Considerations on the neoblasts of planarians on the basis of certain karyological evidence

The problem of the sexual differentiation of the neoblasts of planarians is approached on the basis of karyological evidence assembled by the author and by his collaborators. In the polyploid and pseudogamic biotypes of Dugesia benazzii, D. lugubris and Polycelis nigra, the transformation of the neoblasts into oogonia or spermatogonia is accompanied by variations in the chromosome set. These variations may be considered to be dependent upon the gradient of sexuality — that is, of the territorial influences that determine the evolution in a female or a male direction. Significant evidence is also inferred from the chromosome cycle of hybrids between amphigonic diploid biotypes and polyploid biotypes. A new direction of research, based on the comparison between anterior and posterior regenerated segments of experimental polyploids with variable chromosome sets aims at establishing possible regional differences in the set of the neoblasts.Dedicated to Prof. Dr. J. Seiler on the occasion of his eightieth birthday.     

Flow cytometry analysis of DNA ploidy levels and protein profiles distinguish between populations of Lumbriculus (Annelida: Clitellata)

Variations in DNA ploidy have been observed in Lumbriculus, a freshwater annelid, as well as in other clitellates. Interpretation and application of experimental results using these animals may be impacted as ploidy levels affect the protein expression, reproductive behavior, and response to stressors. Ploidy is typically determined by chromosome spreads, a time‐consuming and inefficient method. We adapted flow cytometry protocols used on vertebrates and plants to determine the ploidy levels in different populations of Lumbriculus, including a laboratory strain (Environmental Protection Agency), a commercial strain (Aquatic Foods), and worms collected from natural habitats. To isolate nuclei, worms were homogenized, filtered to remove cell debris, and centrifuged through Optiprep? density gradients. Nuclei were recovered, treated with RNAse, and stained with propidium iodide. Flow cytometry of the labeled nuclei showed that Lumbriculus from natural habitats in Minnesota and Iowa were diploid, with an estimated genome size of 2.7 pg. Populations from natural habitats in California and Oregon were highly polyploid, as were the laboratory and commercial strains. Chromosome spreads verified the high ploidy levels indicated by flow cytometry results, but also suggested that flow cytometry may be underestimating the DNA content levels. Staining of nuclei with diamidino‐2‐phenylindole indicated that this may be due to high levels of heterochromatin in nuclei from polyploid forms of Lumbriculus. To further compare the populations, proteins in worm homogenates were subjected to isoelectrofocusing gel electrophoresis. Distinct protein profiles were seen; one was shared in common by the diploid worms, the other was characteristic of polyploid populations. Diploid worms could also be distinguished from polyploid worms based on differences in hemoglobin linker proteins. The results further support taxonomic classification of the diploid and polyploid forms of Lumbriculus as distinct species.     

Attack of the clones: reproductive interference between sexuals and asexuals in the Crepis agamic complex

Negative reproductive interactions are likely to be strongest between close relatives and may be important in limiting local coexistence. In plants, interspecific pollen flow is common between co‐occurring close relatives and may serve as the key mechanism of reproductive interference. Agamic complexes, systems in which some populations reproduce through asexual seeds (apomixis), while others reproduce sexually, provide an opportunity to examine effects of reproductive interference in limiting coexistence. Apomictic populations experience little or no reproductive interference, because apomictic ovules cannot receive pollen from nearby sexuals. Oppositely, apomicts produce some viable pollen and can exert reproductive interference on sexuals by siring hybrids. In the Crepis agamic complex, sexuals co‐occur less often with other members of the complex, but apomicts appear to freely co‐occur with one another. We identified a mixed population and conducted a crossing experiment between sexual diploid C. atribarba and apomictic polyploid C. barbigera using pollen from sexual diploids and apomictic polyploids. Seed set was high for all treatments, and as predicted, diploid–diploid crosses produced all diploid offspring. Diploid–polyploid crosses, however, produced mainly polyploidy offspring, suggesting that non‐diploid hybrids can be formed when the two taxa meet. Furthermore, a small proportion of seeds produced in open‐pollinated flowers was also polyploid, indicating that polyploid hybrids are produced under natural conditions. Our results provide evidence for asymmetric reproductive interference, with pollen from polyploid apomicts contributing to reduce the recruitment of sexual diploids in subsequent generations. Existing models suggest that these mixed sexual–asexual populations are likely to be transient, eventually leading to eradication of sexual individuals from the population.     

