Chemotaxonomical investigations of theSymphytum officinale polyploid complex andS. asperum (Boraginaceae): The pyrrolizidine alkaloids

By means of thin layer chromatography in conjunction with mass spectrometry the pyrrolizidine alkaloid patterns derived fromSymphytum asperum, several cytotypes ofS. officinale agg. and the artificial hybrids of the former taxa, were compared. The obtained patterns were not essentially affected by variation in cytotype, harvesting times and -location of plants. Lycopsamine, acetyl-lycopsamine and symphytine or their isomers were generally found in theS. officinale cytotypes, echimidine and symphytine inS. asperum. The interspecific hybrids contained all alkaloids mentioned. The definite lack of echimidine in the 2 n=40 cytotype proves that it is conspecific withS. officinale and does not belong to a hybrid-swarmS. asperum × S. officinale with 2 n=48. Part I of this series of contributions.     

Ribosomal DNA variation within and among individuals ofLisianthius (Gentianaceae) populations

Restriction endonuclease fragment analysis of nuclear ribosomal DNA (rDNA) was completed on 25 individuals each from seven populations of theLisianthius skinneri (Gentianaceae) species complex in Panama. Seven restriction enzymes were used to determine the amount and type of rDNA variation within and among individuals of the populations. No restriction site variation was seen within populations or individuals although site differences were seen among populations. Spacer length variation within and among individuals of populations was mapped to the internal transcribed spacer (ITS) region between the 18S and 5.8S rRNA genes, a region inLisianthius rDNA that previously was shown to exhibit length differences among populations. This is the first reported case of such variation within and among individuals of populations for the ITS region. Presence or absence of ITS spacer length variation is not correlated with levels of isozymic heterozygosity within populations. No detectable length variation within individuals or populations was seen in the larger intergenic spacer (IGS). Although populations varied with respect to IGS length, all individuals of a given population had a single and equivalent IGS length.     

Molecular phylogenetics of the subtribeGentianinae (Gentianaceae) inferred from the sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA from representatives of 23 species of the subtribeGentianinae and one outgroup species (Centaurium capitatum) were analyzed by polymerase chain reaction amplification and direct DNA sequencing. Within the taxa analyzed, the length of the ITS1 region varied from 221 to 233 bp, ITS2 from 226 to 234 bp. Of the aligned sequences of 497 positions, 151 sites involved gaps or nucleotide ambiguity, 133 were invariable and 213 showed divergence. In pairwise comparisons among the taxa of the subtribeGentianinae and the outgroup, sequence divergence ranged from 1.3% to 34.1% in ITS1, from 0 to 28.1% in ITS2 and from 0.6% to 27.5% in combined ITS1 and ITS2. Phylogenetic trees generated from ITS sequences were highly resolutive and principally concordant with morphological classifications for the major phylogenetic divisions in the subtribe. An ancient divergence leading to two evolutionary lines was suggested in the subtribe by both DNA sequence and morphological data. One line encompasses the generaGentiana, Crawfurdia andTripterospermum, morphologically characterized by their glands on the base of ovary and their plicate corolla, while the other line involves all other members of the subcribe surveyed, characterized by their epipetalous glands and simple corolla without plicae.Megacodon, with glands on the base of ovary but without plicae on its corolla, was revealed to be more related to the latter group than to the former.Comastoma, Gentianella andGentianopsis were shown to be well-defined monophyletic genera.Pterygocalyx showed much closer affinity toGentianopsis than to any other genus. Some conflictions were detected in the genusSwertia.     

Aestival and autumnal vicariads ofGentianella (Gentianaceae): A myth?

Plant samples were collected from 13 populations of the two speciesGentianella germanica andG. campestris. In addition, two samples from populations of grazed plants lacking their main stems were collected. A number of relevant morphological characters were measured, the flowering time, and the habitats of each population were studied. Some ecotypes ofG. germanica with clear differences in flowering time were recognized. Differences in flowering time and morphology between populations ofG. campestris seem to be clinal. The generally accepted theory of seasonal dimorphism with the assumption of separate aestival and autumnal vicariads proves to be inadequate. A hypothesis based upon the effect of human influences on the origin of this ecotypes is suggested.     

