Restriction enzyme analysis of the chloroplast and nuclear 45s ribosomal DNA ofAllium sectionsCepa andPhyllodolon (Alliaceae)

Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of molecular characters. Restriction enzyme analysis of the chloroplast DNA (cpDNA) of the bulb onion (Allium cepa), Japanese bunching onion (A. fistulosum), wildAllium species in sect.Cepa andPhyllodolon, and the outgroupsA. ampeloprasum andA. tuberosum detected 39 polymorphisms.Allium cepa andA. vavilovii were identical for all characters. Cladistic analysis generated three most-parsimoniousWagner trees of 44 steps differing only in a zero-length branch.Allium fistulosum andA. altaicum (sect.Phyllodolon) comprised a monophyletic lineage separated from theA. cepa andA. vavilovii of sect.Cepa. The unresolved node was composed ofA. galanthum, A. roylei, and the lineage containingA. cepa, A. vavilovii, A. fistulosum, andA. altaicum. The clade containingA. altaicum, A. cepa, A. fistulosum, A. galanthum, A. roylei, andA. vavilovii remained resolved for strict consensus ofWagner trees of 48 steps or less.Allium pskemense andA. oschaninii were increasingly distant.Allium oschaninii has been proposed as the progenitor of the bulb onion, but was more closely related to the common progenitor of all species in sect.Cepa andPhyllodolon. Phylogenies estimated from cpDNA characters usingDollo parsimony resulted in a single most-parsimonious tree of 46 steps and agreed with phylogenies based onWagner parsimony. Polymorphic restriction enzyme sites in the 45s ribosomal DNA were not used to estimate phylogenies because of uncertain homologies, but are useful for identifying interspecific hybrids. The maternal phylogenies estimated in this study help to distinguish wildAllium species closely related to the bulb onion. Although not in agreement with classifications based on morphology, the phylogenies closely reflected crossability among species in sect.Cepa andPhyllodolon.     

The cytogeographical distribution pattern ofAllium (Alliaceae) in the Greek Peninsula and Islands

Greece is considered as a secondary centre of evolution for the genusAllium since it possesses about 50% of the species known from the whole Flora Europaea area. In the present investigation 44 GreekAllium spp. have been studied and new chromosome counts are reported from 40 populations and 17 species. The distribution of the different cytotypes (x = 7, x = 8, x = 11 and 2n = 2x, 3x, 4x, 5x, 6x, 7x) in Greece is discussed. From the four phytogeographical subdivisions recognized, South continental Greece shows the greatest species and karyotype diversity. This phenomenon is probably due to the geographical position and to the geological history of this area which has received species and populations from different directions. Subsequently, hybridization apparently has been of evolutionary importance.The genusAllium in Greece I.     

Restriction enzyme analysis of the nuclear 45s ribosomal DNA of six cultivated Alliums (Alliaceae)

Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of objective molecular characters. Restriction fragment length polymorphisms in the nuclear 45s ribosomal DNA (rDNA) were identified among two of five accessions of each of six cultivated Alliums. Restriction enzyme sites forBamHI,DraI,EcoRI,EcoRV,SacI, andXbaI were mapped. Different lengths of the rDNA repeat unit among the cultivated Alliums were due to sizes of the intergenic spacer. Nineteen polymorphic restriction enzyme sites were discovered and used to estimate phylogenetic relationships. Cladistic analyses based on Wagner parsimony were completed without an outgroup and resulted in two equally most parsimonious trees of 22 steps. A combined analysis of differences at RE sites in the ribosomal (19 characters) and chloroplast (15 characters) DNA generated a single most parsimonious tree of 39 steps. Single trichotomies were observed at 40 and 41 steps. Strict consensus of the three trees of 41 or fewer steps consisted of a lineage forA. tuberosum, a second forA. ampeloprasum andA. sativum, and a third forA. cepa, A. fistulosum, andA. schoenoprasum. Estimates of phylogenetic relationships based on variability at restriction enzyme sites in the rDNA and chloroplast DNA agree with the classification scheme ofTraub. Because of the predominance of autapomorphies, restriction enzyme analysis of the nuclear 45s rDNA is of limited use in estimating phylogenies amongAllium sections. However it is useful in the establishment of interspecific hybridity.     

