Rare recent natural hybridization in Hieracium s. str. – evidence from morphology, allozymes and chloroplast DNA

The first proven data on natural hybridization in the genus Hieracium s. str. are presented. Plants with intermediate morphological characters between the diploids H. alpinum and H. transsilvanicum were found in the Muntii Rodnei (Romanian Eastern Carpathians) in 2001 and in the Chornohora Mts (Ukrainian Eastern Carpathians) in 2003. While plants of intermediate morphology between usually so called basic species are usually tri- or tetraploid in Hieracium s. str., these plants were diploid (2n=18) like both parental species in this region. The Romanian plant did not produce fertile achenes in free pollination and in control backcrosses with H. transsilvanicum, two hybrids from Ukraine were completly seed sterile in free pollination and reciprocal crosses. Pollen stainability as an indirect measure of male fertility was quite high in the studied Ukrainian hybrid plants and similar to the parental taxa. Evidence from allozyme analysis also confirmed the hybrid origin of the studied plants. Sequencing and PCR-RFLP analyses of the trnT-trnL intergenic spacer revealed that all hybrid plants had the H. transsilvanicum chloroplast DNA haplotype. Maternal inheritance of chloroplast DNA in this particular cross was proved with artificial hybrids from reciprocal experimental crosses between H. alpinum and H. transsilvanicum. In both localities, the natural hybrid plants were found in disturbed habitats, exceptionally allowing contact of the otherwise ecologically vicariate parental species. Morphologically, the hybrid plants belong to H. × krasani Woł.     

Tetraploid induction of Mitracarpus hirtus L. by colchicine and its characterization including antibacterial activity

Tetraploid plants were successfully induced for the first time in Mitracarpus hirtus L., by overnight immersion of shoot meristems in 0.1 % colchicine solution, followed by in vitro culture leading to plant regeneration. Examination of ploidy level by flow cytometric analysis and counting chromosome number at metaphase confirmed that original diploid plant (WT1) contained chromosome number as 2n = 2x = 28, whereas 2n = 4x = 56 was observed in the tetraploids induced with colchicine treatment (CC102 and CC110). Thicker root formation, larger stomata (1.3–2 times), and lower density of stomata (1.7–4 times) were observed in these tetraploid plants. After transplantation to the pot, tetraploid plant (CC110) showed higher fresh weights of aerial part and leaves (1.5 and 1.4 times respectively) than diploid. However, the methanolic extracts from leaves of tetraploid line CC102 showed inhibition against human pathogenic bacterium, S. aureus while WT1 and CC110 showed no activity. GC–MS revealed 40 unique compounds present in CC102, but absent in WT1 and CC110. Through hierarchical clustering analysis the 40 unique compounds in CC102 formed a cluster group found to correlate with anti-S. aurens activity. These results suggested that tetraploid M. hirtus CC102 created in this study provides a novel source of compounds useful in fighting infectious disease.     

A CYTOGENETIC ANALYSIS OF CERTAIN POLYPLOIDS IN GRINDELIA (COMPOSITAE)

Two diploid taxa, Grindelia procera and G. camporum, and 3 tetraploid ones, G. camporum, G. hirsutula, and G. stricta, have been studied to ascertain their interrelationships. Meiosis in diploid parental strains was regular, the common chromosome configuration being 5 rod bivalents and 1 ring bivalent. The average chiasmata frequency per chromosome was 0.60. Pollen fertility was about 90% in all strains examined. Diploid interspecific hybrids had normal meiosis with an average chiasmata frequency of 0.56 per chromosome. No heterozygosity for inversions or interchanges was detected, and pollen fertility was above 85%. Meiosis in parental tetraploid strains was characterized by the presence of quadrivalents in addition to a complementary number of bivalents. The average chiasmata frequency per chromosome was 0.59 and pollen fertility was generally about 80%. Tetraploid interspecific hybrids also had quadrivalents, normal meiosis, and high pollen fertility. Close genetic relationships between the diploids and between the tetraploids are indicated, and geographical, ecological, and seasonal barriers to gene exchange exist. Attempts to obtain hybrids between diploids and tetraploids were successful in a few cases. The hybrids were tetraploid and had normal meiosis and fertility similar to parental and F₁ tetraploids. Their origin was by the union of unreduced gametes of the diploid female parent and normal pollen from the tetraploid parent. On the basis of chromosome homology, normal meiosis, plus high fertility exhibited in the diploid, tetraploid, and diploid X tetraploid interspecific hybrids, these species of Grindelia are considered to be a part of an autopolyploid complex. Gene exchange between diploids and diploids, tetraploids and tetraploids, and diploids and tetraploids is possible. Tetraploid G. camporum may have originated by hybridization between G. procera and diploid G. camporum with subsequent doubling of chromosomes and selection for the combined characteristics of the diploids.     

