Phylogenetic relationships of the enigmatic species Serratula chinensis and Serratula forrestii (Asteraceae - Cardueae)

The phylogenetic relationships of the Chinese species Serratula forrestii and S. chinensis within the subtribe Centaureinae (Asteraceae – Cardueae) were investigated based on nuclear ribosomal DNA external and internal transcribed spacer sequences. Results indicated that Serratula s. l. is clearly polyphyletic, none of the two species showing close affinities to either Serratula s. str. or Klasea, a genus traditionally included in Serratula. Serratula forrestii has a basal position within the subtribe and has retained most plesiomorphic character states. Serratula chinensis is related to the genera of the Rhaponticum group. Its chromosome number was determined as 2n = 26, and the relationship between this species and the Rhapon-ticum group is discussed from a morphological and karyological point of view.     

Phylogenetic analyses and chromosome evolution in Convallarieae (Ruscaceae sensu lato), with some taxonomic treatments

The tribe Convallarieae, comprising 10 genera and 95 species, has recently been transferred from its own family to Ruscaceae sensu lato. In this study, sequence data from trnK and rbcL were analyzed for 19 species in 8 genera, and chromosome morphology was analyzed for 17 species in 7 genera. The parsimony analysis of trnK and rbcL sequences showed that Convallarieae are monophyletic. Although early branches did not receive strong bootstrap support, Convallaria diverged at the first branch, followed by Speirantha. The rest of the tribe was split into three, well-supported clades: one with Reineckea, the second with Campylandra and Rohdea, and the third with Tupistra, Tricalistra, and Aspidistra. Two monotypic genera, Rohdea and Tricalistra, were embedded in a clade of Campylandra and of Tupistra, respectively. Three karyotypes were distinguished in the tribe on the basis of the basic number and morphology of metaphase chromosomes: Convallaria type (with x=19 and unimodal chromosome length), Tupistra type (with x=19 and trimodal chromosome length), and Aspidistra-elatior type (with x=18 and trimodal chromosome length). The character-state distribution in the molecular tree showed that the Convallaria type is plesiomorphic, from which was derived the Tupistra type and subsequently the Aspidistra-elatior type. Taxonomic treatments to transfer Campylandra to Rohdea and Tricalistra to Tupistra are also given.     

Paraphyly of Veronica (Veroniceae; Scrophulariaceae): Evidence from the Internal Transcribed Spacer (ITS) Sequences of Nuclear Ribosomal DNA

Veronica (Veroniceae; Scrophulariaceae) and segregated genera, such as Hebe from New Zealand has been debated intensively in the past. We conducted an analysis of sequence data from the internal transcribed spacer region of nuclear ribosomal DNA (ITS) to evaluate the validity of segregate genera and the monophyly of Veronica. According to the results presented here, Veronica is paraphyletic, with the Hebe complex, Synthyris, and Paederota nested within the larger Veronica clade. Pseudolysimachion is in a basal polytomy of the expanded Veronica clade in the strict consensus tree and might be nested within Veronica as well. Clades within Veronica do not correspond to sections traditionally recognized. This study provides a first estimation of the phylogeny of Veroniceae using molecular data and can serve as a starting point for future investigations of Veronica and relatives. Received 24 July 2000/ Accepted in revised form 19 October 2000     

Phylogenetic relationships of JapaneseAster (Asteraceae, Astereae) sensu lato based on chloroplast-DNA restriction site mutations

RFLPs of cpDNA were examined for 18 species ofAster, six species ofKalimeris, two species ofMiyamayomena and one species and one variety ofHeteropappus from Japan, using 16 restriction endonucleases. Approximately 275 restriction sites were surveyed, and a total of 74 restriction site mutations was detected, and 31 of these were phylogenetically informative. Sixteen most parsimonious trees constructed from Wagner parsimony analysis indicated the polyphyly ofKalimeris andMiyamayomena sensu Kitamura;K. miqueliana belongs to a different clade from the remaining species ofKalimeris, and two species ofMiyamayomena did not make a single clade. This result suggests that the shortening or loss of pappus have happened parallelly in different evolutionary lineages. We must be careful to assess the pappus character in taxonomy and phylogeny, and it is desirable to examine their phylogenetic relationships using a molecular data.     

