Variation of cell cycle duration within suspension cultures ofDaucus carota and its consequence for the induction of ploidy changes with colchicine

Summary Continuous exposure to colchicine was used to estimate the variation in cell cycle time between cells within suspension cultures ofDaucus carota. Observations were made of the pattern of disappearance of cells of the initially predominant ploidy levels in diploid and tetraploid cultures having markedly different aggregation patterns. Both cultures showed a similar range of cycle times, normally distributed about the culture mean. Shorter colchicine treatments, followed by regrowth in colchicine-free medium, showed that spread of cycle times in the diploid culture prevented uniform induction of tetraploidy, and that the resulting mixoploid suspensions showed a gradual reversion to diploidy during subsequent subculture.     

Triploidy and mosaicism in Liolaemus chiliensis (Sauria: Tropiduridae).

A remarkable diversity of ploidy, i.e., diploidy, triploidy, and diploid-triploid mosaicism, was found among the somatic and testicular cells in a natural population of the lizard Liolaemus chiliensis from central Chile. Intra pop ulation, intersexual, and intraindividual ploidy variation is reported. In contrast with other species of polyploid reptiles, 86% of L. chiliensis males were mosaics (2n/3n) and 14% were diploids; 33% of females were triploid, 57.1% were mosaics, and 9.5% were diploid. Among 21 specimens, no triploid males were found in this sample. In the mosaic males, the diploid and triploid spermatogonia both enter meiosis, producing both reduced and unreduced metaphase II spermatocytes, most of them euploids. We discuss the origin for this ploidy in this iguanid lizard.     

Hermaphroditism in Meloidogyne hapla

Hermaphrodites were detected in diploid and polyploid isolates of population 86-Va of Meloidogyne hapla. Young hermaphrodites are indistinguishable from normal females. Initially, hermaphrodite ovaries are filled with oocytes at various stages of development. Hermaphroditism is expressed later when young oocytes in the early pachytene region of the growth zone suddenly advance to diakinesis and proceed with maturation divisions, resulting in spermatid production. Spermatogenesis may be initiated shortly after the fourth molt, or later, after a female has produced some eggs. Spermatogenesis may occur in one or both gonads, and it may be initiated in one gonad before the other. Once initiated, spermatogenesis continues for the entire reproductive life of the hermaphrodite. Several thousand spermatozoa accumulate in the ovotestis. Because they do not pass through the oviduct into the spermatotheca, they do not take part in reproduction (nonfunctional hermaphroditism). Among the progeny of hermaphrodites, ca. 50% are hermaphroditic, and the remainder are apparently normal females which, however, produce about 50% hermaphroditic progeny. Two temperature regimes (20-23 C and 27-30 C) did not influence the percentage of hermaphrodites among the progeny. Hermaphroditism could not be transmitted to nonhermaphroditic isolates following attempted crosses between males of hermaphroditic and females of nonhermaphroditic isolates. Although this result suggests cytoplasmic rather than nuclear inheritance, this conclusion is not definitive.     

Polyploidy in an Amphimictic Population of Heterodera glycines

A tetraploid single-cyst isolate of Heterodera glycines from a field population from Indiana has been propagated in the greenhouse on Lee soybeans since its discovery, in 1973. The tetraploid isolate has n = 18 chromosomes, compared with n = 9 of the diploid H. glycines; it has larger cysts and larvae, but shows the same level of parasitism and host range as the diploid population from which it apparently evolved. Association of chromosomes is irregular at metaphase I, with quadrivalents, trivalents, and univalents often observed in addition to the bivalents. The second maturation division is usually normal. About 80% of the mature oocytes (just before fertilization) have n = 18, and the other 20% have n = 17 or 19. Reproduction of the tetraploid isolate is exclusively by cross-fertilization. The discovery of such a tetraploid provides an experimental tool for the study of polyploidy in nematodes. Many amphimictic plant-parasitic nematodes are suspected of representing polyploids.     