Taxonomic studies of theMuscari botryoides complex in Hungary

The ploidy level and karyotype ofMuscari botryoides (s.l.) samples from Hungary (25 localities) and Romania (1 locality:locus classicus ofM. transsilvanicum) were determined. The Romanian sample proved to be diploid (2n=18), while in Hungary both diploid and tetraploid (2n=36) populations occurred. The karyotypes of all diploid populations were similar: 2 pairs of long acrocentric (one of them usually with satellites) + 3 pairs of medium-sized submetacentric-metacentric + 4 pairs of short ± metacentric chromosomes. All diploid populations in Hungary can be identified asM. transsilvanicum. There is no reason to support the taxonM. botryoides subsp.hungaricum because it does not differ from the sample collected at thelocus classicus ofM. transsilvanicum (Romania, Sibiu-Gu?teri?a) in any of the characteristics mentioned in its protologue. Its karyotype also corresponds to that ofM. transsilvanicum. Contrary to the former assumptions, the tetraploidM. botryoides is also native to Hungary. The tetraploid karyotype seems to be somewhat of a duplication of the diploid one. Morphological characters used in the identification keys are not suitable for unambiguous separation of the taxa mentioned above, though morphometric analyses revealed some quantitative differences between diploids and tetraploids. Their separation on species level can only be supported by the supposed reproductive barriers caused by different ploidy level and chorology. In HungaryM. transsilvanicum is restricted mostly to the Eupannonian Region, the Mecsek and Villány Mts.M. botryoides does not occur in the Eupannonicum, instead it inhabits the subatlantic hilly W and SW part of Hungary and the Northern Mountain Range. The latter territory (including also the Slovak localities) seems to be the easternmost extension of the area ofM. botryoides.     

In vitro induction of tetraploids in an interspecific hybrid of Calanthe (Calanthe discolor × Calanthe sieboldii) through colchicine and oryzalin treatments

Tetraploids were successfully produced from diploid seeds obtained through interspecific crossing between Calanthe discolor and Calanthe sieboldii by treating with colchicine or oryzalin. Colchicine was tested at concentrations of 0.05 and 0.1 % for 0, 3, or 7 days and oryzalin was tested at a concentration of 0.003 % for 1, 2, 4, and 7 days, and the ploidy of the seedlings was determined by flow cytometry. Tetraploids (4×) were obtained from the interspecific hybrid seeds treated with all colchicine and oryzalin concentrations. The most efficient condition for inducing tetraploids seemed to be treated with 0.003 % oryzalin for 1 or 2 days. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The stomatal density and sizes of the tetraploid plants were significantly higher and larger than those of the diploid plants. Differences in leaf shape were found between the tetraploid and diploid plants under the same growing conditions: the leaves of the diploids were elongated and those of the tetraploids were round.     

