DEVELOPMENT OF APOGAMIC AMOEBAE FROM HETEROTHALLIC LINES OF A MYXOMYCETE,DIDYMIUM IRIDIS

By making appropriate crosses between heterothallic sexual clones of Didymium iridis we can recover apogamic lines in the F₁ generation. In this organism, heterothallic forms typically produce a haploid myxamoebal stage, but recently two diploid myxamoebal clones homozygous for mating types were discovered. When these are crossed, A²A² x A⁵A⁵, tetraploid Plasmodia are produced which later yield diploid F₁ meiospores. Sixty-four percent of the single-spore-derived clones produce both myxamoebae and Plasmodia, while the remainder do not progress past the myxamoebal stage. These results are consistent with the predictions that from tetraploid nuclei, mating types should segregate in the meiospores in a ratio of 1A²A²:4A²A⁵:1A⁵A⁵, and that myxamoebae heterozygous, A²A⁵, for mating type should yield Plasmodia apogamously. As the means for verifying relative ploidy levels of myxamoebae and Plasmodia, nuclear DNA was measured with a scanning microspectrophotometer.     

CYTOPHOTOMETRIC MEASUREMENT OF NUCLEAR DNA IN SEVEN HETEROTHALLIC ISOLATES OF DIDYMIUM IRIDIS,A MYXOMYCETE

Absorption cytophotometry was used to measure nuclear Feulgen-DNA content of myxamoebae and Plasmodia in seven heterothallic isolates of Didymium iridis. Measurements of myxamoebal nuclei from clones of four isolates (Hon 1, Pan 1, Pan 2, and CR 5) gave a mean DNA value of 0.34, whereas the nuclei of Plasmodia which develop from each of the four intraisolate crosses had a mean value of 0.63. These values correspond to the 2C haploid level in myxamoebae and the 4C diploid level in Plasmodia. DNA values in two additional isolates (Pan 3 and CR 2) are much higher than the mean for the other five. Accordingly, it is proposed that these may be polyploid. The question of polyploidy in D. iridis and in other myxomycetes is evaluated. The seventh isolate, Ky 1, is taxonomically very close to D. nigripes and was not included in calculations of mean values for D. iridis.     

GROWTH AND DEVELOPMENT OF THE MYXOMYCETE PERICHAENA VERMICULARIS. II. CHROMOSOME NUMBERS AND NUCLEAR CYCLES

Chromosome counts of dividing nuclei of the myxomycete Perichaena vermicularis indicate a number of 25 ± 2 in the amoebae and 50 ± 4 in the Plasmodia, confirming earlier reports that amoebae are haploid and plasmodia diploid. Chromosome numbers obtained from nuclei during sporangial development indicate a fluctuation in the location of meiosis influenced by environmental conditions. The implications of these observations are discussed in reference to past conflicting evidence of the location of meiosis.     

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.     

COMPARATIVE MEASUREMENTS OF NUCLEAR DNA IN A HETEROTHALLIC AND A SELF-FERTILE ISOLATE OF THE MYXOMYCETE,DIDYMIUM IRIDIS

Comparative measurements were made of the nuclear Feulgen-DNA content of a heterothallic and a self-fertile isolate of the myxomycete Didymium iridis. Plasmodial nuclei of both isolates contain the diploid amount of DNA. The replicated diploid (4C) values for the heterothallic and the self-fertile isolates are 5.66 and 5.95, respectively. Myxamoebae, however, are quite dissimilar in their nuclear DNA content. Those of the heterothallic isolates, Honduran 1–2 (A¹) and Panamanian 2–4 (A⁷), have mean values of 3.81 and 3.69, whereas myxamoebae of the self-fertile Philippine-1 isolate were found to have a mean value of 6.07. Myxamoebae of the Ph-1 isolate are, therefore, at the same ploidy level as the Ph-1 Plasmodium. Mean DNA values for Ph-1 sporangial nuclei were in category 4C. Measurement of the DNA content of mitotic metaphases in sporangia at T = 6 hr confirmed that the mean DNA content of both Ph-1 myxamoebae and plasmodial nuclei is equivalent to 4C. It is concluded that nuclear phase alternance is lacking in the Ph-1 isolate and that the Plasmodium of this isolate develops by apogamy.     

