Endopolyploidy inHelianthus annuus (Asteraceae), A scanning cytophotometric study

Endopolyploidy has been detected in some varieties ofHelianthus annuus L. (Asteraceae/Compositae) by means of scanning photometry of Feulgen-stained nuclei and analysis of nuclear structure. In the hypocotyl cells of seedlings, ploidy levels reach respectively 8 C and 16 C in different varieties, in the root cells 8 C and 16 C; in the cotyledons of ripening seeds 4 C to 8 C values have been found, while all nuclei of the inflorescence axis of one variety exhibit a DNA content of 4 C.—This is the first report of endopolyploidy in a non-succulentAsteraceae species. The characteristic distribution of the endopolyploidy levels in different varieties suggests a strong genetic and/or hormonal control of the final nuclear DNA content in differentiated cells.     

Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.     

Distribution of parental DNA markers inEncelia virginensis (Asteraceae: Heliantheae), a diploid species of putative hybrid origin

Morphological, geographical and ecological evidence suggests thatEncelia virginensis is a true-breeding diploid species derived from hybrids ofE. actoni andE. frutescens. To test this hypothesis, we examined the chloroplast and nuclear DNA of severalEncelia species. PCR amplification targeted three separate regions of chloroplast DNA:trnK-2621/trnK-11,rbcL/ORF106, andpsbA3/TrnI-51, which amplify 2600bp, 3300bp and 3200bp fragments respectively. Restriction fragment analysis of chloroplast DNA revealed no variation that could be used to discriminate between the parent species. A RAPD analysis using 109 dekamer primers was used to analyze the nuclear genome.Encelia actoni andE. frutescens were distinguished by several high-frequency RAPD markers. In populations ofE. virginensis, these markers were detected in varying proportions, and no unique markers were found. Evidence from the nuclear genome supports the hypothesis thatE. virginensis is of hybrid origin. ThatE. virginensis may have arisen by normal divergent speciation followed by later introgression remains a possibility, however, and is not formally ruled out here. Diploid hybrid speciation inEncelia differs from other documented cases in that there are no discernible chromosome differences between the species, and all interspecific hybrids are fully fertile. In addition, apparent ecological selection against backcross progeny provides an external barrier to reproduction between F₁ progeny and the parental species. These characteristics suggest that hybrid speciation inEncelia may represent an alternative model for homoploid hybrid speciation involving external reproductive barriers. In particular, this may be the case for other proposed diploid hybrid taxa that also exhibit little chromosomal differentiation and have fertile F₁s.     

Nuclear behaviour of colonies and regenerated buds from protoplasts ofNicotiana plumbaginifolia Viviani haploids

The ploidy level variations of protoplast cultures ofNicotiana plumbaginifolla Viviani (n=10) were investigated from protoplast isolation until regenerated buds, using cytophotometric measurements of nuclear DNA content and chromosome counting. An increase in the average nuclear DNA amount has been found to occur in freshly isolated protoplasts after 15 hours of maceration. Cytological abnormalities like nuclear fragmentation, chromatin connections between interphasic nuclei and micronuclei were observed during the following days. Chromosome counting in 15, 30 and 50-day-old calli and in regenerated buds revealed that nuclei are haploid, diploid or aneuploid.Abbreviations p-cells, p-calli or p-colonies protoplast-derived cells, calli or colonies - BAP 6-benzylaminopurine - NAA 1-naphtaleneacetic acid - 2iP ²-isopentyl-aednine     

Cytophotometrical measurement of nuclear DNA content in some coniferous and deciduous trees

Summary The DNA content of nuclei from meristematic root tip cells of five coniferous and one deciduous tree species and, for comparison, ofVicia faba was cytophotometrically determined. The DNA values of diploid nuclei fromGinkgo biloba are approximately a quarter lower than those fromVicia faba. The nuclear DNA values of the other tree species are merely a third to a ninth part of those ofVicia faba. In three tree species, as well as diploid, we have found nuclei of different polyploid level.The reliability of different cytochemical methods, which are used for determination of the nuclear DNA content, is critically analyzed. The DNA values of the investigated tree species are discussed in connection with the evolution of the DNA content in higher plants.Dedicated to Professor Dr. F. Mechelke in honour of his 60th birthday     

Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry

The technique of DNA flow cytometry was used to study variation in DNA content among different ploidy levels, as well as among diploid species, of Vaccinium section Cyanococcus. In a sample of plants of varying ploidy level, the relative fluorescence intensity (RFI) of nuclei stained with propidium iodide was a function of the number of chromosome sets (x), as represented by the linear equation RFI=3.7x-2.3 (r²=95%). The data indicated that DNA flow cytometry could be useful for the determination of ploidy level at the seedling stage in blueberry. They also suggest that conventional polyploid evolution has occurred in this section of the genus Vaccinium with an increase in nuclear DNA content concurrent with the increase in chromosome number. The nuclear DNA content of diploid species of Vaccinium section Cyanococcus was estimated from the relationship of the observed RFI to an internal known DNA standard (trout red blood cells). A nested analysis of variance indicated significant variation among species, as well as among populations within species, in nuclear DNA content, although this variation was small compared to the variation among ploidy levels. The variation in nuclear DNA content corresponded to the phylogenetic relationships among species determined from previous studies.     

Genome size variation and C-band polymorphism inAlstroemeria aurea, A. ligtu andA. magnifica (Alstroemeriaceae)

Among a total of 43 accessions ofAlstroemeria aurea, A. ligtu andA. magnifica nuclear DNA amounts (2C-values) showed significant intraspecific variation, 1.09, 1.21 and 1.15 fold, respectively, when determined through flow cytometric measurements of fluorescence of propidium iodide (PI) stained nuclei. After staining with another fluorochrome, 4,6-diamidino-2-phenylindole (DAPI), an intraspecific variation of 1.10, 1.11 and 1.12 fold, respectively, was found. C-band polymorphisms were present among and within the accessions of all three species. In some cases only very small differences in C-banding pattern were observed. In other cases, however, differences were more prominent. Besides C-band polymorphism, there were also instances of chromosome length polymorphism, which concerned the total chromosome complement or single chromosomes. The variation in nuclear DNA amount inA. aurea andA. ligtu was more or less continuous, except for one accession ofA. ligtu subsp.simsii. Artificial selection and possibly introgression of chromosomes from other species may have moulded the karyotypes of some of the accessions ofA. aurea, a species that has been under cultivation for more than 160 years. The variation as observed inA. magnifica subsp.magnifica was discontinuous and could be due to a broad species concept.     

An efficient method for flow cytometric analysis of pollen and detection of 2n nuclei in Brassica napus pollen

A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.Abbreviations DAPI 4,6-Diamidino-2-phenylindole - NIB Nuclear isolation buffer     

Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Summary Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G₁). In certain cases, however, the nuclear DNA content of differentiated G₁ cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after³H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.     

Relationships betweenNor-loci from differentTriticeae species

TheNor-loci of polyploid wheats and their putative diploid progenitor species were assayed by probing isolated nuclear DNA with ribosomal DNA spacer sequences (spacer rDNA sequences, isolated by cloning), from theNor-loci of genomes B (Triticum aestivum), G (T. timopheevi), B (syn. S,T. speltoides), A (T. monococcum) and V (Dasypyrum villosum). DNA samples for analysis were digested with the restriction endonuclease Taq 1 and assayed by DNA-DNA hybridization under standard (37°C) and high stringency (64°C) conditions. The assay procedure emphasized differences between the divergent spacer sequences of the polyploid species and allowed relative homologies to the respective sequences in diploid species to be established. — The studies indicated thatT. timopheevi andT. speltoides contain different sets of spacer rDNA sequences which were readily distinguishable and, in the case ofT. timopheevi, assigned toNor-loci on different chromosomes. This contrast with the spacer rDNA sequences of the majorNor-loci on chromosomes 1 B and 6 B inT. aestivum, which were difficult to distinguish and were deduced to contain very similar sequences. Among the diploid progenitor species only the spacer rDNA fromT. speltoides shared close homology with polyploid wheat species. OneNor-locus inT. timopheevi (on chromosome 6 G) did not show close homology with any of the rDNA spacer probes available. — The data suggestsT. speltoides was the origin of someNor-loci for both theT. timopheevi andT. turgidum lines of tetraploid wheats. The possibility that the 6GNor-locus inT. timopheevi may have derived from an unknown diploid species by introgressive hybridization is discussed. The spacer rDNA sequence probe fromT. monococcum shared good homology with some accessions ofD. villosum and a line ofT. dicoccoides; the implications of this finding for evolution of present-day wheats are discussed.     