Production and molecular characterization of two new Citrus somatic hybrids for scion improvement

Somatic hybridization by protoplast fusion from cell suspension cultures and leaf parent has been a well-established technique holding great potential for citrus variety improvement. In this study, somatic hybrid plants were regenerated from the following two fusion combinations: ‘Murcott’ tangor (Citrus reticulata Blanco × C. sinensis (L.) Osbeck) + Hirado Buntan Pink pummelo (HBP) (C. grandis (L.) Osbeck) and ‘Bingtang’ orange (C. sinensis (L.) Osbeck) + Calamondin (C. microcarpa Bunge). Somatic hybrids were selected at an early stage based on their higher capacity for embryogenesis comparing to non-hybrid cells. Flow cytometry analysis showed that all plants from pre-selected lines of the two combinations were tetraploid. SSR analysis confirmed their hybrid nature, with nuclear DNA from both fusion parents, and absence of parental specific bands was also detected. Cytoplasmic compositions of the recovered plants were further revealed by CAPS and cpSSR analysis. The allotetraploid somatic hybrids from the ‘Murcott’ tangor + HBP combination will be applied to develop triploid seedless cultivars by interploid crossing with diploid seedy citrus cultivars, and those from ‘Bingtang’ orange + Calamondin could be valuable for Asiatic citrus canker-tolerant and ornamental citrus breeding.     

Hieracium × grofae — a rediscovered diploid hybrid from the Ukrainian Carpathians

Diploid hybrid plants (2n = 18) between sexual diploid cytotypes of Hieracium alpinum and H. umbellatum were found in the Ukrainian Eastern Carpathians. They were identified with H. × grofae Woł., originally used for the combination H. decipiens × H. umbellatum var. lactaris. As H. decipiens sensu Woł. (non Tausch) does not produce viable pollen grains and is most probably a polyploid apomict, it is unlikely to produce diploid hybrid plants with diploid H. umbellatum. Both parent species, Hieracium alpinum and H. umbellatum are also given by Wołoszczak from the original locality. Thus we conclude that H. × grofae is result of hybridization between H. alpinum and high mountain form of H. umbellatum. Hybrid plants are morphologically intermediate between the parent species, and moreover resemble closely or they are identical with the experimental hybrids of the same parent combination. Hybrids produce rather high amount of homogeneously sized pollen grains (values of standard deviation and coefficient of variation lower then upper limits for diploids — 3μm or 7.5%, respectively), and might probably serve as male parents in further crosses; on the other hand, they are fully seed-sterile. A lectotype of H. × grofae, a second proved nothotaxon in the genus Hieracium s.str., is designated. Localities of H. × grofae are located in subalpine belt of the Marmarosh Mts, the Svydovets’ Mts and the Horhany Mts (all in the Ukrainian Eastern Carpathians). Biotopes of hybrid plants usually represent secondary formed and disturbed pastures allowing close contact of altitudinally vicariant parent taxa.     

Polyphyletic allopolyploid origin ofRanunculus cantoniensis (4x) fromR. silerifolius (2x) ×R. chinensis (2x)

Both diploid and tetraploid experimental interspecific hybrids betweenRanunculus silerifolius (2x) andR. chinensis (2x) exhibit normal bivalent pairing. However, microspores of diploid hybrids do not undergo mitosis and their pollen grains are highly sterile, whereas tetraploid hybrids form good pollen grains after microspore division. Evidence is forwarded for the assumption thatR. cantoniensis (4x) has originated by hybridization between these two diploid parental species and by polyploidization of the diploid hybrids. Parallelisms between the different karyotypes ofR. cantoniensis (4x) andR. silerifolius (2x) suggest that the former is a species of polyphyletic origin.     

Genome synteny and evolution of AABB allotetraploids in Hibiscus section Furcaria revealed by interspecific hybridization, ISSR and SSR markers

Two tetraploid species of Hibiscus section Furcaria, H. acetosella and H. radiatus, have an AABB genomic constitution. The diploid species, H. cannabinus (AA) and H. surattensis (BB), were hybridized to develop interspecific alloploid (AB) hybrids. The synthetic interspecific hybrid exhibited intermediate morphological characters, with expression of domestication-related traits, but exhibited higher genomic association with the B genome donor. Evolution of allopolyploids in section Furcaria was found to be associated with mutations in repetitive sequences, leading to higher variation in the tetraploid genome. Allopolyploidization was observed to be associated with both loss of repetitive sequences and appearance of new alleles. Genetic diversity analysis using ISSR and cross-species SSR markers revealed a closer association of diploid genomes and high variability of tetraploid genomes. The evolution of AABB tetraploids in this section possibly took place by hybridization of the A and B genome in geographically isolated regions.     