Nuclear DNA content and chromosome numbers in the myxomycete Physarum polycephalum

An improved method is described for making chromosome spreads of the plasmodium of the myxomycete, Physarum polycephalum. It consists of isolating metaphase nuclei, spreading the chromosomes with hot lactic acid, and staining with acetic-orcein.Most sublines derived from the Backus Wis 1 sclerotium had about 1 pg of DNA per nucleus, and had nuclei with 50 and 75 chromosomes in both the growing and sporulating plasmodium. Mature spores contained 0.6 pg of DNA, and hatching amoebae had 20–25 chromosomes and 0.6 pg of DNA. Plasmodia of the homothallic Colonia strain had a nuclear DNA content of about 1 pg, and had 35–40 chromosomes during growth and sporulation. Polyploid plasmodial sublines were found which had 1.5 and 3 times the normal DNA content and chromosome number. The polyploid sublines had the same plasmodial protein:DNA and RNA:DNA ratios as normal cultures. DNA content of nuclei varied directly with nuclear surface area. Ploidy was determined by the parent amoebae and therefore can serve as a genetic marker.A simple technique is given for completing the life cycle of P. polycephalum axenically. Germinating spores are plated without bacteria on one-tenth strength semidefined plasmodial growth medium, containing 2% agar. Plasmodia are visible in 2–4 days.     

Analysis of development and growth in a mutant of Physarum polycephalum with defective cytokinesis

Cultures of amoebae of the mutant strain ATS23 isolated from strain CLd of Physarum polycephalum contain multinucleate cells and cells with increased nuclear DNA content. Plasmodia derived from ATS23 clones show abnormal morphology and defective sporulation. All abnormalities are enhanced by high incubation temperature (31 °C). Genetic analysis suggested that all the abnormalities were caused by a single mutation, denoted hts-23. The kinetics of plasmodium formation were followed in cultures of apogamic amoebae carrying hts-23 and hts⁺ (wild type) respectively. Results indicated that, relative to wild type, hts-23 did not increase the rate of plasmodium formation. There was evidence that, in both mutant and wild-type strains, commitment to plasmodium development occurred in uninucleate cells. Analysis of cell pedigrees by time-lapse cinematography indicated that the primary abnormal event in cultures of hts-23 amoebae was failure of cytokinesis; an apparently complete cleavage furrow was formed but cell separation failed, resulting in a binucleate cell. This event occurred randomly in pedigrees in which the majority of divisions were completed normally; its frequency increased during incubation at 31 °C. All other abnormalities in hts-23 amoebal cultures could be attributed to this primary event, assuming that DNA synthesis continued in the absence of cytokinesis and that the binucleate cells underwent the amoebal type of “open” mitosis, allowing the possibility of spindle fusion. This implies that the acquisition of “closed” mitosis is an essential early step in plasmodium development.     

INHERITANCE OF NUCLEAR 2C DNA CONTENT VARIATION IN INTRASPECIFIC AND INTERSPECIFIC HYBRIDS OF MICROSERIS (ASTERACEAE)

Nuclear DNA content varies over 20% within the diploid (2n = 18) species M. douglasii and M. bigelovii. Two different intraspecific crosses were made between M. douglasii biotypes which differed by about 10% in 2C nuclear DNA content. The F₂ progeny of one intraspecific cross showed no striking evidence of segregation for DNA content. The mean DNA contents of F₂ progeny from two sister hybrids from the second intraspecific cross were significantly different at the 1% level. An interspecific cross was made between biotypes of M. douglasii and M. bigelovii that differed by approximately 10% in DNA amount. The 12 F₁ progeny did not cluster around the parental midpoint, but instead encompassed nearly the entire range between the parental means. The five families of F₂ progeny studied each had a mean DNA content corresponding to that of the particular F₁ from which they were derived, indicating that the F₁ plants were not of identical DNA content. The results of this study suggest that DNA sequences which account for the DNA content differences among the plants are unstable and can undergo deletion or amplification in a hybrid. The altered DNA content may be heritably stable and show little or no segregation in the F₂ progeny.     

Multiple Alleles and Other Factors Affecting Plasmodium Formation in the True Slime Mold Physarum polycephalum Schw*

SYNOPSIS. The life cycle of the true slime mold Physarum polycephalum includes 2 vegetative stages: the multinucleate coenocytic plasmodium and the uninucleate amoeba. A clone of amoebae established from a single spore does not normally yield plasmodia. Plasmodia are formed when amoebae from particular clones are mixed; thus plasmodium formation is said to be controlled by a ‘mating-type’ system. Previous work by the author with a sample of P. polycephalum derived from a single source revealed that 2 mating types were present and were determined by a pair of alleles at 1 locus. The present paper reveals the presence of 2 more mating types in a sample of P. polycephalum derived from a different source and provides evidence that these are determined by 2 alleles at the same locus as the other 2. Evidence for the presence of other inherited factors affecting plasmodium formation, the mode of action of these factors and possible explanations for the occurrence of plasmodia in single-spore cultures are also discussed.     

The Life Cycle of Cryptochlora perforans (Chlorarachniophyta)

Observations on the behaviour of different life cycle stages, gamete fusions, and measurements of nuclear DNA contents in Cryptochlora perforans resulted in a first concept concerning life histories in Chlorarachniophyta: the life cycle of Cr. perforans is diplohaplontic (gamete fusion with karyogamy - mitosis - meiosis - mitosis). In the haploid as well as in the diploid life cycle phases amoeboid and coccoid stages occur. The isomorphic gametes are modified amoebae frequently without filopodia. Only haploid flagellate stages are known representing mito- or meiozoospores. Diploid coccoid stages have a granular cytoplasmic structure and may be somewhat larger than haploid ones. Nevertheless, positive identification of haploid (gametophytic) and diploid (sporophytic) stages is only possible on the basis of nuclear DNA contents.     

Pattern of DNA synthesis in nuclei of feeding and starving Amoeba proteus : A cytofluorometric study

The DNA content in isolated nuclei of Amoeba proteus was determined for each of the three groups of synchronized amoebae over different intervals after division. Several nuclei of each amoeba group were fixed 1 h after division, before the amoebae were fed. About h after division, some amoebae in each group were given food (Tetrahymena pyriformis), while the rest were left starving. Samples of the nuclei of fed and starved amoebae were fixed 24 h and (in different groups) 42–55 h after division. In each group from 22 to 48% of the fed amoebae had divided prior to the last nuclei fixation. Starved amoebae did not undergo division. In all three amoeba groups the nuclear DNA content of fed cells by the end of interphase had increased to 280–300% the value for 1 h amoebae. The nuclear DNA content of starved amoebae of all three groups was also increased, and in two groups it exceeded the initial level more than two-fold. However, in all three groups, it was lower than that of fed amoebae. In all the groups the nuclear DNA content in fed amoebae grew after 24 h, i.e. during the second half of interphase, the increase accounting for from 11 to 48% of the total increase. The hypothesis is put forward that the increase in the nuclear DNA content during the cell cycle of Amoeba proteus is the result of two processes: (1) one-time replication of the DNA of the whole genome; and (2) repeated replication of some part of the DNA. In amoebae the relation of the pattern of nuclear DNA synthesis to the diet is considered.     

Mating type and the differentiated state in Physarum polycephalum.

In the acellular slime mold, Physarum polycephalum, the differentiation of amoebae into plasmodia is controlled by a mating type locus, mt. Amoebae carrying heterothallic alleles usually do not differentiate within clones; plasmodia form when two amoebae carrying different alleles fuse and undergo karyogamy. In this paper, we show that amoebae heterozygous for heterothallic alleles can be isolated and maintained as amoebae; the amoebae form plasmodia in clones without a change in ploidy. Plasmodia were also found to be formed, infrequently, by heterothallic amoebae of a single mating type. The plasmodia are healthy and are also formed without a change in ploidy. Thus, the presence of two different heterothallic mating type genes in a single nucleus is compatible with the amoebal state and one heterothallic mating type gene is compatible with the plasmodial state, once established.     

STABLE DIPLOIDS FROM INTRAGROUP ZYGOSPORES OF CLOSTERIUM EHRENBERGII MENEGH. (CONJUGATOPHYCEAE)1

Two pairs of stable diploid clones were obtained as aberrant forms among F₁ progeny of an intragroup (intraspecific) cross between R-11-4 (mating type +) and M-16-4b (mating type -) of Group A of Closterium ehrenbergii Menegh. Each pair was derived from the two germination products of a single zygospore, and both clones were mating type minus. The cell size range of these four diploid minus clones was considerably above that of normal (haploid) Group A clones. Chromosome counts at the second meiotic metaphase indicated that these clones were diploid with approximately 200 chromosomes, which was double the number for normal Group A clones. Diploid minus clones conjugated normally with any haploid Group A plus clones, and yielded many triploid zygospores. Triploid zygospores germinated normally as did intragroup diploid zygospores. In metaphase I preparations, only bivalents were observed except on a few occasions where some uni- and multivalents were also detected. Viability of F₁ progeny from triploid zygospores (55–74%) was somewhat lower than from diploid zygospores of Japanese Group A populations (65–90%), but higher than intergroup (interspecific) hybrid zygospores from Groups A, B and H (0–12%). In addition to lower viability, some F₁ progeny from triploid zygospores exhibited slow vegetative growth. Almost all pairs of F₁ clones from single triploid zygospores were of opposite mating type, similar to normal diploid zygospores of the intragroup cross. Morphological variability of F₁ progeny of triploid zygospores was great. The apparently normal meiosis of triploid zygospores and the high viability of F₁ progeny suggested that the genome of Group A contains several sets of chromosome complements with mechanisms by which bivalents are regularly formed in the first meiotic division.     

Cytogenetical studies in Erianthus: Meiosis and behaviour of B chromosomes in 2n=20 forms

Meiosis was studied in several clones of Erianthus elephantinus, E. ravennae and E. munja. B chromosomes were observed in the pollen mother cells of two clones of E. ravennae and two clones of E. munja. They were absent in root tip cells. The transmission of B chromosomes was studied in S₁ and S₂ progenies. A numerical decrease of B chromosomes was noticed in selfed progeny.     

Nuclear DNA content and chromosome number in somatic hybrid allopolyploids of <Emphasis Type="Italic">Solanum</Emphasis>

An attempt was made to change the proportion of the parental genomes in interspecific hybrids Solanum nigrum + S. tuberosum (ngr + tbr) by means of repeated protoplast fusion. In order to enlarge the potato input into the hybrid genome, the protoplasts of two ngr + tbr hybrids of different ploidy (7x and 8x) were fused with the protoplasts of two different diploid potato clones in three combinations. Protoclonal variability was studied in three populations of new ngr + tbr allopolyploids maintained in vitro. The absolute nuclear DNA content (2C) was measured using flow cytometry to estimate the ploidy of the hybrids. The ploidy level of the selected clones was verified by chromosome counts in root meristems. The newly synthesized allopolyploids (75 clones) showed only a small gain in nuclear DNA content above the mean value determined for the parents, instead of the expected addition of an entire diploid potato genome to the combined parental ngr + tbr genome. An increase in nuclear DNA was observed mostly in the clones having the 7x hybrid as a parent (75% of allopolyploids from two combinations). When the 8x hybrid was used as a parent, only two allopolyploids (5%) exhibited a significantly increased nuclear DNA content. The 8x level of ngr + tbr allopolyploids was shown to be stable and was only occasionally exceeded. Somatic hybrids ngr + tbr offer a model system for studying the molecular mechanism(s) and processes involved in stabilization and establishment of the synthetic Solanum allopolyploids.     

Chromosome numbers, karyotypes and nuclear DNA contents from perennial polyploid groups ofCerastium (Caryophyllaceae)

Somatic chromosome numbers have been determined for the followingCerastium taxa:C. eriophorum (2n = 36),C. alpinum (2n = 72),C. transsylvanicum (2n = 108),C. arcticum (2n = 108),C. latifolium (2n = 36),C. carinthiacum (2n = 36),C. banaticum (2n = 36),C. arvense subsp.glandulosum (2n = 36),C. arvense subsp.arvense (2n = 72) andC. fontanum (2n = 144). Karyotypes of three diploid species (C. eriophorum, C. banaticum andC. latifolium), belonging to three different taxonomic groups, were analysed and found to be similar. The relative nuclear DNA contents of all taxa were determined by flow cytometry and, for five species, also by Feulgen cytophotometry. The values obtained by the two methods are similar. A comparison of nuclear DNA contents among diploids shows that values differ significantly between different taxonomic groups, and are correlated with average chromosome size. Within closely related polyploid groups nuclear DNA amounts increase from 2x- to 4x- and 6x taxa as 1 : 1.4 : 2.4 in theC. alpinum complex, whereas DNA amounts are doubled comparing 2x- and 4x-subspecies in theC. arvense complex.     

Cytogenetic studies of Poecilia (Pisces)

Natural populations of triploid females resembling the gynogenetic teleost, Poecilia formosa (Girard), occur in northeastern Mexico where they intermingle with diploid populations of this species and the members of congeneric bisexual species such as P. mexicana or P. latipinna. Mitotic configurations from gill epithelial cells show 46 chromosomes for the diploid fishes, but 69 chromosomes for members of the triploid clones associated with P. formosa. Triploid females have erythrocytes that are significantly larger than those from diploid specimens and also show a roughly 50% elevation in the average DNA content of their somatic nuclei. Similar analyses of two functionally incompetent males of P. formosa, of a number of bisexual F₁ and F₂ hybrid offpsring from P. latipinna x P. mexicana, and of females from several other poeciliid species consistently show only diploid DNA levels and somatic chromosome complements where 22N=46. Demonstration of cytogenetic criteria by which females from triploid clones may be clearly distinguished from sympatric diploid specimens of P. formosa or P. mexicana leaves unresolved, for the present, problems of an appropriate systematic designation for natural populations of triploid gynogenetic fishes. The role of sympatric speciation in the evolution of poeciliid genomes is discussed in terms of alternative mechanisms to account for the persistence in nature of a vertebrate triploid of hybrid origin.This work was supported by grants from the National Science Foundation (GB 7393) and from the U.S. Public Health Service (GM 14644).Recipient of a Research Career Development Award from the U.S. Public Health Service (1 K3 GM 3455).     

CYTOPHOTOMETRIC DETERMINATION OF THE AMOUNT OF DNA IN ARACHIS L. SECT. ARACHIS (LEGUMINOSAE)

The 2C amounts of DNA for 12 taxa of the section Arachis nom. nud. of the genus Arachis L. were determined using cytophotometric techniques. The diploid taxa ranged from 4.92 to 5.98 pg of DNA per cell. The species of the diploid series Annuae Krap. & Greg. nom. nud. averaged ca. 1 pg less DNA per cell than the taxa of the diploid series Perennes Krap. & Greg. nom. nud. No significant differences were found between taxa within these two series. The tetraploid taxa ranged from 10.36 to 11.35 pg of DNA per cell. Within the tetraploid series Amphiploides Krap. & Greg. nom. nud. differences were found between A. monticola Krap. & Rig. and A. hypogaea L. The two subspecies of A. hypogaea, ssp. hypogaea and ssp. fastigiata Waldron, were found to differ significantly in their 2C amounts of DNA. The implications of the cytophotometric data on the chromosomal evolution of this section are discussed.     

NUCLEAR DNA CONTENT OF MYXAMOEBAE AND PLASMODIA IN SIX NON-HETEROTHALLIC ISOLATES OF A MYXOMYCETE,DIDYMIUM IRIDIS

The nuclear DNA content of six non-heterothallic isolates of the myxomycete Didymium iridis was measured by combining the Feulgen reaction with absorption microspectrophotometry. This allowed us to distinguish between homothallic (sexual) and apogamic (non-sexual) isolates. Four of the isolates studied, Panamanian 4 and 5, California 1, and Missouri 1 are homothallic. Moreover, the average DNA content of the myxamoebal and plasmodial nuclei (0.32 and 0.61 respectively) does not differ significantly from the calculated haploid and diploid values for heterothallic isolates of D. iridis (0.34 and 0.63). Hence, it is concluded that in each of these isolates the myxamoebae are haploid and the plasmodia diploid. In two of the isolates investigated, Georgia 1 and Hawaii 1, the DNA content of the myxamoebal and plasmodial nuclei did not differ significantly. Therefore, in both of these isolates the plasmodia appear to develop apogamically. In addition the mean DNA values recorded for the Ha-1 isolate suggest that it is aneuploid.     

A Diploid,Interspecific, Fertile Hybrid from Cultivated Sorghum, <Emphasis Type="Italic">Sorghum Bicolor</Emphasis>, and the Common Johnsongrass Weed <Emphasis Type="Italic">Sorghum Halepense</Emphasis>

Valuable agronomic traits are often present but inaccessible in the wild relatives of cultivated crop species. Utilization of wild germplasm depends on the production of fertile interspecific hybrids. Several unsuccessful attempts have been made to hybridize cultivated sorghum with its wild relatives to broaden its genetic base and enhance agronomic value. The successful approach used in this study employed the nuclear male sterility gene ms3 to generate a diploid fertile hybrid between the diploid cultivated sorghum (Sorghum bicolor (L) Pers.) and its weedy tetraploid wild relative Johnsongrass (Sorghum halepense (L.) Pers.). Eight sorghum plants were selected from a Nebraska stiff stalk collection that contains the male sterility gene ms3 and were used as the female parent. About 36,000 florets of male sterile sorghum were pollinated with Johnsongrass pollen to produce an average of one well-developed and 180 severely shriveled seed/18,000 crosses. The well-developed seed gave rise to a self-fertile diploid, while none of the shriveled seed were able to germinate. The F₁ hybrid was confirmed by using cultivated sorghum SSR markers and was selfed to produce an F₂ population. A sub-sample of 96 segregating F₂ plants was examined with 36 sorghum polymorphic SSR markers. Thirty-four markers showed a normal 1:2:1 segregation ratio, evidence of normal recombination across the genome. Preliminary results showed that several desirable traits from Johnsongrass, including resistance to greenbug and chinch bug and adaptability to cold temperatures, were expressed in the resulting progenies. These observations suggest that speciation within the genus Sorghum, giving rise to widely divergent phenotypes, is effected largely by ploidy-maintained crossing barriers but apparently not by extensive genomic divergence.