Chloroplast DNA variation and phylogeny of theRanunculaceae

Restriction site mapping of chloroplast DNA from 31 species representing 26 genera of theRanunculaceae was performed using eleven restriction endonucleases. The chloroplast genome varies in length from approximately 152 to 160 kb. Length variants are frequent in theRanunculaceae and range from usually less than 300 bp to rarely 1.5 kb. The inverted repeat is extended into the large single copy (LSC) region by 4–4.5 kb inAnemone, Clematis, Clematopsis, Hepatica, Knowltonia, andPulsatilla. Several inversions are present in the LSC-region of the cpDNA in all these genera and inAdonis. The frequency of restriction site mutations varies within the chloroplast genome in theRanunculaceae between 4 and 32 mutations per kilobase, and is lowest in the inverted repeat and the regions containing the ATPase-genes and the genespsaA, psaB, psbA, rpoB, andrbcL. A total of 547 phylogenetically informative restriction sites was utilized in cladistic analyses of the family using Wagner, Dollo, and weighted parsimony. These three parsimony analyses result in different tree topologies. Four, six, and one equally most parsimonious trees were obtained with Wagner, Dollo, and weighted parsimony, respectively. The amount of support for the monophyletic groups was evaluated using bootstrapping and decay analysis. All three parsimony methods suggest thatHydrastis is the sister group to the remainder of theRanunculaceae, and that theAnemone-Clematis group, which shares several derived cpDNA rearrangements, is monophyletic. Only a few of the traditional groups in theRanunculaceae are supported by cpDNA restriction side data. Only Dollo parsimony provides support for the hypothesis thatThalictroideae andRanunculoideae are monophyletic.     

Chloroplast DNA variation inAnemone s. lato (Ranunculaceae)

Chloroplast DNA of seven species belonging toAnemone (sectt.Omalocarpus, Anemonidium, andAnemonanthea),Hepatica, andPulsatilla have been analyzed by restriction enzymes. According to the dendrogram constructed, the sections ofAnemone and the generaHepatica andPulsatilla seem to be evolutionary approximately equidistant to each others. This supports the concept that these groups should be treated on a similar taxonomic level, either as genera or subgenera.     

Chloroplast DNA restriction site variation and phylogeny of the Berberidaceae

Comparative restriction site mapping of the chloroplast genome was performed to examine phylogenetic relationships among 27 species representing 16 genera of the Berberidaceae and two outgroups. Chloroplast genomes of the species included in this study showed no major structural rearrangements (i.e., they are collinear to tobacco cpDNA) except for the extension of the inverted repeat in species of Berberis and Mahonia. Excluding several regions that exhibited severe length variation, a total of 501 phylogenetically informative sites was mapped for ten restriction enzymes. The strict consensus tree of 14 equally parsimonious trees indicated that some berberidaceous genera (Berberis, Mahonia, Diphylleia) are not monophyletic. To explore phylogenetic utility of different parsimony methods phylogenetic trees were generated using Wagner, Dollo, and weighted parsimony for a reduced data set that included 18 species. One of the most significant results was the recognition of the four chromosomal groups, which were strongly supported regardless of the parsimony method used. The most notable difference among the trees produced by the three parsimony methods was the relationships among the four chromosomal groups. The cpDNA trees also strongly supported a close relationship of several generic pairs (e.g., Berberis-Mahonia, Epimedium-Vancouveria, etc.). Maximum likelihood values were computed for the four different tree topologies of the chromosomal groups, two Wagner, one Dollo, and one weighted topology. The results indicate that the weighted tree has the highest likelihood value. The lowest likelihood value was obtained for the Dollo tree, which had the highest bootstrap and decay values. Separate analyses using only the Inverted Repeat (IR) region resulted in a tree that is identical to the weighted tree. Poor resolution and/or support for the relationships among the four chromosomal lineages of the Berberidaceae indicate that they may have radiated from an ancestral stock in a relatively short evolutionary time.     