Karyological studies onGentiana sect.Chondrophyllae (Gentianaceae) from China

Nineteen populations of fifteen species ofGentiana sect.Chondrophyllae from China were observed cytologically.Gentiana alsinoides, G. anisostemon, G. asterocalyx, G. exigua, G. heterostemon, G. intricata, G. praticola, G. pseudoaquatica, G. spathulifolia, andG. subintricata all had the same chromosome number of 2n = 20 (or n = 10), whereasG. piasezkii had 2n = 36,G. squarrosa 2n = 38,G. prattii 2n = 18,G. aristata 2n = 14 (n = 7), andG. heleonastes 2n = 12. All these chromosome numbers are documented here for the first time, except forG. squarrosa, where it is a new number report. The basic numbers of x = 6, x = 7 and x = 19 are new for the section. Karyotype analyses of some species revealed that, except for a few cases, the species examined mainly had metacentric chromosomes. 2n = 20 = 2m(SAT) + 18m was found to be the main type of karyotype for the species with 2n = 20. Chromosomal evolution and its mechanism in this section are also discussed.     

The evolution of approach herkogamy from protandry in New ZealandGentiana (Gentianaceae)

Most mainland New Zealand species ofGentiana are protandrous as are manyGentianaceae elsewhere in the world. Selfing has evolved in some species confined to areas where pollinator availability is limited by weather, for example, inG. lineata. InG. chathamica, endemic to the Chatham Islands, dichogamy has been lost without the evolution of selfing; instead the flowers are approach herkogamous. It is suggested that as herkogamous blossoms can both receive and dispatch pollen during a single insect visit, whereas dichogamous systems usually require two visits, such a system may have evolved inG. chathamica to maintain outcrossing in an area where prospects for insect pollination are poor.     

Chemotaxonomic study of the genusTabernaemontana (Apocynaceae) based on their indole alkaloid content

According to their alkaloidal products species of the new genusTabernaemontana can be partly differentiated. This differentiation is in agreement with the old genera classification. From the chemotaxonomic point of view a subdivision of subfam.Plumerioideae of theApocynaceae is proposed.     

The phylogenetic relationships and evolution of the Canarian laurel forest endemicIxanthus viscosus (Aiton) Griseb. (Gentianaceae): Evidence frommatK and ITS sequences, and floral morphology and anatomy

The phylogenetic relationships of the Canarian laurel forest endemicIxanthus viscosus (Aiton) Griseb. (Gentianaceae) are investigated through a cladistic analysis of sequence variation of parts of the chloroplast genematK and the ITS region of nuclear ribosomal DNA. The floral anatomical and morphological characteristics ofIxanthus based on paraffin sections and SEM studies are also discussed. In the molecular analysesIxanthus is part of a clade of mostly temperate Erythraeinae and Chironiinae sensu Gilg, in which it is sister to the mostly Mediterranean annualBlackstonia. This relationship is supported by farreaching similarities in flower morphology and anatomy, and the shared possession of at least some perfoliate leaves. This finding contradicts most former classifications in whichIxanthus was placed either in subtribe Gentianinae which never includedBlackstonia, or in a subtribe of its own which was suggested to connect woody tropical with herbaceous temperate taxa. The phylogenetic relationships found here show that 1) the sister group ofIxanthus is distributed in the Mediterranean area, and that 2) its habit as a basally woody suffrutescent herb is best interpreted as an advanced character state.     

川东獐牙菜(龙胆科)的胚胎学研究

利用石蜡切片技术,对川东獐牙菜(Swertia davidii)的胚胎发育过程进行显微观察,并根据现有资料,对獐牙菜属的几种植物进行了比较胚胎学研究。结果表明:川东獐牙菜花药四室,药壁发育为基本型,绒毡层异型起源,为腺质绒毡层,发育后期药室内观察到的绒毡层核是早期该层细胞有丝分裂凸入药室并原位退化形成的,中层细胞3层,药室内壁退化,花药壁表皮宿存,细胞柱状伸长,纤维状加厚;小孢母细胞减数分裂为同时型,四分体排列方式主要为四面体形和左右对称型,少数为"T"形和十字交叉形,成熟花粉为2-细胞类型;子房2心皮、1室,侧膜胎座;薄珠心,单珠被,倒弯生胚珠,大孢子母细胞减数分裂形成4个大孢子直线形排列,合点端的大孢子具功能,雄配子体发育为蓼型;2个极核在受精前融合为1次生核,合点端3个反足细胞宿存,每个细胞均多核和异常膨大,形成明显的反足吸器,并在胚乳之外形成染色较深的类似"外胚乳"结构;珠孔受精,受精作用属于有丝分裂前类型;胚乳发育为核型,胚胎发育为茄型;果实成熟时,种子发育至心形胚阶段;反足细胞在龙胆科一些短命植物中的宿存与分裂具有重要的生殖适应与进化意义。     