The position ofCastrilanthemum Vogt & Oberprieler and the phylogeny of Mediterranean Anthemideae (Compositae) as inferred from nrDNA ITS and cpDNAtrnL/trnF IGS sequence variation

Sequences of the nrDNA internal transcribed spacer (ITS) region and the cpDNAtrnL/trnF intergenic spacer (IGS) region were analysed for 41 Mediterranean and Eurasian representatives of the Anthemideae (Compositae) to ascertain the systematic position of the unispecific genusCastrilanthemum Vogt & Oberprieler and to study the phylogeny of the Anthemideae in the Mediterranean region. Both markers consistently point to the close relationship ofCastrilanthemum with the W Mediterranean generaLeucanthemopsis (Giroux) Heywood andProlongoa Boiss., forming a strongly supported monophyletic group (theLeucanthemopsis-group which also comprises the unispecific genusHymenostemma (Kunze) Willk.). Results also demonstrate that subtribes Achilleinae, Leucantheminae, and Matricariinae sensu Bremer & Humphries are non-monophyletic. Besides results from sequence variation, a marked 5bp-deletion intrnL/trnF IGS divides all these subtribes into more basal subgroups which are related to each other and to the large Eurasian generaTanacetum L. andAnthemis L., and a monophyletic group of closely related and more advanced subgroups which also contain the monophyletic Chrysantheminae sensu Bremer & Humphries. For this second group a W Mediterranean centre of diversification is suggested, however, its sister-group relationships within the basal grade of generic groups remain unclear.     

Molecular authentication of Anthemis deserti Boiss. (Asteraceae) based on ITS2 region of nrDNA gene sequence

The dried plant material of medicinally important Anthemis deserti Boiss. (family: Asteraceae) especially when it remains in the powdered form often look similar to Anthemis melampodina Del.; and therefore, difficult to distinguish, finally lead to chances of adulteration. The adulteration in medicinal plants effects on the efficacy of the drugs. The molecular authentication of herbal plant materials such as based on the internal transcribed spacer 2 (ITS2) sequences of nuclear ribosomal DNA (nrDNA) is considered as more reliable method compared to other the biochemical or histological methods. The present study aims to molecular authentication ofA. deserti based on molecular phylogenetic analyses of ITS2 gene sequence of nrDNA region. The ITS2 region of nrDNA of A. deserti were sequenced, and the molecular phylogenetic analyses were performed together with the GenBank sequences. The Maximum Parsimony tree revealed the close relationships of A. deserti with A. melampodina; however, the Neighbor-Joining and Maximum Likelihood tree clearly revealed that A. deserti is distinct from A. melampodina, which is also supported by the differences in nucleotides at five diffident positions (i.e. 22, 28, 87, 175 and 198) in the DNA sequence alignment.     

Internal transcribed spacer sequence analyses indicate cytoevolutionary patterns within Brachycome Cass. (Asteraceae)

The sequences of the Internal Transcribed Spacer regions (ITS1 and ITS2) within the genes coding for cytoplasmic ribosomal (r) RNAs on the A chromosome complement of 34 members of the higher plant genus Brachycome (synonym Brachyscome) have been compared. The ITS1 sequence of species within the B. lineariloba complex contains a 56 bp tract that is absent from at least 12 Brachycome species but is present in other species within Brachycome as well as other Asteraceae. Phylogenetic data support the suggestion that the number of chromosomes reduced in several independent Brachycome lineages during speciation. Comparisons with the B chromosome ITS2 of B. dichromosomatica cytodeme A1 suggests an origin of the B chromosome at a time prior to the divergence of the four cytodemes of B. dichromosomatica.     

Karyomorphology of Maianthemum sensu lato (Polygonatae, Ruscaceae)

We report results of karyotype analyses using nine species of Maianthemum from China. The species, except M. atropurpureum (with 2n=72), had 2n=36, and the results support the earlier suggestion that Maianthemum has x=18 with 2n=36 in most species. The species examined, however, showed marked differences in karyotype, particularly in the numbers of metacentric, submetacentric, and acrocentric chromosomes as well as in the number of satellites. In addition, we distinguished three different modes based on the number of clear gaps in chromosome length variation: unimodal, bimodal, and trimodal. The unimodal variation (with no gap) was found in M. dahuricum and M. atropurpureum, the bimodal variation (with one gap) in M. tatsienense, and the trimodal variation (with two gaps) in M. bifolium, M. forrestii, M. japonicum, M. henryi, M. purpureum, and M. lichiangense. In the trimodal variation, the positions of the two gaps may differ from species to species. In addition, the frequency of acrocentric chromosomes per complement was generally higher in the trimodal variation than in the unimodal and bimodal variations. Results of our analyses, which had not been clearly presented prior to this, may provide a better understanding of species evolution in the tribe Polygonatae.     