Parasexual Ploidy Reduction Drives Population Heterogeneity Through Random and Transient Aneuploidy in Candida albicans

The opportunistic pathogen Candida albicans has a large repertoire of mechanisms to generate genetic and phenotypic diversity despite the lack of meiosis in its life cycle. Its parasexual cycle enables shifts in ploidy, which in turn facilitate recombination, aneuploidy, and homozygosis of whole chromosomes to fuel rapid adaptation. Here we show that the tetraploid state potentiates ploidy variation and drives population heterogeneity. In tetraploids, the rate of losing a single heterozygous marker [loss of heterozygosity (LOH)] is elevated ∼30-fold higher than the rate in diploid cells. Furthermore, isolates recovered after selection for LOH of one, two, or three markers were highly aneuploid, with a broad range of karyotypes including strains with a combination of di-, tri-, and tetrasomic chromosomes. We followed the ploidy trajectories for these tetraploid- and aneuploid-derived isolates, using a combination of flow cytometry and double-digestion restriction-site-associated DNA analyzed with next-generation sequencing. Isolates derived from either tetraploid or aneuploid isolates predominately resolved to a stable euploid state. The majority of isolates reduced to the conventional diploid state; however, stable triploid and tetraploid states were observed in ∼30% of the isolates. Notably, aneuploid isolates were more transient than tetraploid isolates, resolving to a euploid state within a few passages. Furthermore, the likelihood that a particular isolate will resolve to the same ploidy state in replicate evolution experiments is only ∼50%, supporting the idea that the chromosome loss process of the parasexual cycle is random and does not follow trajectories involving specific combinations of chromosomes. Together, our results indicate that tetraploid progenitors can produce populations of progeny cells with a high degree of genomic diversity, from altered ploidy to homozygosis, providing an excellent source of genetic variation upon which selection can act.     

Ploidy reduction in Saccharomyces cerevisiae

Large-scale transitions in genome size from tetraploid to diploid were observed during a previous 1800-generation evolution experiment in Saccharomyces cerevisiae. Whether the transitions occurred via a one-step process (tetraploid to diploid) or through multiple steps (through ploidy intermediates) remained unclear. To provide insight into the mechanism involved, we investigated whether triploid-sized cells sampled from the previous experiment could also undergo ploidy loss. A batch culture experiment was conducted for approximately 200 generations, starting from four triploid-sized colonies and one contemporaneous tetraploid-sized colony. Ploidy reduction towards diploidy was observed in both triploid and tetraploid lines. Comparative genomic hybridization indicated the presence of aneuploidy in both the founder and the evolved colonies. The specific aneuploidies involved suggest that chromosome loss was not haphazard but that nearly full sets of chromosomes were lost at once, with some additional chromosome mis-segregation events. These results suggest the existence of a mitotic mechanism allowing the elimination of an entire set of chromosomes in S. cerevisiae, thereby reducing the ploidy level.     

Occurrence of ploidy shift in a strain of the imperfect yeast Candida albicans

A clinical isolate of Candida albicans, a member of the Fungi Imperfecti, was polyploid as shown by the fact that it contained two kinds of nuclei, one of diploid and one of tetraploid DNA content. These determinations were made by fluorescence microscopy-photometry. The nucleus-associated organelles (NAOs), or spindle pole bodies, of yeast cells in this isolate were classified into two groups, one diploid and the other tetraploid, according to their dimensions as determined by serial thin-sectioning electron microscopy. A ploidy shift from diploid to tetraploid was found in individual cells of a culture of this isolate undergoing diphasic growth in minimal salts medium. A process of shift-down or reduction of ploidy from tetraploid to diploid was also observed by electron microscopy during these growth conditions: this appeared to occur in large cells which showed multiple spindle formation during nuclear division, a phenomenon apparently similar to the process of meiosis II during sporogenesis of Saccharomyces cerevisiae, but differing in that it produces diploid daughter nuclei by the vegetative process.     

