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.     

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.     

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.     

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.     

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.     

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.     

Plastid transformation in greening scales of the onion bulb (Allium cepa,Alliaceae)

The greening of the upper part of the outerAllium cepa L. bulb scales, in particular along the vascular regions, is limited to the hypodermal cells in which typical leucoplasts are transformed to normal and functional chloroplasts. This process is light dependent and cannot afterwards be reversed or modified by darkness. The changes in fine structure are described and briefly discussed.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday and 55 years after the publication of his Grundriß der Cytologie.     

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     

New cytogenetic information on Allium iranicum (Alliaceae) from Iran

Karyotype analysis and chromosome behaviour in tetraploid Allium iranicum is reported. The somatic karyotype 2n = 32, consists of 12 pairs of metacentric chromosomes, two pairs of submetacentric chromosomes and two pairs of submetacentric satellite chromosomes. Chromosome complement follows two sets of 16 pairs of homologous chromosomes. A detailed analysis of Pachytene, Diplotene and Metaphase I of meiosis in pollen mother cells in this taxon showed that the most common chromosome configurations were bivalents at all subphases mentioned. It is concluded that A. iranicum is most likely a natural allotetraploid and certainly differs from related species A. ampeloprasum, A. commutatum and A. porrum.     

Karyology of theScorzonera (Compositae) species from the Iberian Peninsula

A karyological study of 15 taxa ofScorzonera L. from the Iberian Peninsula has been made. The chromosome numbers found inS. hispanica var.pinnatifida, S. baetica, S. reverchonii, S. angustifolia, S. laciniata var.calcitrapifolia and var.subulata (2n = 14) are new. Diploid cytotypes with 2n = 14 and 2n = 12 prevail, andS. hispanica var.crispatula is the only taxon which exhibits autopolyploidy (2n = 14, 28). x = 7 is considered to be the base chromosome number within the genus, with x = 6 being derived from it by translocation. This and detailed karyotype analyses allow to group the Iberian Peninsula species ofScorzonera into three groups.     

Floral rewards in someScrophularia species (Scrophulariaceae) from the Iberian Peninsula and the Balearic Islands

The genusScrophularia in the Iberian Peninsula and Balearic Islands comprises two sections,Scrophularia andCaninaG. Don. Analyses were carried out on flower production, flower duration and their sexual phases, pollen and nectar production together with observations on their pollinators. Nectar production is correlated with corolla size and pollen production with anther size. The taxa of sect.Scrophularia show greater nectar and pollen production than those of sect.Canina. Also, those of the first section produce more ovules per ovary than those of the second group, production being correlated with the ovary size.Some observations on floral and reproductive biology in some species ofScrophularia from the Iberian Peninsula and the Balearic Islands. I.     

Evolution and systematic relationships in theTriticeae (Poaceae)

The evolution and taxonomic relationships in theTriticeae are discussed with the view to highlight aspects of this agronomically important group of plants, which may be of interest to molecular biology. Some of these aspects are addressed in more detail in adjoining papers in which specific genomic loci have been examined at the DNA sequence or isozyme level. Aspects discussed include the systematics and geographic distribution of theTriticeae species, isozyme and chromosome pairing studies on some of the species as well as more recent developments in DNA analyses. A survey of the systematics of theTriticeae indicated that the genomic system ofLöve is probably the most useful starting point for interpreting molecular data even though the system has many problems from a taxonomic point of view. The geographical distribution ofTriticeae species, using both published and unpublished data, suggested that information of this type taken together with the theory of continental drift provides a broad time-span for considering data from DNA sequence studies. The significance, and modes of analyses, of isozyme studies were assessed because they often provide valuable characters in determining relationships between species. The main character underlyingLöve's andDewey's analyses of theTriticeae, namely chromosome pairing, is discussed with particular reference to isozyme studies to show that in some cases, such as species ofHordeum sensu lato, consistent relationships are obtained. Finally, new developments in understanding chromosome structure are considered in relation to the above variables in the taxonomy and evolution of theTriticeae.     

Multivariate and cladistic analyses of the purple-flowered species ofErysimum (Cruciferae) from the Iberian Peninsula

A taxonomically difficult purple-flowered group within the genusErysimum, restricted to the Iberian Peninsula, is analyzed by multivariate and cladistic analyses. 51 specimens have been scored for 14 characters. Both principal components and discriminant analyses provide support to the recognition of the five species considered by the author, namelyE. linifolium, E. lagascae, E. baeticum, E. popovii, andE. cazorlense. Cladistic analysis, using 7 characters resulted in a single most parsimonious cladogram containing no homoplasies. The pattern of morphologic divergence follows a clear NW.-SE. trend, which is congruent with the topology of the cladogram. This trend significantly affects growth-form as well as fruit characters, both providing the main grounds for species recognition. The different behavior and significance of several characters in both kinds of analysis is discussed. The co-occurrence of morphologically similar individuals differing in the flower color is discussed, too. Possible explanations for this phenomenon involve hybridization in a wide sense or, alternatively, rejecting the assumption of monophyly for the group.     