A protocol for fertility restoration of F<Subscript>1</Subscript> hybrid derived from <Emphasis Type="Italic">Lilium</Emphasis> × <Emphasis Type="Italic">formolongi</Emphasis> ‘Raizan 3’ × Oriental hybrid ‘Sorbonne’

The present study was an attempt to develop an in vitro colchicine chromosome doubling protocol to restore the fertility of an F₁ interspecific hybrid in Lilium. Basal scale segments of Lilium?×?formolongi?×?Oriental hybrid (FO) bulblets were pre-cultured for three durations (6, 15 and 25 days) and soaked in three colchicine concentrations (0.00, 1.25 and 2.50 mM) for 18, 24 and 36 h. To separate mixoploids, three cycles of adventitious bud induction were performed. The ploidy levels of the surviving plantlets were detected by flow cytometry at 30–31 weeks after induction and were confirmed by chromosome counts. The results indicated that the pre-culture duration, colchicine concentration and exposure time all had significant impacts on the tetraploid induction rate. The preferred procedure was to pre-culture the segments for 6 days and then treat them with 1.25 mM colchicine for 24 h. The morphological traits of rosette leaves were significantly different between the tetraploid and diploid plants. The adult tetraploid plants had considerably longer and wider leaves, larger flowers, and delayed flowering time (8 days later) than did diploid plants. Pollen viability tests and backcross trials of FO hybrids demonstrated fertility restoration at the tetraploid level. This protocol provides a feasible method for inducing fertile tetraploid FO hybrids for further breeding.     

Genomic relationships between hexaploid Helianthus resinosus and diploid Helianthus annuus (Asteraceae)

Genus Helianthus comprises diploid and polyploid species. An autoallopolyploid origin has been proposed for hexaploid species but the genomic relationships remain unclear. Mitotic and meiotic studies in annual Helianthus annuus (2n = 2x = 34) and perennial Helianthus resinosus (2n = 6x = 102) as well as the F₁ hybrids between both species were carried out. Chromosome counting confirmed the hybrid origin of the latter plants and their tetraploid condition. Bivalents in hybrids ranged from 12 to 28 ( $ \bar{x} $  = 20.8). Univalents, trivalents and quadrivalents were also observed. Meiotic products comprised dyads, triads and normal tetrads and pollen grains were heterogeneous in size. These observations suggest the occurrence of 2n pollen in addition to the expected n. Genomic in situ hybridization (GISH) of total H. annuus DNA on H. resinosus chromosomes rendered weak but uniform signals; similar hybridization pattern was observed using three other annual species. Hybridization with H. annuus probe performed on root tip cells of F₁ H. annuus × H. resinosus hybrids revealed 17 chromosomes with a strong hybridization signal. GISH in hybrid meiocytes distinguished chromosomes from parental species and revealed autosyndetic pairing of H. resinosus chromosomes, allosyndetic pairing in bivalents, trivalents and quadrivalents, and the presence of univalents derived from parents, H. annuus and H. resinosus. Results obtained from classical and molecular cytogenetics do not support H. annuus as a direct ancestor of H. resinosus. The occurrence of allosyndetic pairing and the relatively high fertility of the F₁ hybrids point to the possibility that useful genes could be transferred from H. resinosus to cultivate sunflower, although the effective rate of recombination has not been evaluated. GISH method proved effective to recognize parental chromosomes in H. annuus × H. resinosus progeny.     