Parallel changes in mate-attracting calls and female preferences in autotriploid tree frogs

For polyploid species to persist, they must be reproductively isolated from their diploid parental species, which coexist at the same time and place at least initially. In a complex of biparentally reproducing tetraploid and diploid tree frogs in North America, selective phonotaxis--mediated by differences in the pulse-repetition (pulse rate) of their mate-attracting vocalizations--ensures assortative mating. We show that artificially produced autotriploid females of the diploid species (Hyla chrysoscelis) show a shift in pulse-rate preference in the direction of the pulse rate produced by males of the tetraploid species (Hyla versicolor). The estimated preference function is centred near the mean pulse rate of the calls of artificially produced male autotriploids. Such a parallel shift, which is caused by polyploidy per se and whose magnitude is expected to be greater in autotetraploids, may have facilitated sympatric speciation by promoting reproductive isolation of the initially formed polyploids from their diploid parental forms. This process also helps to explain why tetraploid lineages with different origins have similar advertisement calls and freely interbreed.     

Haematological characterization of loach Misgurnus anguillicaudatus: comparison among diploid, triploid and tetraploid specimens

The purpose of this study was to determine whether diploid, triploid and tetraploid loach (Misgurnus anguillicaudatus) differed in terms of their main haematological and physiological characteristics. Diploid and tetraploid fish were produced by crossing of natural diploids (2n x 2n) and natural tetraploids (4n x 4n), respectively. Triploid fish were produced by hybridization between diploid males and tetraploid females. The blood cells were significantly larger in polyploids, and the volumetric ratios of erythrocytes and leucocytes (thrombocyte and neutrophil) in tetraploids, triploids and diploids were consistent with the ploidy level ratio of 4:3:2. No significant differences were observed in haematocrit among polyploids. The erythrocyte count decreased with increased ploidy level, while total haemoglobin, mean cell volume, mean cellular haemoglobin content, and mean cell haemoglobin concentration all increased with increase in ploidy level. Erythrocyte osmotic brittleness declined in polyploids so that polyploid erythrocytes were more resistant to osmotic stress than diploid ones. Overall, loach with higher ploidy levels showed evidence of some advantages in haematological characteristics.     

Ploidy of somatic embryos and the ability to regenerate plantlets in melon (Cucumis melo L.)

Summary The number of chromosomes in cells of callus, somatic embryos and regenerated plantlets during somatic embryogenesis were examined in two cultivars of melon (Cucumis melo L.). Somatic embryos were diploid (50.0%/32.1%), tetraploid (38.5%/57.5%) and octoploid (11.5%/10.4%) whereas in callus cells diploidy (41.9%/43.3%), tetraploidy (27.9%/25.8%), octoploidy (11.6%/15.5%) and a low frequency of other types of ploidy and aneuploidy were observed. Mixoploid somatic embryos were not observed. These results suggest that the somatic embryos were selectively differentiated from diploid, tetraploid and octoploid cells, and that endopolyploidization of cultured cells occurred before the start of cell division leading to somatic embryogenesis. The ratio of diploid to tetraploid (1.30/0.55) in somatic embryos was less than that in callus cells (1.50/1.68) while ratios of diploid to octoploid (4.35/3.09) and tetraploid to octoploid (3.35/5.52) in somatic embryos were greater than those in callus cells (3.61/2.80 and 2.40/1.67). Therefore, it appears that the ability of callus cell to differentiate into somatic embryos increases in the following order: octoploid < diploid < tetraploid. Regenerated plantlets were diploid (65.5%/55.1%) and tetraploid (34.5%/44.9%). No octoploid plantlets were observed. The ratio of diploid to tetraploid in regenerated plantlets (1.72/1.23) was greater than that in somatic embryos. Therefore, it appears that the ability of somatic embryos to develop into plantlets increases in the following order: octoploid < tetraploid < diploid.     