The chromosomes ofGrahamia (Portulacaceae)

The somatic and gametic chromosomes of the monotypical genusGrahamia (G. bracteata) have been studied for the first time: 2n = 18, n = 9. The karyotype is symmetrical; of the nine m pairs one has microsatellites. The basic chromosome number x = 9 is considered to be primitive within the family.     

Chromosomal variations in the callus tissues ofAllium tuberosum andA. cepa

Summary Chromosome studies ofAllium tuberosum andA. cepa were made from one month to eighteen months old calluses. Different types of chromosomal variations like aneuploid number ranging from 28 to 31, tripolarity, lagging, micronuclei, haploid number etc. were noted inA. tuberosum, whereas inA. cepa the cells showed high chromosome numbers such as 32, 64 or more. The normal chromosome number (2n=16) occurred rarely. The selective pressure of the culture media may have led to the manifestation of the genetic control of differential response to chromosome behaviour and growth in the two species of the same genus.     

The karyotype ofLimnobium spongia (Hydrocharitaceae)

The somatic chromosomes ofLimnobium spongia have been studied for the first time. Its number is 2n = 24; the karyotype is composed of 5 m pairs + 3 sm pairs + 4 st pairs. Pairs 3 and 11 bear microsatellites. Results are compared with previous chromosome reports for the family, mainly as regards the taxonomic status ofLimnobium versusHydromystria; our findings seem to support the point of view of those who favour the independence of both generic entities.The first author is member of the Carrera del Investigador (CONICET, Argentina) and the second one fellowship from the same institution.     

The reproductive biology of the genusBaldellia (Alismataceae)

The reproductive biology of the genusBaldellia was studied under cultivation and in nature.B. ranunculoides subsp.repens is self-incompatible whereasB. ranunculoides subsp.ranunculoides andB. alpestris are self-compatible. Bud-pollination occurs in theB. alpestris. Self-incompatibility correlates with the production of fewer, larger, more showy flowers, higher pollen production (but reduced pollen grain size), fewer carpels and smaller seeds. Resource allocation to sexual reproduction is inversely correlated with the degree of vegetative growth.B. ranunculoides subsp.repens produces more ramets than subsp.ranunculoides, which reproduces mainly by seeds.B. alpestris is intermediate between the two since it is self-compatible, produces fewer, larger seed than subsp.ranunculoides and is also able to propagate by vegetative means.     

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.     

Microtubules and coated vesicles in guard-cell protoplasts ofAllium cepa L.

Protoplasts were prepared from the guard cells ofA. cepa. Epidermal peels taken from expanding green leaves and largely free of mesophyll were treated with Cellulysin, and protoplasts were harvested after 18 h of digestion. That the protoplasts were derived from guard cells was ascertained from their characteristic vacuolar autofluorescence and from observations showing that all other epidermal cells are killed in the peeling procedure. The protoplasts proved to be a good system with which to view the cell cortex and inner surface of the plasmalemma. The lysis of cells adhering to polylysine-treated, Formvar-coated grids, followed by negative staining in uranyl acetate, showed that many microtubules normally present in ordered arrays in situ remain closely applied to the inner surface of the plasmalemma in protoplasts. In addition, numerous vesiculate elements including coated vesicles and/or pits are present amongst the microtubules. Similar vesicles are evident in thin sections of fixed, embedded guard cells and protoplasts. The significance of these structures in the cell cortex is discussed.     

A molecular phylogeny of the wild onions (Allium; Alliaceae) with a focus on the western North American center of diversity

Nuclear ribosomal DNA (ITS and ETS) sequences from 39 native Californian (USA) Allium species and congeners were combined with 154 ITS sequences available on GenBank to develop a global Allium phylogeny with the simultaneous goals of investigating the evolutionary history (monophyly) of Allium in the Californian center of diversity and exploring patterns of adaptation to serpentine soils. Phylogenies constructed with ITS alone or ITS in combination with ETS provided sufficient resolution for investigating evolutionary relationships among species. The ITS region alone was sufficient to resolve the deeper relationships in North American species. Addition of a second marker (ETS) further supports the phylogenetic placements of the North American species and adds resolution within subgenus Amerallium, a clade containing many Californian endemics. Within the global phylogeny, the native North American species were found to be monophyletic, with the exception of Allium tricoccum and Allium schoenoprasum. All native Californian species included in the analysis fell into a monophyletic subgenus Amerallium section Lophioprason, although endemic Californian species were not monophyletic due to the inclusion of species with ranges extending beyond the California Floristic Province. The molecular phylogeny strongly supports previous morphology-based taxonomic groupings. Based on our results, serpentine adaptation appears to have occurred multiple times within section Lophioprason, while the ancestor of the Californian center of diversity may not have been serpentine-adapted.