Phylogeny ofCyclamen L. (Primulaceae): Evidence from morphology and sequence data from the internal transcribed spacers of nuclear ribosomal DNA

A molecular phylogenetic study of the genusCyclamen L. (Primulaceae) has been undertaken, based on sequence data from the 5.8S gene in the ribosomal nuclear DNA and its flanking internal transcribed spacers ITS1 and ITS2. Sequence data from 15 species ofCyclamen and seven outgroup taxa selected from Primulaceae and Myrsinaceae were analyzed phylogenetically. A second analysis based on a combined morphological and molecular dataset was performed to evaluate earlier hypotheses of character evolution in the genus. The results indicate that four monophyletic subgroups may be recognized in the genus, viz.Cyclamen, Psilanthum Schwarz,Eucosme Schwarz andGyrophoebe Schwarz. Each of the four subgenera is diagnosed by distinct basic chromosome number, as well as by morphological and molecular characteristics.     

Biogeographical and evolutionary insights onErigeron and allies (Asteraceae) from ITS sequence data

Complex geographical distribution patterns and agamospermy have united to obscure systematic relationships in the genusErigeron (400+ species). Sequence data were analyzed from the internal transcribed spacer region of nrDNA (ITS) and 5.8S cistron for 77 taxa including 63Erigeron species. Results support the North American origin ofErigeron and document thatAphanostephus, Conyza, and three small genera restricted to South America (Apopyros, Hysterionica, Neja) are all derived from withinErigeron. Phylogenetic data indicate that agamospermy has arisen at least three times inErigeron and that autogamy has evolved independently inConyza and inE. sect.Trimorpha.     

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.     

The evolution of Dactylorhiza (Orchidaceae) allotetraploid complex: insights from nrDNA sequences and cpDNA PCR-RFLP data

Sequence data from a portion of the external transcribed spacer (ETS) and from the internal transcribed spacers (ITS1 and ITS2) of 18S-26S nuclear ribosomal DNA were used together with chloroplast DNA PCR-RFLP data to unravel patterns of allotetraploid speciation within the Western European Dactylorhiza polyploid complex. A maximum likelihood tree based on combined ETS and ITS sequences suggests that the Western European Dactylorhiza allotetraploids have evolved by hybridization between four main diploid lineages. Cloned sequences and the topology of the ITS plus ETS tree indicate that the allotetraploid species D. elata, D. brennensis, and D. sphagnicola have originated from the autotetraploid D. maculata together with the diploid D. incarnata, while D. majalis, D. traunsteineri, and D. angustata seem to have evolved by hybridization between the D. fuchsii s.str and D. incarnata lineages. Finally, the diploid D. saccifera lineage seems to have been involved together with the D. incarnata lineage in the formation of the allotetraploid D. praetermissa. The observed congruence between the chloroplast tree and the ITS/ETS tree suggests a directional evolution of the nrDNA after polyploidization in favor of the maternal genome. Considered together with morphological, biogeographical, and ecological evidence, the molecular analysis leads us to recognize four species within the investigated allotetraploid complex, namely D. majalis, D. praetermissa, D. elata, and D. sphagnicola.     

The anthers of Senecio vulgaris (Asteraceae): saltatory evolution caught in the act

Anthers of the common annual weed, Senecio vulgaris, show an incomplete development of the two adaxial pollen sacs (microsporangia, MS). One or both adaxial MS can be missing, or they are replaced by sterile lobes. The reduction is stronger in the derived subspecies, S. vulgaris var. vulgaris than in the ancestral subspecies, S. vulgaris ssp. denticulatus. This character in S. vulgaris differs from the usual complete reduction of adaxial MS in other, independent instances of disporangiate anthers in the Asteraceae. It corresponds to the transition phenotypes associated with various recombinant genotypes derived from artificial crosses between tetrasporangiate (4 MS) and disporangiate (2 MS) species in the Asteracean genus Microseris. Senecio vulgaris could be a rare natural instance of homozygosity for a major gene permitting reduction of the adaxial MS in which the expression of the reduced phenotype is determined by different numbers of modifiers in the two subspecies.     