Reproductivity of early males of the temperate paper wasp Polistes rothneyi iwatai

In Polistes paper wasps, haploid early males can mate with early emerging females and leave viable offspring. In contrast, diploid early males are eventually sterile because they contribute triploid offspring via diploid sperm. Clarifying the ploidy of early males is important for determining whether early male production is a reproductive strategy for the species. We examined the mating behavior and the ploidy of early males in the Japanese paper wasp, Polistes rothneyi iwatai van der Vecht. Thirteen early males from four colonies were all diploid. Two of the nine early males (22.2%) attempted to mate with females, but only one individual (11.1%) was successful (the female's spermatheca contained spermatozoa). These results suggest that although most early males of P. rothneyi iwatai do not produce offspring, their mating may be linked to the occasional production of triploid females.     

Variability in diet and foraging behaviour between sexes and ploidy forms of the hybridogenetic Squalius alburnoides complex (Cyprinidae) in the Guadiana River basin, Portugal

Samples of the Squalius alburnoides complex, a hybridogenetic Iberian minnow composed of diploid, triploid and, less commonly, tetraploid forms, were collected from a tributary of the Ardila River (Guadiana River basin) between February 1999 and January 2001. Seasonal variation in diet was evident, and was probably linked to prey availability. Distinct foraging behaviours between ploidy forms were found towards several prey items, suggesting that diploid adult males fed mostly near the surface, whereas diploid adult females tended to feed near the bottom and submerged vegetation. Triploid females exhibited an intermediate foraging behaviour, although there was greater affinity towards diploid male feeding behaviour. Diploid males which, in contrast to diploid and triploid females, have non‐hybrid genomes in the Guadiana drainage, exhibited a higher specialization for food. Despite considerable dietary overlap, there appeared to be spatial segregation of feeding niches between the three forms, especially during dry periods when prey availability was lower, which may be a strategy for diminishing competition for food. Therefore, considering asexual generalist and specialist hypotheses, it appears that the different ploidy levels are generalist, opportunistic feeders that partition the resources when limited.     

Reproduction and the origin of polyploids in hybrid salamanders of the genus Ambystoma

Eggs and larvae produced by diploid, triploid, and tetraploid females collected from breeding ponds on Pelee Island in Lake Erie were studied to examine the reproductive mechanism. No instance of parthenogenesis was found as all examined females required sperm to produce viable progeny. Diploid females produced diploid and triploid larvae, triploid females produced triploid and tetraploid larvae, and tetraploid females produced triploid and tetraploid larvae. The majority of the eggs produced by hybrid females do not develop or do not complete embryogenesis. Electrophoretic examination of females and their offspring demonstrate that the male genome is being incorporated in reduced as well as unreduced eggs produced by all three ploidy classes of females. The elevation of ploidy among Pelee Island Ambystoma is attributed to sperm incorporation in unreduced eggs. Triploid as well as tetraploid individuals are constantly being produced. A critical examination of the literature on parthenogenetic or gynogenetic modes of reproduction in North America Ambystoma hybrids shows no conclusive evidence supporting these modes and it is suggested that the reproductive mechanism found among Pelee Island female hybrids may be more generally applied to other hybrid Ambystoma populations.     