Chloroplast DNA restriction site study of Verbesina (Asteraceae: Heliantheae)

Chloroplast DNA variation was examined for 79 species of Verbesina and 24 outgroups. Two independent analyses of the data were performed. An intergeneric study to elucidate the phylogenetic relationships of Verbesina used 22 genera, most of which are regarded in the literature as closely related to Verbesina. Coreopsis and Hymenoxys served as outgroups for this analysis. The 16 6-bp (base pair) restriction endonucleases used in the intergeneric study revealed 263 phylogenetically informative sites. Wagner analyses of these characters resulted in four equally parsimonious trees with a length of 857 steps and a Consistency Index of 0.492. Results from this study indicate that Verbesina is monophyletic, a member of the tribe Heliantheae, and that its sister taxa are the mostly Mexican genera Podachaenium, Squamopappus, and Tetrachyron. The infrageneric study of Verbesina included species belonging to all of its infrageneric taxa, except the monotypic sect. Stenocarpha. The 17 6-bp restriction endonucleases used in the infrageneric study revealed 137 sites 77 of which were phylogenetically informative. Wagner analyses of these characters generated 180 equally parsimonious trees with a length of 158 steps and a Consistency Index of 0.786. The genera Podachaenium, Squamopappus, and Tetrachyron served as outgroups. Two major clades, which correspond to traditional divisions of the genus based on leaf arrangement, are supported by the study. No support was found for the monophyly of seven of the 11 sections examined; further sampling of sections Lipactinia, Ochractinia, Verbesina, and Ximenesia is needed. Results support a major reassessment of the traditional infrageneric classification of the genus and provide the basis for the reevaluation of the sectional taxonomy of Verbesina. These studies support a North American origin for the genus with several independent introductions into South America producing significant diversity, especially in the Andean region.     

Chloroplast DNA variation among five species ofRanunculaceae: Structure,sequence divergence,and phylogenetic relationships

A restriction site map of the chloroplast genome ofCaltha palustris L. (Ranunculaceae) has been constructed for 13 restriction endonucleases using filter hybridization with cloned tobacco chloroplast DNA fragments. A size of 153.8 kb has been estimated for theCaltha chloroplast genome. Forty-six chloroplast genes and four open reading frames have been mapped using small tobacco chloroplast gene probes. Chloroplast DNA sequence divergence has been estimated for all pairs of five species ofRanunculaceae, Caltha palustris, Ranunculus bulbosus, R. fascicularis, R. recurvatus, andTrollius ledebourii, and ranges between 0.2% and 9.6% for the total genome. Divergence values are much higher in the small and large single copy regions than in the inverted repeat. Phylogenetic relationships between the five species have been hypothesized using chloroplast DNA restriction site mapping. One hundred and six informative restriction site mutations have been detected using eleven restriction endonucleases. Cladistic analyses of the restriction site mutations have been performed using Wagner and Dollo parsimony algorithms, and confidence intervals have been calculated for the resulting monophyletic groups using bootstrapping. It is demonstrated that restriction site comparisons are applicable to theRanunculaceae on intergeneric level, with the exception of groups having extensive genomic rearrangements. Moreover, sequence divergence is low enough at the interspecific level to allow phylogenetic analyses within genera such asRanunculus.     

Chloroplast DNA variation inDesmodium subgenusPodocarpium (Leguminosae): Infrageneric phylogeny and infraspecific variations

Chloroplast DNA (cpDNA) restriction site variation was examined in five species ofDesmodium subgenusPodocarpium (Leguminosae; Papilionoideae; Desmodieae). Twenty four phylogenetically informative cpDNA mutations were scored. The cladistic analysis of characters based on the 24 mutations resulted in the most parsimonious tree which supports the monophyly of the subgenus.Desmodium elegans of subgenusDollinera was the sister group of subgenusPodocarpium in this tree. The groupings obtained from the cpDNA characters were consistent with the present infrageneric classification system for the subgenus except for the infraspecific taxa ofD. podocarpum. Three groups withinD. podocarpum, which were incongruent with the infraspecific classification of the species, were distinguished by a total of four site mutations. The first group consisted of subsp.podocarpum, subsp.fallax, and subsp.oxyphyllum var.oxyphyllum; the second subsp.oxyphyllum var.oxyphyllum; and the last subsp.oxyphyllum var.oxyphyllum and var.mandshuricum.     

Chloroplast DNA restriction site data support a narrowed interpretation ofEupatorium (Asteraceae)

Chloroplast DNA (cpDNA) restriction site analysis was used to assess relationships among samples of Eupatorieae from eastern North America. A total of 270 cpDNA variants was recorded from 35 species using 13 restriction enzymes. Phylogenetic analysis usingGalinsoga, Flaveria, andHelianthinae as outgroups indicated that samples ofAgeratina, Hofmeisteria, Mikania, andStevia, each of which have relatively high base chromosome numbers, formed an unresolved basal polytomy. The remaining samples examined formed a well-supported clade, within which there was a split betweenBrickellia, which hasx = 9, and a number of genera withx = 10. Within thex = 10 clade, there was an unresolved trichotomy, with one group ofAgeratum, Conoclinium, Fleischmannia, andCritonia, a second ofLiatris andTrilisa, and a third ofEupatorium s.s. Within theEupatorium s.s. clade there were three further clades, withE. sect.Verticillata diverging first, and a subsequent split between species originating from North America and those from Asia. The cpDNA restriction site data provided support for a relatively narrow interpretation ofEupatorium, and indicated that a high chromosome base number is plesiomorphic for Eupatorieae.     