Hybridisation between oilseed rape (Brassica napus) and tetraploid Brassica rapa under field conditions

Cross-compatible relatives of crop species contribute to the uncertainty regarding the potential risk of transgene escape from genetically modified varieties. The most successful crossing partner of oilseed rape (Brassica napus L.) is diploid Brassica rapa L. Variation of ploidy level among B. rapa cultivars has, until recently, been neglected in the context of gene flow and hybridisation with oilseed rape. We estimated the extent of hybridisation between autotetraploid B. rapa varieties (female) and B. napus (pollen donor) under experimental field conditions. Morphology, variation of relative DNA amount, and microsatellite markers were used to distinguish between intraspecific offspring of tetraploid B. rapa and interspecific hybrids with B. napus. Of 517 seed progenies of tetraploid B. rapa, 45 juvenile plants showed species specific morphological traits of oilseed rape. The detection of putative hybrids based on variation in relative DNA amounts was problematic due to the occurrence of aneuploidy. In total, 84 offspring showed relative DNA amounts deviating from tetraploid B. rapa, four of which were hexaploids. Of the 205 offspring analysed at three microsatellite loci, 67 had oilseed rape alleles. Based on molecular evidence a minimum hybridisation rate of 13.0% was estimated. A few mother plants accounted for the majority of hybrids. The mean pollen viability of hybrids between B. napus and tetraploid B. rapa (80.6%) was high in comparison with mean pollen viability of triploid hybrids between B. napus and diploid B. rapa. Therefore, the occurrence of tetraploid B. rapa should be taken into consideration when estimating the likelihood of gene flow from oilseed rape to close relatives at the landscape level. Tetraploid B. rapa is a common component of several seed mixtures and establishes feral populations in northwest Germany. Assuming a similar abundance of diploid and tetraploid B. rapa, gene flow from B. napus to tetraploid may be more likely than gene flow to diploid B. rapa.     

Somatic hybrid plants of Nicotiana x sanderae (+) N. debneyi with fungal resistance to Peronospora tabacina

Background and Aims

The genus Nicotiana includes diploid and tetraploid species, with complementary ecological, agronomic and commercial characteristics. The species are of economic value for tobacco, as ornamentals, and for secondary plant-product biosynthesis. They show substantial differences in disease resistance because of their range of secondary products. In the last decade, sexual hybridization and transgenic technologies have tended to eclipse protoplast fusion for gene transfer. Somatic hybridization was exploited in the present investigation to generate a new hybrid combination involving two sexually incompatible tetraploid species. The somatic hybrid plants were characterized using molecular, molecular cytogenetic and phenotypic approaches.

Methods

Mesophyll protoplasts of the wild fungus-resistant species N. debneyi (2n = 4x = 48) were electrofused with those of the ornamental interspecific sexual hybrid N. × sanderae (2n = 2x = 18). From 1570 protoplast-derived cell colonies selected manually in five experiments, 580 tissues were sub-cultured to shoot regeneration medium. Regenerated plants were transferred to the glasshouse and screened for their morphology, chromosomal composition and disease resistance.

Key Results

Eighty-nine regenerated plants flowered; five were confirmed as somatic hybrids by their intermediate morphology compared with parental plants, cytological constitution and DNA-marker analysis. Somatic hybrid plants had chromosome complements of 60 or 62. Chromosomes were identified to parental genomes by genomic in situ hybridization and included all 18 chromosomes from N. × sanderae, and 42 or 44 chromosomes from N. debneyi. Four or six chromosomes of one ancestral genome of N. debneyi were eliminated during culture of electrofusion-treated protoplasts and plant regeneration. Both chloroplasts and mitochondria of the somatic hybrid plants were probably derived from N. debneyi. All somatic hybrid plants were fertile. In contrast to parental plants of N. × sanderae, the seed progeny of somatic hybrid plants were resistant to infection by Peronospora tabacina, a trait introgressed from the wild parent, N. debneyi.

Conclusions

Sexual incompatibility between N. × sanderae and N. debneyi was circumvented by somatic hybridization involving protoplast fusion. Asymmetrical nuclear hybridity was seen in the hybrids with loss of chromosomes, although importantly, somatic hybrids were fertile and stable. Expression of fungal resistance makes these somatic hybrids extremely valuable germplasm in future breeding programmes in ornamental tobacco.     