Cytogenetic investigations onOenothera wolfii (Onagraceae)

Oenothera wolfii, endemic in coastal Northern California and Southern Oregon, has been analysed cytogenetically. It is a permanent structural heterozygote composed of two nearly identical genomic complexes which are closely related to the neighboring maritime ecotype of the homozygousOe. elata subsp.hookeri. — Oe wolfii is believed to have evolved recently from its homozygous ancestor by the accumulation of reciprocal translocations and the acquisition of balanced lethals. Forms such asOe. wolfii represent an important connecting link in our understanding of the evolution of the complexheterozygous species ofOenothera. Dedicated to Prof. Dr.Josef Straub for His 75th birthday.     

Chromosomal evolution ofGentiana andJaeschkea (Gentianaceae), with further documentation of chromosome data for 35 species from western China

Chromosome numbers were recorded for 63 populations of 34 species belonging to the genusGentiana from the high altitude regions of western China. Counts for 22 species were reported for the first time and new numbers were found forG. heleonastes (2n = 36),G. prattii (2n = 20) andG. pseudoaquatica (2n = 40). Incorporating previous data, a complete series of gametic chromosome numbers from n = 6 to 24 and 26 was established for the genus, suggesting rapid karyotypic evolution by a combination of dysploidy and polyploidy. The cytotype 2n = 20 is proposed as the ancestral type in sect.Chondrophyllae s. l. The chromosome number 2n = 16 was found forJaeschkea microsperma for the first time which, with previous reports of 2n = 18, 20 and 22, indicates thatJaeschkea is a typically dysploid genus.     

Generic delimitation and phylogenetic relationships within the subtribe Chironiinae (Chironieae: Gentianaceae), with special reference to Centaurium: evidence from nrDNA and cpDNA sequences

To better understand the evolutionary history of the genus Centaurium and its relationship to other genera of the subtribe Chironiinae (Gentianaceae: Chironieae), molecular analyses were performed using 80 nuclear ribosomal ITS and 76 chloroplast trnLF (both the trnL UAA intron and the trnL-F spacer) sequences. In addition, morphological, palynological, and phytochemical characters were included to a combined data matrix to detect possible non-molecular synapomorphies. Phylogenetic reconstructions support the monophyly of the Chironiinae and an age estimate of ca. 22 million years for the subtribe. Conversely, both molecular data sets reveal a polyphyletic Centaurium, with four well-supported main clades hereafter treated as separate genera. The primarily Mediterranean Centaurium s.s. is closely related to southern African endemics Chironia and Orphium, and to the Chilean species Centaurium cachanlahuen. The resurrected Mexican and Central American genus Gyrandra is closely related to Sabatia (from eastern North America). Lastly, the monospecific genus Exaculum (Mediterranean) forms a monophyletic group together with the two new genera: Schenkia (Mediterranean and Australian species) and Zeltnera (all other indigenous American centauries). Several biogeographical patterns can be inferred for this group, supporting a Mediterranean origin followed by dispersals to (1) North America, Central America, and South America, (2) southern Africa (including the Cape region), and (3) Australia and Pacific Islands.     

Karyological investigations inRanunculus subg.Batrachium (Ranunculaceae) on the Aegean islands

This paper is the first report of an investigation on differentiation and evolutionary pattern inRanunculus subg.Batrachium mainly on the Aegean islands and treats the karyology in the subgenus. The following taxa and chromosome numbers are recorded.R. peltatus subsp.peltatus: 2n = 32, subsp.baudotii: 2n = 32, subsp.saniculifolius: 2n = 16, 32, and 40;R. peltatus s.l.: 2n = 32 and 48;R. penicillatus subsp.pseudofluitans: 2n = 16;R. rionii: 2n = 16;R. sphaerospermus: 2n = 16;R. trichophyllus: 2n = 32, 40, and 48;R. tripartitus: 2n = 48. The karyotypes show certain interspecific differences in chromosome size, in number of chromosome types according to centromere position and in occurrence and position of satellite chromosomes. r-Index and RCL have been calculated.     