Isofemale Line Analysis of Meloidogyne incognita Virulence to Cowpea Resistance Gene Rk

Isofemale lines (IFL) from single egg masses were studied for genetic variation in Meloidogyne incognita isolates avirulent and virulent to the resistance gene Rk in cowpea (Vigna unguiculata). In parental isolates cultured on susceptible and resistant cowpea, the virulent isolate contained 100% and the avirulent isolate 7% virulent lineages. Virulence was selected from the avirulent isolate within eight generations on resistant cowpea (lineage selection). In addition, virulence was selected from avirulent females (individual selection). Virulence differed (P ≤ 0.05) both within and between cohorts of IFL cultured for up to 27 generations on susceptible or resistant cowpea. Distinct virulence profiles were observed among IFL. Some remained avirulent on susceptible plants and became extinct on resistant plants; some remained virulent on resistant and susceptible plants; some changed from avirulent to virulent on resistant plants; and others changed from virulent to avirulent on susceptible plants. Also, some IFL increased in virulence on susceptible plants. Single descent lines from IFL showed similar patterns of virulence for up to six generations. These results revealed considerable genetic variation in virulence in a mitotic parthenogenetic nematode population. The frequencies of lineages with stable or changeable virulence and avirulence phenotypes determined the overall virulence potential of the population.     

Genetic differentiation and spatiotemporal history of diploidy and tetraploidy of Clintonia udensis

Polyploidy is an important factor shaping the geographic range of a species. Clintonia udensis (Clintonia) is a primary perennial herb widely distributed in China with two karyotypic characteristics—diploid and tetraploid and thereby used to understand the ploidy and distribution. This study unraveled the patterns of genetic variation and spatiotemporal history among the cytotypes of C. udensis using simple sequence repeat or microsatellites. The results showed that the diploids and tetraploids showed the medium level of genetic differentiation; tetraploid was slightly lower than diploid in genetic diversity; recurrent polyploidization seems to have opened new possibilities for the local genotype; the spatiotemporal history of C. udensis allows tracing the interplay of polyploidy evolution; isolated and different ecological surroundings could act as evolutionary capacitors, preserve distinct karyological, and genetic diversity. The approaches of integrating genetic differentiation and spatiotemporal history of diploidy and tetraploidy of Clintonia udens would possibly provide a powerful way to understand the ploidy and plant distribution and undertaken in similar studies in other plant species simultaneously contained the diploid and tetraploid.     

Higher rate of tissue regeneration in polyploid asexual versus diploid sexual freshwater snails

Characterizing phenotypic differences between sexual and asexual organisms is a critical step towards understanding why sexual reproduction is so common. Because asexuals are often polyploid, understanding how ploidy influences phenotype is directly relevant to the study of sex and will provide key insights into the evolution of ploidy-level variation. The well-established association between genome size and cell cycle duration, evidence for a link between genome size and tissue regeneration rate and the growing body of research showing that ploidy influences growth rate and gene expression led us to hypothesize that healing and tissue regeneration might be affected by ploidy-level variation. We evaluated this hypothesis by measuring the rate of regeneration of antenna tissue of Potamopyrgus antipodarum, a New Zealand snail characterized by frequent coexistence between diploid sexuals and polyploid asexuals. Antennae of triploid and presumptive tetraploid asexuals regenerated more rapidly than the antennae of diploid sexuals, but regeneration rate did not differ between triploids and tetraploids. These results suggest either that ploidy elevation has nonlinear positive effects on tissue regeneration and/or that factors associated directly with reproductive mode affect regeneration rate more than ploidy level. The results of this study also indicate that the lower ploidy of sexual P. antipodarum is unlikely to confer advantages associated with more rapid regeneration.     

A Complementary Method for Production of Tetraploid Crassostrea gigas Using Crosses Between Diploids and Tetraploids with Cytochalasin B Treatments

We present a new method to produce tetraploid Crassostrea gigas by cytochalasin B inhibition of polar body 2 expulsion in diploid females crossed with tetraploid males. This offers a means of direct introgression of genetic characters from selected diploid to tetraploid lines, avoiding a triploid step. Offspring larval ploidy shifted over time and depended on size, with tetraploids more frequent among the smaller larvae and triploids among the large. Viable tetraploids were found at 4 and 6 months, indicating the technique was successful. The possibility that gynogenesis occurred was tested by microsatellite analysis to confirm the presence of paternally inherited alleles. These were present in all animals of the 2n × 4n + CB (female first) cross. However, a 4n × 2n + CB cross produced triploids, including some gynogens. Our method illustrates for the first time that diploid C. gigas eggs, if selected for large size, can give viable tetraploid offspring.     