Minor alkamides from Heliopsis longipes S.F. Blake (Asteraceae) fresh roots

From the fresh roots of Heliopsis longipes three new minor alkamides: longipinamide A (N-isobutyl-8,10-diynoic-3Z-undecenamide), longipenamide A (N-isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide) and longipenamide B (N-isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide); three known alkamides: affinin (spilanthol, N-isobutyl-2E,6Z,8E-decatrienamide), N-isobutyl-2E,6Z-decadienamide and N-isobutyl-2E-decenamide; and 11 other known compounds were isolated. The structures of the three new minor alkamides were established by 1D and 2D NMR spectroscopy including ¹H, ¹³C, DEPT, COSY, HSQC, and HMBC experiments, as well as by EI and FAB⁺ mass spectrometry. To our knowledge, this is the first report of the isolation of linear dihydroxyalkamides as natural products.     

The taxonomic relationships within the genus Excoecaria L. (Euphorbiaceae) based on leaf morphology and rDNA sequence data

The tropical Indo-Pacific genus Excoecaria L. (Euphorbiaceae) has several closely related species in Australia whose taxonomic relationships are unclear. The most widely reported species in Australia is the mangrove species Excoecaria agallocha L. (type species), whose taxonomic and geographic limits are difficult to define from its closely related species or sub-species. Two additional taxa have also been described but not clearly differentiated from the type species: Excoecaria dallachyana Baillon and Excoecaria ovalis Endl. This project aimed to determine the taxonomic relationships of the Australian Excoecaria species using both leaf morphological data and DNA sequence data from the internal transcribed spacer (ITS) region of ribosomal genes. The nucleotide differences in the examined ITS1 region show that E. agallocha from eastern Australia and E. ovalis from Western Australia respectively, are genetically uniform within species but differ from each other consistently, thus supporting species status. The leaf morphological data also support this view: single factor analysis of variance consistently separated E. ovalis from E. agallocha on the basis of leaf width, leaf length and length of petiole. In contrast, E. ovalis from the Gulf of Carpentaria differs only slightly from E. ovalis in Western Australia, but no evidence was found to suggest any leaf morphological differentiation within this species. The analysis also suggests that E. dallachyana is not closely related to either mangrove species E. agallocha or E. ovalis, despite superficial morphological similarities.     

Morphological evolution and ecological diversification of the forest-dwelling poppies (Papaveraceae: Chelidonioideae) as deduced from a molecular phylogeny of the ITS region

Sequences of the ITS region of nrDNA were analyzed for the seven genera of Papaveraceae subf. Chelidonioideae s.str. Three major clades can be recognized. These are 1.Chelidonium/Hylomecon/Stylophorum, 2.Eomecon/Sanguinaria, and 3.Bocconia/Macleaya. The monophyly of genera in the first of these three clades is doubtful, and clades two and three are sister to each other. Use of the ITS phylogeny of the subfamily to trace its morphological and ecological evolution shows that morphological change is concentrated in theBocconia/Macleaya clade, and probably related to the evolution of wind-pollination from insect-pollination in these two genera after habitat shift.     

Molecular phylogeny and systematics of Genista (Leguminosae) and related genera based on nucleotide sequences of nrDNA (ITS region) and cpDNA (trnL-trnF intergenic spacer)

Phylogenetic relationships of Genista and related genera (Teline, Chamaespartium, Pterospartum, Echinospartum, Ulex, Stauracanthus and Retama) were assessed by the analysis of sequences of the nrDNA internal transcribed spacer (ITS region), and the cpDNA trnL-trnF intergenic spacer. The tree obtained by combining both sets of data indicates the existence of three lines of diversification within Genista, that correspond to three subgenera: Genista, Phyllobotrys and Spartocarpus, however, each of these lineages encompass also species of the related genera Echinospartum, Teline, Retama, Chamaespartium, Pterospartum, Ulex, Stauracanthus. The molecular data do not support division of these subgenera into taxonomical units at the sectional level; only sections Genista and Spartocarpus are monophyletic groups. The sequences of both regions are also informative at the specific level, grouping morphologically related species (e.g. the G. cinerea aggregate). The molecular data have also helped to clarify the position of taxa whose relationships were not well established (e.g. G. valdes-bermejoi). The relationships of related genera that belong to the Genista lines of diversification have also been investigated. Echinospartum splits into two separate clades matching the separation of two ecological and caryological differentiated groups. Teline also forms two groups, both placed near to Genista subgenus Genista, but that separated from the main core of the group. Retama, morphologically well differentiated from Genista, is close to Genista subgenus Spartocarpus. Chamaespartium and Pterospartum do not form a monophyletic group. Chamaespartium is closer to Genista subgenus Genista, whereas Pterospartum stands close to: 1) Genista subgenus Spartocarpus (particularly, sect. Cephalospartum); and 2) the Ulex-Stauracanthus clade (a terminal derivative of Genista subgenus Spartocarpus). Cases of incongruence (e.g. Echinospartum, Chamaespartium, Teline) between the trees obtained from the two molecular markers, may be indicating hybridisation and/or introgression between different lines of Genisteae.     