Persistence,dispersal and genetic evolution of recently formed <Emphasis Type="Italic">Spartina</Emphasis> homoploid hybrids and allopolyploids in Southern England

In Southampton Water, UK, the recent (c. 150 years ago) interspecific hybridisation between Spartina alterniflora (2n = 6x = 62; A-genome) and S. maritima (2n = 6x = 60; M-genome) gave rise to the homoploid hybrid (S. × townsendii, 2n = 6x = 62), and subsequently to the invasive allododecaploid species S. anglica (2n = 12x = 120–124) that has since spread worldwide. To address the question of dynamics of mixed ploidy populations involving these plants, we analysed several Spartina populations (fifty one individuals) in Southern England, UK, one of which was the presumed place of origin of the homoploid hybrid (Hythe). Using a combination of flow cytometry and ribosomal DNA (rDNA) genotyping we were able to identify the genomic composition and ploidy level of each individual analysed. The data show that the homoploid hybrid still dominates the population at Hythe (82 % of individuals collected in that locality) since its origin in the nineteenth century. We also identified S. × townsendii for the first time on Hayling Island (66 % individuals), indicating dispersal beyond its likely origin. The fertile allododecaploid S. anglica was mainly found in populations outside the initial hybridisation site, on Hayling Island and at Eling Marchwood. Quantification of the rDNA contributions from each parental genome showed that the ratios were mostly balanced in S. × townsendii. However, two (3 %) S. anglica individuals analysed have lost nearly all M-genome homeologs, indicating extensive repeat loss. Such variation indicates that despite the presumed single allopolyploid origin of S. anglica and genetic uniformity at other loci, it has undergone substantial changes at the rDNA loci following genome duplication.     

Genetic Analysis of Putative Triploid Miscanthus Hybrids and Tetraploid M. sacchariflorus Collected from Sympatric Populations of Kushima, Japan

Miscanthus ?×giganteus, a triploid hybrid between tetraploid M. sacchariflorus and diploid M. sinensis, has considerable potential as a bioenergy crop. Currently only one genotype is widely cultivated, increasing its vulnerability to diseases during production. Finding new hybrids is important to broaden genetic resources of M. ×giganteus. Three putative triploid hybrids were discovered in a sympatric population of tetraploid M. sacchariflorus and diploid M. sinensis in Kushima, Japan. The hybrid nature of the triploids was determined by morphological analysis and sequencing the ribosomal DNA internal transcribed spacer (ITS) region. The triploids had awns on their florets, which is a common characteristic of diploid M. sinensis, and sheath hairs, which is typical of tetraploid M. sacchariflorus. All triploids showed heterozygosity in their ribosomal DNA ITS sequences. Based on these results, it is confirmed that the triploids are hybrids and novel genotypes of M. ×giganteus. Natural crossing between tetraploid M. sacchariflorus × diploid M. sinensis may also lead to the production of tetraploid hybrids. ITS analysis of tetraploid plants showed that one maternal parent of the triploid hybrids, K-Ogi-1, had heterozygous ITS, which was different than the other analyzed tetraploid, M. sacchariflorus. Thus, K-Ogi-1 was likely of hybrid origin. These tetraploid hybrids can also be utilized as parents in M. ×giganteus breeding. Since all hybrids identified in this study had tetraploid M. sacchariflorus as maternal parents, collecting and analyzing seeds from tetraploid M. sacchariflorus in sympatric areas could be an effective strategy to identify natural Miscanthus hybrids that can be used as bioenergy crops.     

CHROMOSOME PAIRING AND POLLEN FERTILITY IN INTERSPECIFIC HYBRIDS OF SPECIES OF PARTHENIUM (ASTERACEAE)

Meiotic analyses and pollen viability tests were performed on F, hybrids between diploid guayule (Parthenium argentatum Gray 2n = 36), P. rollinsianum Rzedowski (2n = 36), P. alpinum var. tetraneuris Barneby (2n = 36), and P. alpinum var. alpinum Nutt. (2n = 36). Parthenium chromosomes are small and karyomorphologically similar, and meiotic analysis is difficult because of chromosome clumping. However, cytogenetic studies at metaphase I indicated univalents can be seen in a lateral view of the metaphase plate. Chromosome pairing and the number of univalents varied within and between the interspecific hybrids, with an average univalent number of 1.54 for the P. rollinsianum hybrids, 2.36 for the P. alpinum var. tetraneuris hybrids, and 2.46 for the P. alpinum var. alpinum hybrids. Pollen viability tests for the parental species and the hybrids were conducted by germination of pollen grains on stigmas. The percent of viable pollen recorded for the diploid guayule hybrids with P. rollinsianum, P. alpinum var. tetraneuris, and P. alpinum var. alpinum are 21.94, 13.47, and 11.17, respectively. The degree of chromosome pairing and pollen viability is striking because there are many morphological differences between the parents. The chromosome homology of these species based on their pairing behavior allows for the design of a backcross breeding program that would permit the transfer of the desirable characteristics from these species into diploid guayule.     

