the morphologically distinct Leionema ellipticum from northern Queensland. Leionema also includes one species from New Zealand, but this species (as with some others) proved difficult to sequence and its phylogenetic position remains unknown. Taxonomic changes at the generic level are recommended.The authors wish to thank Paul G.Wilson, PERTH, for advice and discussion, and Paul Forster, BRI, for collecting and providing material of Leionema ellipticum. The project was supported by a Melbourne University Postgraduate Award (to BM), the Australian Biological Resources Study (ABRS), Australian Systematic Botany Society and Wolf Den (Australia) Investments.     

Molecular phylogeny of Armillaria from the Patagonian Andes

A number of species in the plant pathogen genus Armillaria are known from South America where they cause root rot disease on a wide variety of hosts. Knowledge pertaining to phylogenetic relationships of these species with those of other Armillaria species is almost non-existent. In addition, very few cultures representing these species are available, making DNA-based phylogenetic analyses impossible. The aim of this study was to characterise a collection of Armillaria isolates from the Patagonian Andes using DNA sequences and to determine their phylogenetic relationships with other Armillaria species. DNA sequences were obtained from the internal transcribed regions (ITS1, 5.8S and ITS4) and ribosomal large subunit (LSU) gene and used in phylogenetic analyses. Phylogenetic trees generated from the sequences separated the Armillaria isolates into four lineages. Lineages I and II represented A. novae-zelandiae and A. luteobubalina, respectively. Isolates belonging to A. novae-zelandiae from Malaysia, New Zealand, Australia and South America showed considerable intra-clade sub-structure. Lineages III and IV are probably distinct species and are most closely related to A. hinnulea and an unnamed species isolated from New Zealand and Kenya. This is the first comprehensive study of the phylogenetic relationships of Armillaria species from Patagonia and it provides a foundation for future research in this region.     

四照花亚属的nrDNA ITS致同进化不完全

四照花亚属(Cornus subg.Syncarpea)隶属于山茱萸科山茱萸属(Cornus),我国该亚属共有5种8亚种。为探讨四照花亚属nrDNA ITS序列的致同进化不完全现象及假基因产生的可能原因,分析了该亚属4种(每种1~2个居群)共21个个体的nrDNA ITS序列。结果表明,这些类群的nrDNA ITS存在多态性,通过分析这些nrDNA ITS克隆序列的G+C含量、5.8S保守基序和二级结构最小自由能,推测其可能存在假基因。系统发育研究结果显示所有nrDNA ITS序列分成5个分支,同一个体的不同拷贝被分别置于两个甚至多个分支中,且不同分支显示了不同种间关系。四照花亚属物种个体内部存在nrDNA ITS不完全致同进化,可能归咎于不完全的世系分选(incomplete lineage sorting)、种间杂交或多倍化等进化事件,从而导致基因组内nrITS区序列出现多态性,同时也导致难以通过外部形态来划分亚属内种间界限。     

Phylogenetics of the slipper orchids (Cypripedioideae, Orchidaceae): Nuclear rDNA ITS sequences

Cypripedioideae (Orchidaceae) have been the subject of numerous taxonomic treatments with conflicting interpretations of relationships among the five genera and the 150–170 species. We have produced nuclear ribosomal ITS nucleotide sequences for nearly 100 slipper orchid species and used parsimony analysis to investigate their relationships. Our results demonstrate that each genus, as currently circumscribed, is monophyletic (Mexipedium andSelenipedium being represented by a single taxon). LikerbcL data, ITS sequences placeMexipedium sister toPhragmipedium. Relationships at the sectional level inPaphiopedilum are largely as described byCribb. However, the division ofPaphiopedilum into subgg.Brachypetalum andPaphiopedilum is not supported; subg.Brachypetalum is paraphyletic to subg.Paphiopedilum. Phragmipedium species are divided into the same three major clades as in the taxonomic scheme ofMcCook. The plicate-leaved genera,Cypripedium andSelenipedium, are successive sister groups to the rest of the subfamily, confirming generally held opinions that they display plesiomorphic characters compared to the conduplicate-leaved genera. A survey of karyotypes in the context of the ITS tree reveals a general trend toward increased chromosome number, probably brought about by centric fission. These data also accord with a previously suggested biogeographic hypothesis of a widespread Northern Hemisphere distribution, followed by range fragmentation due to Miocene cooling.     

Molecular authentication of ginseng cultivars by comparison of internal transcribed spacer and 5.8S rDNA sequences

Molecular authentication among three Panax species and within cultivars and accessions of P. ginseng was investigated using the DNA sequence in the ribosomal ITS1–5.8S–ITS2 region. Four single-nucleotide polymorphisms were identified between P. ginseng and other Panax species. In the electrophoresis profile, obtained after digestion with the enzyme TaqI, three fingerprinting patterns were obtained from cultivars and accessions of Panax species. Consequently, this authentication procedure based upon the restriction fragment length polymorphism in the ribosomal ITS1–5.8S–ITS2 region can now be utilized to differentiate these Panax species as well as major Korean cultivars such as Gopoong and Kumpoong from other cultivars and accessions in Panax species at the DNA level. O. T. Kim and K. H. Bang contributed equally to this paper.