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.     

Evolution of genome size inAllium (Alliaceae)

The 4C DNA amounts of 86 species fromAllium subgg.Allium, Rhizirideum, Bromatorrhiza, Melanocrommyum, Caloscordum andAmerallium show a 8.35-fold difference ranging from 35.60 pg (A. ledebourianum, 2n = 16) to 297.13 pg (A. validum 2n = 56). At diploid level the difference is 3.57-fold betweenA. ledebourianum (35.60 pg) andA. ursinum (127.14 pg). This shows that a significant loss and/or gain of DNA has occurred during evolution. On average subgg.Rhizirideum andAllium have less DNA amount than subgg.Melanocrommyum andAmerallium. The distribution of nuclear DNA amounts does not show discontinuous pattern and regular groups. The evolution of genome size has been discussed in relation to polyploidy and genomes, heterochromatin, adaptive changes in morphological characteristics, phenology and ecological factors, and infrageneric classification.     

Chloroplast DNA restriction site mapping and the phylogeny ofRanunculus (Ranunculaceae)

A chloroplast DNA restriction site map forRanunculus sceleratus (Ranunculaceae) was constructed using 14 restriction endonucleases. The total size of the chloroplast genome is 152.4kb. No inversions were detected relative to the tobacco chloroplast DNA. Cladistic analyses of chloroplast DNA restriction site polymorphism were employed in order to elucidate the phylogeny among 76 species of the genusRanunculus in a wide sense and one species ofTrautvetteria. A total of 341 informative restriction site changes were detected. Parsimony jackknifing, bootstrapping and decay analysis were undertaken in order to evaluate the amount of support for the monophyletic groups. The results suggest that the analysed species ofRanunculus are divisible into two main clades. Only few of the traditional sections and subgenera ofRanunculus are monophyletic. The genusTrautvetteria is nested within a clade comprising, e.g.Ranunculus cymbalaria, R. andersonii, R. lapponicus andR. ficaria. SubgenusBatrachium lies within a larger clade containing, e.g.R. sceleratus andR. hyperboreus. Contractions of the inverted repeat due to parallel deletions of 200–300 bp close to the J_SB have occurred in many clades and the phylogenetic distribution of this size reduction was mapped among the species.     

Giemsa C-banded karyotypes ofAllium ericetorum andA. kermesinum (Alliaceae)

The widely distributedAllium ericetorum and the local endemic of the Steiner Alps (Slovenia),A. kermesinum, are two closely related species of sect.Rhizirideum, whose main distinguishing character is perianth colour. To obtain further evidence for species separation, karyotype morphology and C-banding patterns were examined in 10 populations. The chromosome number was 2n = 16. In some populations ofA. ericetorum a B-chromosome occurred. Arm and satellite lengths and C-banding patterns were subjected to cluster analysis. Three different karyotype classes were observed and described. Karyotypes did not clearly discriminate between plants with different colours of perianth segments and therefore did not provide evidence for a taxonomic separation ofA. ericetorum and A. kermesinum. There is polymorphism in number and patterns of C-bands within the populations. No correlation between B-chromosomes and particular banding patterns was observed.     

Phylogeny ofTyphonium (Araceae) inferred from restriction fragment analysis of chloroplast DNA

The interrelationship of the ten species of the genusTyphonium and related genera in subtribe Arinae of the Araceae was inferred by chloroplast DNA restriction fragment analysis. A total of 42 site mutations were observed and 26 site mutations were shared by two or more species. A majority rule consensus tree was made by performing 100 bootstrap replicates using Wagner Parsimony. Two groups ofTyphonium were recognized significantly as monophyletic groups, i.e. 1)Typhonium larsenii andT. kunmingense, and 2)T. trilobatum, T. blumei andT. flagelliforme.     