Cytological relationships in theConvolvulus pluricaulis complex

InConvolvulus pluricaulis Chois. two forms (2n=18, 36) along with one colchicine-autotetraploid have been investigated morphologically and cytologically. The diploid regularly formed 9_II although in the “off-season” plants certain irregularities were observed including formation of unreduced pollen grains. The natural tetraploid and the colchicine-autotetraploid had mean frequencies of configurations of 1.56_IV+14.04_II+1.48_I and 2.95_IV+0.56_III+11,43_II+0.8_I, respectively. The mode of chromosome association in the two types of tetraploids was comparable. A comparison of the morphological characters of the two tetraploid types, further, suggested close similarity. In addition they were indistinguishable from the diploid from except being gigas. From these data it is inferred that the natural tetraploid presumably arose as a result of direct duplication of the diploid form, through the chance fusion of unreduced spores. The lower quadrivalent frequency of the natural polyploid is ascribed to a gradual shift towards bivalent association accompanying natural selection for fertility. The taxonomic status of the two forms (2x, 4x) is discussed and the varietal status accorded to the tetraploid form is supported.     

Cytogenetic studies following high dosage paternal irradiation in the mealy bug,Planococcus citri

Summary In the mealy bug, Planococcus citri, following high dosage paternal irradiation (60,000–120,000 rep), the survivors are mostly female (about 30–40% of the unirradiated control value) whereas very few males survive (about 5% of control value). After lower doses of paternal irradiation (P. I.), however, few or no females survive while the normal number of males (never less than the control value) survive.The females developing after high dosage P. I. are gynogenetic and are triploid or diploid or 3N/2N or 2N/N mosaics (Chandra 1963).The cytology of X₁ embryos following 90,000 rep is described in this report, in comparison with data from embryos following lower doses (8,000 r) of P. I. and unirradiated controls, to illustrate the chromosomal mechanisms leading to the production of gynogenetic females and the probable reasons for lethality of X₁ males after heavy P. I.It has been shown that triploid females stem from a fusion nucleus of the first and second polar bodies. This triploid polar nucleus, which normally participates in the formation of a polyploid sector in the young embryo, undertakes a successful embryogeny in many embryos when the zygote nucleus is unable to do so because of the heavily damaged paternal complement of chromosomes. Since the chromosomes are characterized by holokinetic activity, the irradiated paternal set manages to divide with the maternal complement but did not always segregate as successfully. Restitution divisions of the zygotic nuclei result in haploid, hyperhaploid, diploid and polyploid nuclei. Most of the diploid gynogenetic females probably originate from diploid nuclei of zygotic origin although it is possible that a few diploid females and the 2N/N mosaic females develop from polar bodies.From a dissertation submitted to the University of California, in partial satisfaction of the requirements for the degree of Doctor of Philosophy.Supported in part by a National Science Foundation Grant (No. G-9772) to Professor Spencer W. Brown.N. I. H. Predoctoral Trainee in Genetics 1961–1962.     

RECONSTRUCTING RETICULATION HISTORY IN A PHYLOGENETIC FRAMEWORK AND THE POTENTIAL OF ALLOPATRIC SPECIATION DRIVEN BY POLYPLOIDY IN AN AGAMIC COMPLEX IN CRATAEGUS (ROSACEAE)

Polyploidy plays a prominent role in the speciation process in plants. Many species are known to be part of agamic complexes comprising sexual diploids and more or less exclusively asexual polyploids. However, polyploid formation has been studied in very few cases, primarily because of the challenges in examining these cases phylogenetically. In this study, we demonstrate the use of a variety of phylogenetic approaches to unravel origins and infer reticulation history in a diploid–polyploid complex of black‐fruited Crataegus. The tree approaches are shown to be useful in testing alternative hypotheses and in revealing genealogies of nuclear genes, particularly in polyploid organisms that may contain multiple copies. Compared to trees, network approaches provide a better indication of reticulate relationships among recently diverged taxa. Taken together, our data point to both the autopolyploid and allopolyploid origins of triploids in natural populations of Crataegus suksdorfii, whereas tetraploids are formed via a triploid bridge, involving the backcross of allotriploid offspring with their diploid C. suksdorfii parent, followed by gene introgression from sympatric C. douglasii. Our findings provide empirical evidence for different pathways of polyploid formation that are all likely to occur within natural populations and the allopatric establishment of neopolyploids subsequent to their formation.     