Spatial fragrance patterns within the flowers ofRanunculus acris (Ranunculaceae)

Floral scents emitted from different flower parts ofRanunculus acris were investigated by trapping headspace volatiles onto Porapak Q followed by solvent desorption and GC-MS analysis. Isoprenoids, strongly dominated bytrans--ocimene, constituted the principal class of volatiles in all flower parts except pollen; sesquiterpenes were especially diverse. Odors collected separately from petals, stamens, and sepals + gynoecium comprised the same volatiles, but these were present in disparate proportions among the flower parts, thereby creating subtle contrasts within the flower. The main sources of volatiles were the petals and stamens, which made equal contributions to the whole-flower fragrance. Emissions from the petals differed quantitatively between the apical and basal petal regions, thereby paralleling optical nectar-guide patterns. Pollen odor was markedly unlike that of other flower parts, with only few volatiles, a high representation of 5-methylene-2(5H)-furanone (protoanemonin), and no detectabletrans--ocimene. The distinctiveness of the pollen's volatile profile suggests that it may serve a signalling role to pollen-feeding insects.     

Molecular phylogeny ofIva (Asteraceae,Heliantheae) based on chloroplast DNA restriction site variation

Iva s.str. (comprising ten species) was examined by cpDNA restriction site variation to determine phyletic relationships within the group. The results were compared with relationships proposed from other data. A total of 86 restriction site mutations was detected, 47 of which proved phylogenetically informative. A single most parsimonious tree was obtained using both Wagner and Dollo parsimony. The tree revealed three main lineages that are congruent with the three chromosome lineages (base numbers of x = 16, 17, 18). The monophyly of the x = 16 and 18 groups was supported strongly by molecular data, while the monophyly of x = 17 lineage was only supported moderately. Relationships among the three lineages indicate that the sect.Iva is paraphyletic because sect.Linearbractea is nested within it. Both morphological data and the secondary chemical data are in agreement with the proposed cpDNA phylogeny. Because of this agreement, sect.Iva is revised such that,I. axillaris was excluded and positioned within the newly proposed sect.Rhizoma. Patterns and rates of cpDNA evolution were also examined. The results indicated an uneven evolution in the chloroplast genome with different rates of cpDNA evolution in at least a few species ofIva. However, the evolutionary clock hypothesis can not be rejected within most of the lineages inIva.     

Molecular phylogeny and evolution of the AsianAconitum subgenusAconitum (Ranunculaceae)

Classification of the genusAconitum (Ranunculaceae) has long been considered quite difficult because its species show high levels of morphological and ecological variability. The molecular phylogeny of Asian aconites,Aconitum subgenusAconitum was, therefore, studied based on RFLP and sequences of the intergenic spacer between thetrnL (UAA) 3′ exon andtrnF (GAA), and of thetrnL intron, of the chloroplast DNA. UsingAconitum subgenusLycoctonum as an outgroup, we obtained a statistically reliable molecular tree composed of six clades branched radiatively at the very base. There are three clades of Japanese aconites, a single clade of the species of Yunnan and Himalayas, and two clades of Siberian plants. All the tetraploid taxa of Japan we studied did not show any difference based on the molecular characters analyzed, though they have been classified into many taxa. Evolution and phytogeography of the Asian aconites as well as the phylogeny are discussed.     

Chloroplast DNA variation and the phylogeny ofAsplenium sect.Hymenasplenium (Aspleniaceae) in the New World tropics

Asplenium sect.Hymenasplenium is a well-defined group in Aspleniaceae, distinguished by several morphological and cytological characters. However, interspecific relationships in the section were not clear. In this paper, we report the phylogenetic relationships of 9 Neotropical species of sect.Hymenasplenium determined by chloroplast DNA restriction site variation analyses. From the obtained phylogenetic tree, two major clades: one withA. obtusifolium, A. riparium, A. volubile andA. repandulum and another withA. delitescens, A. ortegae, A. purpurascens, A. laetum andA. hoffmannii were recognized.Asplenium delitescens was shown to have a polyphyletic origin. It was also shown that the epiphytic habit evolved only once in the New World species of sect.Hymenasplenium.     