Closely relatedAllium species (Alliaceae) share a very similar satellite sequence

A satellite sequence repeat ofAllium cepa was tested by fluorescent in situ hybridization (FISH) for cross-hybridization to chromosomes of 27 species (in 37 accessions) belonging to 14 sections of four subgenera ofAllium. All investigated species of sect.Cepa, with the two subsects.Cepa andPhyllodolon, revealed clear satellite-specific hybridization signals mainly at their chromosome termini. The tested species belonging to other sections/subgenera revealed no hybridization signals. An exception wasA. roylei, assigned to sect.Oreiprason. Its chromosomes also showed strong terminal hybridization signals. This and other features suggest a close relationship ofA. roylei to the species of sect.Cepa in spite of deviating morphological characters. The divergence between the satellite repeats to species to which theA. cepa repeat cross-hybridized was determined and revealed high degrees of similarity. Therefore, we conclude that this satellite sequence had evolved already in progenitor forms of sect.Cepa and remained unusually well conserved during speciation. This might indicate selection pressure exerted on a secondarily acquired telomere function of the satellite sequence.Dedicated to Dr habil.Peter Hanelt on the occasion of his 65th birthday.     

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.     

Distribution of a 375 bp repeat sequence inAllium (Alliaceae) as revealed by FISH

Information about evolutionary relationships between species of the genusAllium is desirable in order to facilitate breeding programmes. One approach is to study the distribution of repetitive DNA sequences among species thought on taxonomic grounds, to be closely related. We have used fluorescent in-situ hybridisation (FISH) to examine seven species within sect.Cepa of the genus (A. altaicum, A. cepa, A. fistulosum, A. galanthum, A. pskemense, A. oschaninii andA. vavilovii), one species from sect.Rhizirideum (A. roylei), two species from sect.Allium (A. sativum andA. porrum) and one species from sect.Schoenoprasum (A. schoenoprasum). Each species was probed using a 375 bp repeat sequence isolated fromA. cepa (Barnes & al. 1985), which was generated and labelled by polymerase chain reaction (PCR). No signals were detected in anyAllium species not belonging to sect.Cepa with the exception ofA. roylei, whose designation in sect.Rhizirideum is now questioned. Within sect.Cepa the probe was found to hybridize to the terminal regions of the chromosome arms of all the species examined. In addition a number of interstitial bands were detected. Use of FISH reveals a more detailed map of the location of the repeat sequences than has previously been obtained by C-banding and other staining procedures. The distribution of the terminal and interstitial sites when compared, allow us to identify three species groups namely,A. altaicum andA. fistulosum; A. cepa, A. roylei, A. oschaninii andA. vavilovii; andA. galanthum andA. pskemense.     

Allotetraploid origin ofAllium altyncolicum (Alliaceae, Allium sect.Schoenoprasum) as investigated by karyological and molecular markers

The tetraploidAllium altyncolicum (2n = 4x = 32) is considered to be of hybrid origin, because most of its morphological characters are intermediate between those of its putative parents,A. schoenoprasum andA. ledebourianum. In the present work an attempt has been made to ascertain its parentage by several methods: Giemsa C-banding, genomic in situ hybridization (GISH), PCR-RFLP of cpDNA, restriction enzyme mapping of the rDNA, and RAPDs. C-banding and GISH indicates clearly thatA. altyncolicum is a segmental allopolyploid.Allium schoenoprasum andA. ledebourianum are the most likely the parental species and the larger part of the genome ofA. altyncolicum (26 chromosomes) is derived fromA. schoenoprasum. The low genetic divergence between these three species was confirmed by the lack of sequence variation in the ITS sequences of nuclear rRNA genes and of the plastid rbcL-atpB intergenic spacer. Both parental species andA. altyncolicum could be distinguished by RFLP of the rDNA repeats. The geographic origin of the putative parental species was investigated using RAPDs.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Phylogenetic relationships of turfgrasses as revealed by restriction fragment analysis of chloroplast DNA