A phenetic study of Cassia sensu lato (Leguminosae-Caesalpinioideae: Cassieae: Cassiinae) in Thailand

Cassia L. sensu lato, a large heterogeneous genus of flowering plants, occurs naturally in the tropics around the world. Recent works based on floral morphology have supported a division of this genus into three genera, namely Cassia L. s. str., Chamaecrista Moench and Senna Mill. In order to investigate this new classification, 508 specimens of 18 taxa of the genus Cassia s.l. grown in Thailand were analyzed using cluster analysis and canonical discriminant analysis. The total 32 vegetative and reproductive morphological characters were employed in these analyses. In cluster analysis, Cassia s.l. can be separated into four groups, respectively viz. Chamaecrista, Senna alata, Senna and Cassia s /str. The four-cluster grouping is discussed. From a canonical discriminant analysis using the four-cluster grouping as a priori groups, it can be concluded that Cassia s. str., Senna, and Chamaecrista are indeed distinct taxa. The three most important characters that separate the three genera are filament length, fruit length, and ovary stalk length. These quantitative characters, together with some qualitative characters, were useful in constructing an identification key to these genera. Among the three genera, it was also found that Senna is rather a heterogeneous taxon. The difference between the studied species was discussed.     

Examining morphological and molecular diagnostic character states of Cichorium intybus L. (Asteraceae) and C. spinosum L.

The inter- and intraspecific variability of Cichorium intybus L. was examined to evaluate potential morphological and molecular diagnostic character states. Two diagnostic and one overlapping morphological character clearly delimit the two species C. intybus and C. spinosum. All applied molecular methods (ITS, AFLP, Microsatellites) failed to significantly discriminate between these accepted species. As the morphological traits are fixed and heritable, criteria for species delimitation are fulfilled. These traits, however, are apparently due to mutations of a few crucial loci affecting the morphological diagnostic character states. Intraspecific variability within C. intybus revealed to be highly influenced by plastic response to local environmental factors and subspecific delimitation cannot be supported.     

Phylogeny of the GenusTrichodermaBased on Sequence Analysis of the Internal Transcribed Spacer Region 1 of the rDNA Cluster

Sequences of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA were used to determine the phylogenetic relationships of species ofTrichodermasect.Pachybasium.To this end, 85 strains—including all the availableex-type strains—were analyzed. Parsimony analysis demonstrated that the section is nonmonophyletic, distributing the 85 strains among three main groups that were supported by bootstrap values. Group A comprises two clades (A1 and A2), with A1 includingT. polysporum, T. piluliferum,andT. minutisporum,while A2 includedT. hamatum, T. pubescens,andT. strigosumin addition to species previously included in sect.Trichoderma(i.e.,T. viride, T. atroviride,andT. koningii). Theex-type strain ofT. fasciculatumformed a separate branch basal to clade A. Clade B contained the sect.PachybasiummembersT. harzianum, T. fertile, T. croceum, T. longipile, T. strictipile, T. tomentosum, T. oblongisporum, T. flavofuscum, T. spirale,and the anamorphs ofHypocrea semiorbisandH. cf. gelatinosa.Sequence differences among clades A1, A2, and B were in the same order of magnitude as between each of them andT. longibrachiatum,which was used as an outgroup in these analyses. Sequence differences within clades A1, A2, and B were considerably smaller: in some cases (i.e.,T. virensandT. flavofuscum; T. strictipileandH. cf. gelatinosa), the ITS1-sequences were identical, suggesting conspecifity. In other cases (e.g.,T. crassumandT. longipile; T. harzianum, T. inhamatum, T. croceum, T. fertile,andH. semiorbis; T. hamatumandT. pubescens;andT. viride, T. atroviride,andT. koningii) differences were in the range of 1–3 nt only, suggesting a very close phylogenetic relationship. The sequence of a previously described aggressive mushroom competitor group ofT. harzianumstrains (Th2) was strikingly different from that of theex-type strain ofT. harzianumand closely related species and is likely to be a separate species.