Discovery of highly apomictic and highly amphimictic dihaploids in Allium tuberosum

 To discover highly apomictic and amphimictic Allium tuberosum diploids, we evaluated the degree of apomixis in three dihaploids (2n=16, 2x), KaD2, TeD1 and GMD1, derived from highly apomictic tetraploids. The degree of apomixis, calculated as the percentage of diploid seedlings in the progeny obtained after cross-pollination with tetraploid cultivars, was 96% in KaD2, 7% in TeD1 and 39% in GMD1. In addition to this general index of apomictic nature, two analytical indices were evaluated in KaD2 and TeD1. The degree of diplospory, calculated as the percentage of endoreduplicated embryo-sac mother cells, was 96% in KaD2 and 2% in TeD1. The degree of parthenogenesis, calculated as the percentage of ovules with the egg cell developing parthenogenetically, was 98% in KaD2 and 10% in TeD1. Among angiosperms with gametophytic apomixis, KaD2 is the first diploid apomict whose reproductive mode has been fully described by these three quantitative indices of apomictic nature. And TeD1 is the first highly amphimictic plant found in the A. tuberosum complex. Although TeD1 is poorly fertile, the present results encourage further screening trials for highly fertile, highly amphimictic dihaploids, which may be effective counterparts to KaD2 in diploid-level cross experiments to genetically analyze apomixis in A. tuberosum. Received: 4 December 1995 / Revision accepted: 8 May 1996     

Karyology of a marine population of Gyratrix hermaphroditus (Turbellaria,Rhabdocoela) and chromosomal evolution in this species complex

Karyology and reproductive biology of a marine population of the species complex Gyratrix hermaphroditus, from Roscoff (Brittany, France), have been investigated. A diploid complement of six chromosomes was determined from spermatogonial mitotic figures. One chromosome pair is metacentric, the second is intermediate between meta- and submetacentric, and the third is subtelocentric.In this population, regular meiosis occurs in both female and male germ lines, and the animals reproduce only by means of amphimictic eggs. Certain specimens of the population showed the elimination of one of the three bivalents during the first meiotic division in spermatogenesis. It seems that such animals produce normal and aneuploid sperm simultaneously; the aneuploid sperm are not capable of fertilization.The Roscoff population differs in its karyotype (2n = 6) from freshwater populations, which are either diploid (2n = 4) or polyploid (3n = 6, 4n = 8). These results suggest that aneuploidy played a role in the differentiation of freshwater populations from an originally marine species complex.     

A chromosome-specific sequence common to the B genome of polyploid wheat and Aegilops searsii

A low-copy, non-coding chromosome-specific DNA sequence, isolated from common wheat, was physically mapped to the distal 19% region of the long arm of chromosome 3B (3BL) of common wheat. This sequence, designated WPG118, was then characterized by Southern hybridization, PCR amplification and sequence comparison using a large collection of polyploid wheats and diploid Triticum and Aegilops species. The data show that the sequence exists in all polyploid wheats containing the B genome and absent from those containing the G genome. At the diploid level, it exists only in Ae. searsii, a diploid species of section Sitopsis, and not in other diploids including Ae. speltoides, the closest extant relative to the donor of the B genome of polyploid wheat. This finding may support the hypothesis that the B-genome of polyploid wheat is of a polyphyletic origin, i.e. it is a recombined genome derived from two or more diploid Aegilops species.     

Synergids and filiform apparatus in the sexual and apomictic dandelions from section Palustria (Taraxacum, Asteraceae)

An evolutionary trend to reduce “unnecessary costs” associated with the sexual reproduction of their amphimictic ancestors, which may result in greater reproductive success, has been observed among the obligatory apomicts. However, in the case of the female gametophyte, knowledge about this trend in apomicts is not sufficient because most of the ultrastructural studies of the female gametophyte have dealt with amphimictic angiosperms. In this paper, we tested the hypothesis that, in contrast to amphimictic plants, synergids in apomictic embryo sacs do not form a filiform apparatus. We compared the synergid structure in two dandelions from sect. Palustria: the amphimictic diploid Taraxacum tenuifolium and the apomictic tetraploid, male-sterile Taraxacum brandenburgicum. Synergids in both species possessed a filiform apparatus. In T. brandenburgicum, both synergids persisted for a long time without any degeneration, in spite of the presence of an embryo and endosperm. We propose that the persistent synergids in apomicts may play a role in the transport of nutrients to the embryo.     