Chloroplast DNAs (cpDNAs) were analyzed in order to clarify the phylogenetic relationships among turfgrasses. Physical maps of cpDNAs from Agrostis stolonifera and Zoysia japonica, which are representative species of cool (C3 type) and warm (C4 type) season turfgrasses, respectively, were constructed with four restriction enzymes, i.e., PstI, SalI, SacI, and XhoI. The genome structures of these cpDNAs were found to be similar to each other in terms of genome size and gene orders, showing thereby a similarity to other grass cpDNAs. CpDNAs of 5 species of cool season turfgrasses and 6 species of warm season turfgrasses as well as four species of cereals, distributed among 14 genera of Gramineae, were digested with PstI, XhoI, and BamHI, and their restriction fragment patterns were compared. Their genome sizes were estimated to be 135–140 kbp. Each species showed characteristic RFLP patterns. On the basis of the frequency of commonly shared fragments, a dendrogram showing the phylogenetic relationships among their cpDNAs was constructed. This dendrogram shows that turfgrasses can be divided into three major groups; these correspond to the subfamilies. Cool and warm season turfgrasses are clearly distinguishable from each other, and the latter can be further classified into two subgroups that correspond to Eragrostoideae and Panicoideae. Our classification of turfgrasses and cereals by RFLP analysis of cpDNA agreed in principal with their conventional taxonomy, except for the location of Festuca and Lolium.Contribution no. 101 from the Kihara Institute for Biological Research, Yokohama City University, Yokohama 232, Japan     

Phylogenetic relationships among cultivated Allium species from restriction enzyme analysis of the chloroplast genome

Summary The genus Allium contains many economically important species, including the bulb onion, chive, garlic, Japanese bunching onion, and leek. Phylogenetic relationships among the cultivated alliums are not well understood, and taxonomic classifications are based on relatively few morphological characters. Chloroplast DNA is highly conserved and useful in determining phylogenetic relationships. The size of the chloroplast genome of Allium cepa was estimated at 140 kb and restriction enzyme sites were mapped for KpnI, PstI, PvuII, SalI, XbaI, and XhoI. Variability at restriction enzyme sites in the chloroplast DNA was studied for at least three accessions of each of six cultivated, old-world Allium species. Of 189 restriction enzyme sites detected with 12 enzymes, 15 mutations were identified and used to estimate phylogenetic relationships. Cladistic analysis based on Wagner and Dollo parsimony resulted in a single, most-parsimonious tree of 16 steps and supported division of the species into sections. Allium species in section Porrum were distinguished from species in sections Cepa and Phyllodolon. Two species in section Rhiziridium, A. schoenoprasum and A. tuberosum, differed by five mutations and were placed in separate lineages. Allium cepa and A. fistulosum shared the loss of a restriction enzyme site and were phylogenetically closer to each other than to A. schoenoprasum. This study demonstrates the usefulness of restriction enzyme site analysis of the chloroplast genome in the elucidation of phylogenetic relationships in Allium.     