Nuclear DNA content in the genus Hepatica (Ranunculaceae)

Using flow cytometry, we measured the nuclear DNA contents of all known taxa in Hepatica. Nuclear DNA content of Hepatica falconeri (diploid, crenate leaf lobes) was significantly lower than that of diploid entire species. Among the tetraploid species, crenate species had lower DNA contents than the entire taxon H. nobilis var. pubescens. The DNA content of the tetraploid species was more than double that of the diploid species among the same leaf-type groups.     

Cytological studies inCyclamen subg.Cyclamen (Primulaceae)

Although theCyclamen subg.Cyclamen spp. are morphologically variable, previous studies suggest a superficial cytological uniformity. New chromosome numbers and an indication of karyotypic instability are reported inC. hederifolium andC. africanum that reveal that the cytology of the subgenus is more complicated than previous accounts suggest. The possible significance of these phenomena is discussed. The diploid status ofC. purpurascens has been confirmed and a distinction between the three diploid karyotypes has been described. The cytological variation may help to explain the well documented morphological variation exhibited in these species.     

Conservation of rDNA inPolystichum (Dryopteridaceae)

Restriction site variation in the nuclear 18S–25S ribosomal RNA genes (rDNA) was analyzed hierarchically in a species complex in the fern genusPolystichum. Two distinct rDNA repeat types were present in all individuals ofPolystichum examined. No variation was detected among individuals within a population ofP. munitum, among populations ofP. munitum orP. imbricans, or among the six diploid species ofPolystichum from North America, including the circumborealP. lonchitis. The identity of rDNA repeats across all six North American species ofPolystichum may reflect an overall similarity of the nuclear genomes of these species, an observation supported by isozyme data as well. However, this nuclear similarity contrasts sharply with the highly divergent chloroplast genomes of these six species. The conservative nature of the rDNA inPolystichum also is in contrast to the much more variable rDNAs of most angiosperms investigated. Perhaps the tempo and mode of evolution of rDNA in ferns differ from those of angiosperms; however, the data base for fern rDNA is very small. Furthermore, the number of repeat types per individual is consistent with a diploid, rather than polyploid, condition despite the high chromosome number (n = 41) of these plants, although homogenization of multiple, divergent rRNA genes cannot be disproven.     

Nuclear DNA content of some important plant species

Nuclear DNA contents of more than 100 important plant species were measured by flow cytometry of isolated nuclei stained with propidium iodide.Arabidopsis exhibits developmentally regulated multiploidy and has a 2C nuclear DNA content of 0.30 pg (145 Mbp/1C), twice the value usually cited. The 2C value for rice is only about three times that ofArabidopsis. Tomato has a 2C value of about 2.0 pg, larger than commonly cited. This survey identified several horticultural crops in a variety of families with genomes only two or three times as large asArabidopsis; these include several fruit trees (a pricot, cherry, mango, orange, papaya, and peach). The small genome sizes of rice and the horticultural plants should facilitate molecular studies of these crops.     

Hybridization and evolution inCardamine (Brassicaceae) at Urnerboden, Central Switzerland: Biosystematic and molecular evidence