Meiotic behavior and pollen fertility in triploid and tetraploid natural populations of Campuloclinium macrocephalum (Eupatorieae,Asteraceae)

Campuloclinium macrocephalum DC. is a perennial herb widely distributed in the New World and introduced in South Africa, where it is commonly called “pompom weed”. This species is considered one of the most important weeds of Brazil and one of the problematic invasive plants of South Africa. The meiotic system can be studied to assess the ability of a weed to spread, but only few studies on C. macrocephalum have been realized. In this study, we examined the meiotic behavior and pollen fertility of 14 natural populations of C. macrocephalum from Argentina and Uruguay. Meiotic analysis revealed 2 triploid (2n = 3x = 30), 11 tetraploid (2n = 4x = 40) and 1 mixed population (2n = 2x = 20, 2n = 4x = 40). Both, triploid and tetraploid specimens showed a widely variable meiotic behavior with irregular chromosome pairing showing univalents, bivalents, trivalents (in triploids) and tetravalents (in tetraploids) at diacinesis of first meiotic division. Different abnormalities were observed, such as: laggard chromosomes, chromatin bridges, and out of plate chromosomes at metaphase I. During meiosis I (prophase), some cells showed the phenomenon of cytomixis or chromatin transfer between pollen mother cells. The meiotic indexes suggest that only four populations were normally fertile (over 90 % of fertile pollen), indicating meiotically stable plants. The remaining populations share variable pollen fertility, with triploids ranging from 46.64 to 54.83 % and tetraploids varying from 3.54 to 45.30 %. We suggest that polyploidy seems to be recurrent in C. macrocephalum, promoting partial sterility of pollen grains, generating large numbers of individuals by apomixis promoting invasion of crop fields. This study presents the meiotic behavior of this weed, these could be useful for future studies of biological control in areas with no natural enemies.     

Interspecific hybridization among Hieracium species in New Zealand: evidence from flow cytometry

Hieracium pilosella: (Asteraceae) was accidentally introduced to New Zealand about 100 years ago. Since then it has become an aggressive weed, and an unexpected degree of genetic and genome size variation has been detected; features that might result from interspecies hybridization. We investigated the possibility that H. pilosella has hybridized with related taxa. Of the four other subgenus Pilosella species introduced to New Zealand, H. praealtum is the most abundant and, on morphological and distributional evidence, most likely to be the other parent. Flow cytometry was used to estimate relative genome size for 156 Hieracium plants collected from the wild. Plants assigned to either parental or hybrid morphotypes were found to comprise tetraploid and pentaploid individuals using genome size measurements, and this was confirmed with direct mitotic chromosome counts for a subset of plants. The haploid DNA content of H. praealtum was approximately 22% larger than that of H. pilosella. Putative hybrids that were tetraploid had mean genome sizes equivalent to two H. pilosella and two H. praealtum haploid chromosome sets, implying they were hybrids arising from the fertilization of two reduced gametes. Similar results were obtained from tetraploid hybrids produced by controlled pollination. However, the majority of field hybrids were pentaploid with a genome size equivalent to four H. pilosella and one H. praealtum haploid chromosome sets. We infer that these are not first-generation hybrids but represent successful backcrossing with H. pilosella and/or hybrid-hybrid crossing, and that sexual tetraploid hybrids have been the parents. We note that populations putatively of H. pilosella often comprise apomictic pentaploid hybrids. Significantly, our data indicate the emergence of sexual hybrids that provide further opportunity for gene flow among taxa in this complex.