Chloroplast DNA Variation in the genusSesamum

It is widely approved that comparing restriction profiles and maps of chloroplast DMA provides valuable information concerning inter-and/or intra-specific relationships among plant species. Such chloroplast DNA analysis was applied to species and strains inSesamum which is a genus of approximately 38 species and contains a large number of strains of the cultivated sesame,S. Indlcum. Our chloroplast DNA investigations of 22 species and strains showed that; (i) among four species (S. capense, S. radiatum, S. schinzianum andS. indicum), the chloroplast genome ofS. capense was most distantly related to that of the cultivated species,S. indicum, (ii) chloroplast DNA polymorphism was also recognized among eight cultivated stralns collected from various regions in the tropical zone, but not among eight different varieties grown in the temperate zone, and (iii) the chloroplast DNA alterations observed could be attributed to the site gains or losses with the exception of the alterartion detected within the inverted repeat sequences inS. capense chloroplast DNA. These results demonstrate the presence of chloroplast genome diversity amongSesamum species and strains, suggesting the usefulness of chloroplast DNA analysis for elucidating the species relationships in the genusSesamum and the origin and evolutionary process of the cultivated sesame species. The present paper is based on the contribution which was read in a symposium entitled “Organellar DNA Variations in Higher Plants and Their Taxonomic Significance”, at the 50th Annual Meeting of the Botanical Society of Japan in Shizuoka on October 2, 1990, under the auspices of the Japan Society of Plant Taxonomists.     

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.     

Inheritance and restriction fragment length polymorphism of chloroplast DNA in the genus Coffea L.

CpDNA variation among 52 tree samples belonging to 25 different taxa of Coffea and two species of Psilanthus was assessed by RFLP analysis on both the total chloroplast genome and the atpB-rbcL intergenic region. Twelve variable characters were distinguished allowing the identification of 12 different plastomes. The low sequence divergence observed might suggest that Coffea is a young genus. The results were in contradiction with the present classification into two genera. Additionally, cpDNA inheritance was studied in interspecific hybrids between C. arabica and C. canephora, and in an intraspecific progeny of C. canephora, using PCR-based markers. Both studies showed exclusively maternal inheritance of cpDNA.     

Chloroplast DNA diversity in Vicia faba and its close wild relatives: implications for reassessment

To obtain new information on phylogenetic relationships between wild and cultivated broad bean, restriction fragment length polymorphism (RFLP) analysis of chloroplast (cp) DNAs from Vicia faba and eight subspecies/species of its close wild relatives grouped together in the Narbonensis complex was carried out using 14 restriction endonucleases. The molecular sizes of the cpDNAs obtained were similar (122.6–123.4 kbp), indicating that they had all lost one of inverted repeats. Among the more than 300 sites surveyed, the three subspecies within V. narbonensis, which exhibit just as many types of karyotypes, were shown to have identical cp fragment patterns. Genetic distances between all of the pairs of species were calculated from RFLP data. The cpDNA diversity within the Narbonensis complex was found to be more extensive than expected, except for the genetic relationship between V. hyaeniscyamus and V. johannis in which a total of three mutations were detected among the 300 sites sampled, thereby showing their close relatedness. The cpDNA of V. faba vis-a-vis its wild relatives also exhibited startling differences, indicating a clear division of Vicia species into two distinct lineages. This analysis unambiguously provides new evidence that the wild species grouped in the complex did not contribute their plastomes to the evolution of V. faba, and hence none of the species can be considered to be putative allies of broad bean. The present study also demonstrates profound cpDNA diversity among closely related species that have lost one of inverted repeats.     

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 study of the generaAmbrosia s.l. andHymenoclea (Asteraceae): Systematic implications

The relationship ofAmbrosia (ragweed) andFranseria has long been debated. Their treatment as separate genera has been repeatedly challenged. In this study, chloroplast DNA restriction site variation was examined for species from bothAmbrosia andFranseria as well as taxa from the closely related genusHymenoclea. The chloroplast genomes of members of these three genera were examined using 21 restriction endonucleases and the restriction mutations were used to construct phylogenetic trees. Wagner and Dollo parsimony as well as weighted parsimony were employed to compare the different phylogenies. The results support a close relationship betweenAmbrosia andFranseria, but indicate that the two groups are well separated. Compared toFranseria, Ambrosia is a much more strongly supported group, and the results indicate thatHymenoclea is closer toFranseria than toAmbrosia. The cpDNA phylogeny was used as a framework to examine evolutionary trends in morphology and secondary chemistry.