Chromosome size and DNA values in sundews (Droseraceae)

Relative DNA content has been determined Feulgen cytophotometrically and autoradiographically for roottip nuclei of Drosophyllum lusitanicum L., (2n=12), Drosera rotundifolia L. (2x=20), D. intermedia Hayne (2x=20), D. linearis Goldie (2x= 20), D. binata Labill. (3x=32), D. capensis L. (4x= 40), D. spathulata Labill. (8x=80), all Droseraceae. Relative DNA values per diploid genome for Drosophyllum and diploid, triploid, and higher polyploid Drosera were approximately as 16421. These values are terms of a geometric series and are compatible with a multistranded (polyneme) interpretation of chromosome structure.     

Ectopic expression of the<Emphasis Type="Italic"> Suppressor of Underreplication</Emphasis> gene inhibits endocycles but not the mitotic cell cycle in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>

The Suppressor of Underreplication ( SuUR) gene contributes to the regulation of DNA replication in regions of intercalary heterochromatin in salivary gland polytene chromosomes. In the SuUR mutant these regions complete replication earlier than in wild type and, as a consequence, undergo full polytenization. Here we describe the effects of ectopic expression of SuUR using the GAL4-UAS system. We demonstrate that ectopically expressed SuUR exerts qualitatively distinct influences on polyploid and diploid tissues. Ectopic expression of SuUR inhibits DNA replication in polytene salivary gland nuclei, and reduces the degree of amplification of chorion protein genes that occurs in the follicle cell lineage. Effects caused by ectopic SuUR in diploid tissues vary considerably; there is no obvious effect on eye formation, but apoptosis is observed in the wing disc, and wing shape is distorted. The effect of ectopic SuUR expression is enhanced by mutations in the genes E2F and mus209 ( PCNA). Differential responses of polyploid and diploid cells to ectopic SuUR may reflect differences in the mechanisms underlying mitotic cell cycles and endocycles.Communicated by G. P. Georgiev     

Establishment of a tetraploid cytotype in a diploid population: Effect of relative fitness of the cytotypes

The conditions for the establishment of a tetraploid in a diploid population were investigated by means of a deterministic model, on the assumption that the diploid cytotype produces some 2n gametes. If the fertility and viability of both cytotypes were the same and the initial population was diploid, then a mixed population would occur if the production of 2n gametes was below 17.16%. The tetraploid excluded the diploid above this limit. By modifying the fertility and the viability of the polyploid this threshold varied, dropping to 10% when one of the two parameters was twice that of the diploid, and falling to as low as 6% if both fertility and viability were double that of the diploid. The conditions for the establishment of a polyploid are therefore quite restrictive under the assumptions of this model. In nature, such processes would probably allow the spread of the polyploid only if the immigration of polyploids considerably enhanced the frequency of tetraploids, or if genetical or environmental changes, or chance processes in small populations caused a substantial increase in the frequency of 2n gametes produced by the diploid.     

Physical mapping of rDNA sites in possible diploid progenitors of polyploid Festuca species

Festuca species form a polyploid series but only two of the diploid species have been firmly proposed as progenitors of any polyploid. The number and distribution of rDNA sites on the chromosomes of F. scariosa (section Scariosae) and the four diploid species that comprise section Montanae are presented with their relative DNA amounts and key morphological features. Comparisons of the results with those of some polyploid Festuca species from section Bovinae published previously indicate that F. scariosa and F. altissima could be diploid progenitors of the polyploids. It is unlikely that any one of the other three Montanae species is a progenitor of these polyploids.     

Occurrence of B-chromosomes in diploid Allium stracheyi Baker and their elimination in polyploids

Summary In a population of Allium stracheyi Baker (2n=14) growing in Darjeeling both diploids and polyploids occur. The diploids contain B-chromosomes varying from 2–10 in number. Polyploids are conspicuous by absence of B-chromosomes. These in diploids are found in the pollen mother cells and also in the pollen, and some are provided with subterminal constriction.Diploid individuals when brought from Darjeeling to Calcutta (i. e. from temperate to tropical regions) became polyploid within a month and the B-chromosomes were simultaneously lost. In order to confirm this unexpected result, the transfer experiment has been repeated thrice with fresh collections in each case and selection of diploid bulbs after cytological observation. In all cases the result has been the same. In rare cases one or two B-chromosomes were found in the polyploid cells which might represent intermediate steps of the disappearance.B-chromosomes in diploids possibly help the individual to compete with polyploids by enlarging the adaptive capacity.The sudden polyploidisation by transfer from the mountains to the plains might have been the result of a shock due to the temperature difference. The high temperature may be deleterious for the reproduction of B-chromosomes, and their degeneration products possibly contribute to cytoplasmic changes and the spindle disturbances which effect polyploidisation.     