Chloroplast DNA phylogeny of subtribe Dendrobiinae (Orchidaceae): Insights from a combined analysis based onrbcL sequences and restriction site variation

Phylogenetic analyses using two chloroplast DNA data sets, derived from variation of the ribulose-bisphosphate carboxylase gene (rbcL) and restriction sites, were performed to examine relationships among 13 taxa in subtribe Dendrobiinae, one of the most taxonomically complicated groups in Orchidaceae, and its putative sister groups. Owing to a limited number of informative substitutions, therbcL data set did not provide conclusive evidence in itself. The data set combiningrbcL and restriction site mutations, however, provided the following insights: (1)Pseuderia belongs with tribe Podochileae rather than tribe Dendrobieae. (2) Subtribe Dendrobiinae is monophyletic ifPseuderia is excluded. (3) ExcludingPseuderia, Dendrobiinae comprises three major clades: Clade 1 (Dendrobium sectionSpatulata, Cadetia, Diplocaulobium, andFlickingeria); Clade 2 (Dendrobium sectionsDendrobium andCallista); and Clade 3 (Epigeneium). (4)Epigeneium diverged early from the lineage including Clades 1 and 2. (5) Relative toCadetia, Diplocaulobium, andFlickingeria, Dendrobium is shown to be para-/polyphyletic. (6)Diplocaulobium andFlickingeria constitute a monophyletic clade, from which cladeDendrobium sectionSpatulata andCadetia form succesive sister groups. Among these results, (1) and (5) are especially stable in view of the congruence between the separate and combined analyses as well as robust internal support.     

TheUlmaceae, one family or two? Evidence from chloroplast DNA restriction site mapping

The Ulmaceae is usually split into two subgroups, referred to as either tribes or more commonly subfamilies (Ulmoideae andCeltidoideae). The two groups are separated, with some exceptions, on the basis of leaf venation, fruit type, seed morphology, wood anatomy, palynology, chemistry, and chromosome number. Propositions to separate the two groups as distinct families have never gained general acceptance. Recent morphological and anatomical data have suggested, however, that not only is family status warranted but thatCeltidaceae are more closely related toMoraceae and otherUrticales than toUlmaceae. In order to test these alternative sets of relationships, restriction site mapping of the entire cpDNA was done with nine rare cutting enzymes using 11 genera ofUlmaceae s. l., three other families of theUrticales, and an outgroup family from theHamamelidae. Cladistic analysis of the data indicates thatUlmaceae s. l. is not monophyletic and that distinct families (Ulmaceae andCeltidaceae) are warranted; thatUlmaceae is the sister group toCeltidaceae plus all other families in the order; and thatCannabaceae might be nested withinCeltidaceae. Familial placements of various problematic genera (e.g.Ampelocera, Aphananthe) are resolved and character evolution of key morphological, anatomical, chemical, and chromosomal features are discussed.     

cpDNA intergene sequences corroborate restriction site data for reconstructingRubiaceae phylogeny

Representatives of seven genera from five tribes ofRubiaceae have been compared in respect to a non-coding intergene cpDNA region of about 1000 bp, situated between the atpB and the rbcL genes. The resulting most parsimonious PAUP cladogram corresponds very well with one based on total cpDNA restriction site data obtained byBremer & Jansen (1991). The two different molecular analyses thus corroborate each other and contribute to an improved systematic arrangement of the large family, e.g., in respect to placing the tribeHedyotideae clearly into the subfamilyRubioideae, closer toRubieae than toPsychotrieae.     

Chloroplast DNA restriction analysis and the infrageneric grouping ofAllium (Alliaceae)

The utility of chloroplast DNA variation for checking a recently proposed infrageneric classification of the genusAllium was tested. cpDNA restriction patterns of 49 species representing the main subgenera, sections, and subsections of the existing classification were compared. 363 different fragments generated by 4 restriction enzymes were identified and analysed by UPGMA clustering. The resulting phenogram largely confirms the subgeneric classification based on an integration of morphological and other methods.     