Cytogenetic investigations onOenothera nutans (Onagraceae)

Oenothera nutans, common to the Appalachian Mts between 650 and 1 700 m altitude, was investigated cytogenetically and taxonomically. The species is permanently structurally heterozygous. It consists of two genomes of the B-type which are more or less indistinguishable phenotypically. Nearly all of the strains investigated possess a self-incompatibility factor in one of the two complexes. Both complexes show a close relationship to the predominantly homozygousO. grandiflora, a native of the southern lowlands.O. nutans andO. grandiflora possess the same plastid type, plastome III. Probably,O. nutans evolved by an accumulation of reciprocal translocations within an originally structurally homozygous population, which must be regarded ancestral to the present forms ofO. grandiflora.     

Knotenbau und Blattgrundvaskularisation bei einigenGentianaceae

As a rule,Gentianaceae exhibit a double unilacunar nodal pattern. Upon leaving the lacun, the complex leaf trace divides first into three branches, and then into five or more bundles which become the leaf veins. OnlyGentiana lutea (andG. punctata) as well asMenyanthes trifoliata differ from all other species investigated by their multilacunar nodes. BothCentaurium andOrphium within the cortex form a completely closed bundle ring around the nodal flanks from smaller bundles which depart from the lateral branches of the complex leaf traces.

     

Genetic transformation of Centaurium erythraea Rafn by Agrobacterium rhizogenes and the production of secoiridoids

Hairy roots of Centaurium erythraea were obtained by infection with Agrobacterium rhizogenes strain LBA 9402. They spontaneously regenerated adventitious shoots in Woody Plant liquid medium without growth regulators. The shoots were grown continuously in Murashige and Skoog (MS) liquid or agar solidified media supplemented with 0.1 mg l⁻¹ indole-3-acetic acid and 1.0 mg l⁻¹ 6-benzylaminopurine. These shoots produced roots 4 weeks after transfer into agar-solidified MS medium without phytohormones. Regenerated plants grown and flowered under greenhouse conditions. The transgenic value of the regenerated plants was confirmed by the polymerase chain reaction amplification. Transformation by Agrobacterium rhizogenes alters plant morphology and production of secoiridoid glucosides. The level of secoiridoids was also modified by development stage of transformed plants. The total content of the compounds (expressed as the sum of gentiopicroside, sweroside and swertiamarin) in 10-week old pRi-transformed regenerants was 280 mg g⁻¹ dry weight and was 8-times the content in the sample of commercially available C. erythraea herb.     

A new species of Tachia (Gentianaceae) from Suriname

A new species ofTachia,T. longipes, is described and illustrated. It is distinct from the other species ofTachia not only in its leaf and stem morphology but also in its characteristic pollen. A revised key to the neotropical species ofTachia is also provided. Studies for theFlora of the Guianas. No. 87.     

Chemical constituents of the rhizomes and roots of Gentiana scabra (Gentianaceae)

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Taxonomical investigations onHelleborus viridis s. l. (Ranunculaceae) in Northern Italy

The taxon commonly namedHelleborus viridis in Lombardy (NW. Italy) differs from both, subsp.viridis and subsp.occidentalis, and is shown to fall within the variation range ofH. odorus subsp.laxus which has been reported so far for NE. Italy only. TrueH. viridis within Italy grows only in the Maritime Alps.     

A re-evaluation of morphological characters in European Gentianella section Gentianella (Gentianaceae)

Morphological traits were investigated in Gentianella section Gentianella by morphometrics and Scanning Electronic Microscopy (SEM). Variation in vegetative measures and in calyx, corolla, ovary, and gynophor was analyzed in populational samples and herbarium material. Special emphasis was given to micro-morphology and variation of papillae on the calyx using SEM. Three types of papillae on the calyx lobes were found: (A) short conical (G. amarella-group, G. insubrica, G. germanica); (B) long conical often curved (G. campestris, G. anisodonta, G. engadinsesis, and G. liburnica; (C) long cylindrical (G. aspera and G. pilosa). G. austriaca, G. caucasea, G. crispata, G. fatrae, and G. ramosa usually lack any papillae. Plants without and with short conical papillae were found in G. bulgarica and G. lutescens as well as in the intermediate taxa G. bohemica and G. stiriaca. The different types of papillae together with other calyx characters (sinus, shape and margin of lobe) are of high systematic importance and provide more stable characters than morphometric flower measures. Principal component and correlation analysis revealed a strong response of nearly all morphometric traits to the environmental variable altitude. Adaptive and historical causes of morphological variation as well as taxonomical consequences are discussed and a determination key is provided.