Masking and purging mutations following EMS treatment in haploid, diploid and tetraploid yeast (Saccharomyces cerevisiae)

The yeast, Saccharomyces cerevisiae, was used as a model to investigate theories of ploidy evolution. Mutagenesis experiments using the alkylating agent EMS (ethane methyl sulphonate) were conducted to assess the relative importance that masking of deleterious mutations has on response to and recovery from DNA damage. In particular, we tested whether cells with higher ploidy levels have relatively higher fitnesses after mutagenesis, whether the advantages of masking are more pronounced in tetraploids than in diploids, and whether purging of mutations allows more rapid recovery of haploid cells than cells with higher ploidy levels. Separate experiments were performed on asexually propagating stationary phase cells using (1) prototrophic haploid (MAT alpha) and diploid (MATa/alpha) strains and (2) isogenic haploid, diploid and tetraploid strains lacking a functional mating type locus. In both sets of experiments, haploids showed a more pronounced decrease in apparent growth rate than diploids, but both haploids and diploids appeared to recover very rapidly. Tetraploids did not show increased benefits of masking compared with diploids but volume measurements and FACScan analyses on the auxotrophic strains indicated that all treated tetraploid strains decreased in ploidy level and that some of the treated haploid lines increased in ploidy level. Results from these experiments confirm that while masking deleterious mutations provides an immediate advantage to higher ploidy levels in the presence of mutagens, selection is extremely efficient at removing induced mutations, leading growth rates to increase rapidly over time at all ploidy levels. Furthermore, ploidy level is itself a mutable trait in the presence of EMS, with both haploids and tetraploids often evolving towards diploidy (the ancestral state of S. cerevisiae) during the course of the experiment.     

不同倍性大青杨的光合特性及叶片解剖结构比较

通过对二年生二倍体、三倍体和四倍体大青杨叶片结构和光合特性的研究,探讨不同倍性大青杨生长性状差异的原因。结果表明：不同倍性大青杨的净光合速率（Pn）、光饱和点（LSP）、光补偿点（LCP）等光合指标差异显著。三倍体的最大净光合速率（Pmax）高出二倍体21%。叶片厚度、表皮细胞厚度、栅栏组织厚度、栅栏组织与海绵组织厚度的比值,都是三倍体和四倍体大青杨比二倍体高,且均呈现增加的趋势。其中,四倍体大青杨表皮细胞最厚,上下表皮分别高出二倍体64%和17%。三倍体大青杨的栅栏组织厚度及栅栏组织与海绵组织厚度的比值最大,比值高出二倍体50%。通过叶片光合特性和解剖结构比较证明,三倍体和四倍体大青杨对光的适应性和光合作用能力强于二倍体。     

Arrangement of spindle apparatus in mitoses of different ploidy

In non-hypotonically treated mitoses from tissue cultures of Microtus agrestis, both the constitutive heterochromatin of the sex chromosomes and the spindle apparatus were stained by the Giemsa C-banding technique. By means of counting the heterochromatic chromosomes, we determined the cell ploidy and studied the number of centrioles and the spindle arrangement of diploid, triploid, tetraploid and octoploid mitoses. Diploid and triploid prophases contained 2 centrioles in most cases, tetraploid prophases 4, binucleate cells with 2 diploid nuclei likewise 4 and binucleate cells with 2 tetraploid nuclei 8 centrioles. Nearly 99% of diploid and triploid metaphases were bipolar. Of the tetraploid metaphases only 45% were bipolar, 29.5% tripolar, 7.5% quadripolar and 18% formed as a parallel mitosis. In all examined binucleate cells that had had an asynchronous DNA synthesis, a multipolar mitosis was found.