Hybridization between two diploid (2n = 2x = 16) species ofBrassicaceae, Cardamine rivularis andC. amara, at Urnerboden, Central Switzerland, resulted in the rather unusual triploid hybridC. insueta (2n = 3x = 24), and later on in the amphiploidC. schulzii (2n = 6x = 48). The hybrid and the neopolyploid species colonized successfully some man-made biotopes. Plants ofC. insueta are mostly functional females with non-dehiscent anthers, but true hermaphrodite individuals with partly sterile pollen grains also occur within the population. Analyses of cpDNA and nuclear DNA permitted to establish the parentage of the hybrid: the maternal parent which contributed unreduced egg cells proved to beC. rivularis whereas the normally reduced pollen originated fromC. amara. The pronounced genetic variability inC. insueta revealed by isozyme and RAPD analyses, at variance with the polarized segregation, heterogamy and strong vegetative reproduction of the hybrid, is possibly influenced by recurrent formation ofC. insueta which party results from backcrosses betweenC. insueta andC. rivularis but may also proceed by other pathways. The amphiploidCardamine schulzii has normally developed anthers but its pollen is sometimes highly sterile. The surprisingly uniform genetic make-up of the new amphiploid species might be related to its possible monotopic origin and/or young phylogenetic age but should be further assessed. Site management seems to be very important to a further development of hybridogenous populations and their parent species. In conclusion, the evolution at Urnerboden is discussed in the context of the traditional concept of multiple plant origins.     

Quantitative nuclear DNA changes inEleusine (Gramineae)

2C nuclear DNA amounts were determined in 30 collections belonging to 10 species ofEleusine. About a 2.5-fold variation in genome size is evident in the genus. The 2C DNA amount in the diploid species ranged from 2.50 pg inE. verticillata to 3.35 pg inE. intermedia. In contrast, the tetraploid species showed a range from 4.95 pg inE. africana to 6.13 pg inE. floccifolia. At intraspecific level 10 collections ofE. coracana, 6 ofE. indica, 4 ofE. africana, 2 ofE. tristachya, and 2 ofE. kigeziensis did not show any significant variation. However, 2 collections ofE. floccifolia, connected with polyploidy, displayed about 90% variation. Polyploid species showed approximately double the genome size of that of their corresponding diploids. An evolutionary increase in DNA amount is evident inE. coracana during the course of its origin and domestication fromE. africana.     

DNA evidence for multiple origins of intergeneric allopolyploids in annualMicroseris (Asteraceae)

Seventy populations of North American annualMicroseris, Stebbinsoseris, andUropappus species were examined for chloroplast and nuclear ribosomal DNA restriction site variability to determine the origin of the allotetraploid speciesS. heterocarpa andS. decipiens. Previously identified chloroplast DNA restriction site variants were used in concert with restriction site variation forNco I in the nuclear-encoded ribosomal DNA repeat. The presence of two, mutually exclusive restriction site gains were observed in diploid populations ofM. douglasii; these same variants were also found in populations of allotetraploidS. heterocarpa, indicating mutiple origins of this species from different maternal diploid populations ofM. douglasii. Variation in the rDNA repeat between the diploid annual species and the putative paternal genome ofU. lindleyi was found to be additive inS. heterocarpa. A similar relationship was observed for the origin ofS. decipiens; cpDNA restriction site variants found inM. bigelovii andM. douglasii were present inS. decipiens. The rDNANco I variants also were additive in this purported allotetraploid. These results confirm the reticulate evolutionary pattern inStebbinsoseris and provide another example of multiple origins of intergeneric allopolyploids.     

Genomic in situ hybridization inArachis (Fabaceae) identifies the diploid wild progenitors of cultivated (A. hypogaea) and related wild (A. monticola) peanut species

Genomic in situ hybridization offers a powerful tool for investigating genome organisation and evolution of taxa known, or suspected, to be allopolyploids. The question of the diploid progenitors of cultivated peanut (Arachis hypogaea, 2n=4x=40) has been the subject of numerous studies at cytogenetical, cytochemical, biochemical and molecular levels, but no definitive conclusions have been reached. The biotinylated total genomic DNA from potential diploidArachis species were separately hybridized in situ to root tip chromosomes ofA. hypogaea and wild speciesA. monticola (2n=4x=40) without or mixed with an excess of unlabelled DNA from the species not used as a probe. Among the range of different species combinations used, the strong and uniform signals given by labelledA. ipaensis DNA when hybridized toA. hypogaea andA. monticola in combination with unlabelledA. villosa DNA indicates that overall molecular composition of twenty chromosomes ofA. hypogaea andA. monticola is very similar toA. ipaensis chromosomes. ProbingA. hypogaea andA. monticola chromosomes with labelled genomic DNA fromA. villosa mixed with unlabelled DNA fromA. ipaensis likewise labelled strongly and uniformly the other twenty chromosomes. BarringA. ipaensis, all the diploidArachis species presently investigated had characteristic centromeric bands in the twenty chromosomes within the complement indicating a clear division ofA. ipaensis from other species. InA. hypogaea andA. monticola only twenty chromosomes showed centromeric bands. These results (i) confirm the allopolyploid nature ofA. hypogaea andA. monticola, (ii) strongly support the view that wildA. monticola and cultivatedA. hypogaea are very closely related, and (iii) indicate thatA. villosa andA. ipaensis are the diploid wild progenitors of the tetraploid species studied. The present results also reveal that the nucleolus organizing region (NOR) originating fromA. villosa alone is expressed in the two tetraploid species.     