Chloroplast DNA reassociation and grass phylogeny

Sequence variation in chloroplast DNA (cpDNA) as measured by DNA reassociation was examined in 12 grass species to address systematic problems in thePoaceae at the subfamilial and tribal levels. Two species,Petunia (Solanaceae) andGlycine (Leguminosae), were included to determine degrees of sequence divergence in cpDNA between monocots and dicots. The data were analyzed phenetically and phylogenetically. Species were segregated into four major groups that corresponded to the subfamiliesPooideae, Oryzoideae, Chloridoideae, andPanicoideae. Representatives of thePooideae andOryzoideae grouped together as did members of theChloridoideae andPanicoideae. ThePooideae split into two major groups corresponding to the recently recognized supertribesTriticanae andPoanae. Internodes between subfamily branches were short which might indicate a burst of divergence in the family early in its evolution. Sequence similarity values between the monocot grass species and the two dicot taxa ranged from 0.15 to 0.27, representing the highly conserved sequences of the chloroplast genome.     

Phylogenetic relationships inCrossostylis (Rhizophoraceae) inferred from restriction site variation of chloroplast DNA

Phylogenetic relationships among the nine species ofCrossostylis (Rhizophoraceae) were elucidated using cladistic analysis of restriction site variations of chloroplast DNA. As a result, this genus was found to comprise two pronounced monophyletic groups as follows:C. biflora, C. grandiflora, C. multiflora andC. sebertii; andC. cominsii, C. pachyantha, C. parksii, C. richii andC. seemannii. Moreover, the monophyly ofC. biflora, C. grandiflora andC. sebertii in the former group and the monophyly ofC. pachyantha, C. parksii, C. richii andC. seemannii in the latter group were also suggested. The molecular tree corresponded well with that inferred from morphological data and no discrepancy was recognized. Many of the floral morphological characters reflected lineage, but all seed coat characters were homoplasious. Evolutionary trends in some morphological characters were optimized on the cpDNA tree obtained. Species from New Caledonia and Polynesia were monophyletic, as were those from the Solomon Islands, Vanuatu and the Fiji Islands. All species endemic to the Fiji Islands made a cluster, and this suggests that speciation occurred from a single ancestral species on the Islands.     

The phylogeny ofActaea (Ranunculaceae): A biogeographical approach

Phylogenetic analyses of the genusActaea were performed using morphological, ecological and biogeographical characters. Using solely morphological characters, the relationships of the three identified species-groups remain uncertain. Close biogeographical examination and comparison of the areas with ecological peculiarities as well as climate data gave important insight into the phylogeny ofActaea and the whole tribe. Consequently, the obtained biogeographical data were used for phylogenetic reconstructions. Both, from the point of view of morphological and biogeographical data,A. pachypoda andA. asiatica are the most ancestral species. They grow on the east sides of the continents, mainly in broad-leaved forests. In West Eurasia the apomorphicA. spicata andA. acuminata occur under similar climatic and ecological conditions, but these species are adapted to another climate rhythm. The most advanced species (A. erythrocarpa, A. rubra) are to be found in the boreal forests where they are widely distributed. This biogeographical approach revealed that the evolution of the species led to a gradual widening and shifting of their ecological constitutions.     

Chloroplast DNA restriction site polymorphism inGenisteae (Leguminosae) suggests a common origin for European and American lupines

Restriction site polymorphism in cpDNA of 35 legumes was studied in order to address natural relationships and geographic distribution within the tribeGenisteae. 386 sites were studied, 277 were polymorphic, 207 were informative. Phylogenetic inferences with distance and parsimony methods suggest that the American and MediterraneanLupinus species belong to a monophyletic group which arose from a single center of diversification. The data furthermore indicate thatLupinus should not be included in the tribeGenisteae since at the level of cpDNA polymorphismAnagyris foetida (tribeThermopsideae) appears more closely related to otherGenisteae thanLupinus does.     

Molecular phylogeny ofForsythia (Oleaceae) based on chloroplast DNA variation

Phylogenetic relationships of ten wild species and several cultivars ofForsythia were reconstructed based on the chloroplast (cp) DNA variation. A total of 216 cpDNA variants, 44 of which were potentially phylogenetically informative, was detected using 24 restriction endonucleases. Phylogenetic analysis usingFontanesia andAbeliophyllum as outgroups revealed four well defined species groups in the genus: 1)F. suspensa, 2)F. europaea — F. giraldiana, 3)F. ovata — F. japonica — F. viridissima, and 4)F. koreana — F. manshurica — F. saxatilis. The amount of support for each monophyletic group was evaluated by various methods including character number, decay analysis, parsimony bootstrapping, Neighbour-Joining (NJ) — bootstrapping, NJ-jackknifing, and the topology-dependent permutation tail probability (T-PTP) test. The data do not support the hybrid origin ofF. intermedia fromF. suspensa andF. viridissima. The disjunctly distributed European species,F. europaea, was identified as a sister species of the ChineseF. giraldiana and it was probably derived through recent long distance dispersal.