The Phylogeny of Triticum L. and Aegilops L. Inferred from Comparative Analysis of Nucleotide Sequences in rDNA Promoter Regions

The process of accumulation of knowledge on wheat and related wild species during the 20th century is briefly reviewed with special reference to the evidence of the recent years on evolution of polyploid wheats and the role of diploid species. The latter serve as potential donors of the genomes, detection of which is particularly important because of the continuing speciation in the tribe Triticeae and artificial development of synthetic forms. The arguments in favor of the donor role for various diploid wheat and aegilops species from the section Sitopsis are compared. It is stated that in the formation of the both lines of polyploid wheats turgidum–aestivumand timopheevi,diploid Aegilops speltoides acted as a maternal form. In addition to cytoplasmic genomes, this aegilops species introduced into them also the B and G nuclear subgenomes. A comparison of nucleotide sequences in the variable part of the promoter of evolutionary conserved rRNA genes in polyploid wheats with their counterparts in diploid wheats and aegilops species confirmed the accepted wheat phylogenies.     

Genome origins of Triticum cylindricum,Triticum triunciale,and Triticum ventricosum (Poaceae) inferred from variation in restriction patterns of repeated nucleotide sequences: a methodological study

Three methods of phylogenetic inferences on polyploid plants employing variation in restriction sites in repeated nucleotide sequences were compared. Allotetraploid Triticum species of well-established origin were used as a model. Methods based on determination of the proportion of restriction fragments shared between a polyploid and its diploid relatives generated biased results because of uneven numbers of restriction fragments among diploid species and presence of common bands in phylogenetically related diploid species. A method employing restriction fragments unique to a diploid species (marker bands) was not affected by either factor and generated results consistent with cytogenetic inferences. It is shown that the latter method can be used to investigate the origin of a polyploid species even when one of its progenitors is extinct or when the polyploid and its diploid progenitors have diverged.     

Cytologie de la Pseudogamie chez Luffia lapidella Goeze (Lepidoptera Psychidae)

Summary The bisexual species Luffia lapidella has a pseudogamic (or gynogenetic) form, which is thelytokous and very similar to the parthenogenetic L. ferchaultella, but its egg needs the stimulation of the sperm.The inseminated egg restores its diploid chromosomal number in performing the same kind of restitutional first meiotic division as ferchaultella does. The second division, although already diploid, is normal and is followed by the formation of two nuclei, one of them degenerating as a polar body. The sperm starts developing as in the normal egg, but by metaphase II, it stops growing to a pronucleus and remains as a contracted and pycnotic body. Although attracted by the central female nucleus, it does not fuse with it. The male centriole behaves normally, it possibly plays a role in the first cleavage mitosis. The egg divides diploid and without any paternal chromosomes.

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Travail subventionné par le Fonds National Suisse de la Recherche Scientifique.     

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.     

Further Studies on the Role of Polyploidy in the Evolution of Meloidogyne

Two tetraploid isolates of Meloidogyne hapla, 86P and E289P, with haploid chromosome numbers of 34 and 28, respectively, were studied cytogenetically and biologically in relation to the diploid populations, 86-Va (n = 17) and E289-Taiwan (n = 14), from which they had been originally isolated. Both isolates were quite stable, converting to diploidy at the low rate of about 2.5%. The tetraploid isolate 86P maintained itself in competition with its diploid counterpart in mixed cultures, although an initial frequency of 50% polyploidy was reduced to about 9% at the end of the sixth generation. Both tetraploid isolates could maintain themselves in greenhouse cultures without artificial selection for at least 2 years. Crosses between diploid females and tetraploid males resulted in a few triploid females that produced mostly nonviable eggs, suggesting partial reproductive isolation between the two ploidy forms. Ten generations of propagation of only polyploid females of isolate 86P that were associated with males failed to yield an obligatorily amphimictic isolate that would not convert at all to diploidy. If one accepts a previous assumption that the present day amphimictic root-knot nematodes are tetraploids derived from diploid ancestors, results of the present study are not inconsistent with an evolutionary trend toward an even higher level of ploidy in Meloidogyne, presumably octaploidy.