Systematics and biogeography of the Andean genusEriocnemis (Aves: Trochilidae)

Summary With 11 currently recognised species, the genusEriocnemis (Reichenbach, 1849) is one of the most diversified Andean trochilid groups occupying mainly open montane habitats such as the edges of cloud forest or páramos. On the basis of distributional and morphological patterns, this study highlights the geographical variation and biogeography of the taxon. Characteristics common to all these species are the greenish dorsal plumage, the conspicuous and mostly whitish tibial tufts, and a fairly pronounced tail bifurcation. With the help of plumage synapomorphies for a cladistic analysis (PAUP*), several species groups or superspecies can be distinguished: theE. vestitus group (incl.E. vestitus, E. godini, E. nigrivestis), theE. luciani group (incl.E. luciani, E. cupreoventris, E. sapphiropygia), and theE. alinae group (incl.E. alinae, E. mirabilis).E. glaucopoides, E. mosquera, andE. derbyi differ quite widely in morphology and ecological requirements from the other species. Three new subspecies are described,E. vestitus arcosi from southern Ecuador and northern Peru, andE. luciani baptistae from central and southern Ecuador. A previously overlooked specimen ofE. luciani from the Andes of Mérida represents the first species record for Venezuela, about 1100 km northeast of the main population range, and should be recognised taxonomically asE. luciani meridae, subsp. nov., on the basis of its unique plumage morphology and geographical separation. Additionally, the unique type ofE. ventralis (Salvin, 1891) is probably of hybrid origin (E. vestitus × cupreoventris). The genus may have evolved in the northern Andes, subsequently spreading southward and invading the central Andes. Its recent range and phylogenetic patterns indicate vicariance events as the major speciation factor inEriocnemis.In memoriam Dr. Luis F. Baptista (1941–2000)     

Chloroplast DNA and isozyme evidence on the evolution ofSenecio vulgaris (Asteraceae)

Chloroplast DNA (cpDNA) and isozyme variation were analyzed over a range of populations of two infraspecific taxa of the tetraploidSenecio vulgaris. The isozyme data were supportive of the hypothesis that the weedy and cosmopolitanS. vulgaris var.vulgaris is an evolutionary derivative ofS. vulgaris subsp.denticulatus from the coasts of W Europe and montane altitudes in S Spain and Sicily. The two taxa exhibited a very high genetic identity with subsp.denticulatus containing slightly more isozyme diversity than was found in var.vulgaris. — Three cpDNA haplotypes (A, B, C) already known from other Mediterranean diploid species ofSenecio were resolved in var.vulgaris, and an additional fourth haplotype (E) was found in subsp.denticulatus. Two alternative hypotheses were chosen to account for the origin and maintenance of the observed cpDNA composition ofS. vulgaris. It either reflects (1) the retention of an ancestral polymorphism which stems from the recurrent and polytopic formation of ancestral tetraploid lineages; or (2)S. vulgaris originally was characterized by haplotype E, and haplotypes A, B and C were acquired through repeated introgressive hybridization with related diploid species. The finding that very low levels of nuclear (isozyme) diversity were present in both taxa ofS. vulgaris examined supports the second of these two hypotheses; however, more detailed analysis of nuclear genetic diversity is required before a firm conclusion can be reached on this matter.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday