CHROMOSOME NUMBERS IN THE HYPOCREALES 1. NUCLEAR DIVISION IN THE ASCUS OF NECTRIA PEZIZA

El -Ani , Arif S. (Columbia U., New York, N. Y.) Chromosome numbers in the Hypocreales. 1. Nuclear division in the ascus of Nectria peziza. Amer. Jour. Bot. 46(6): 412–417. Illus. 1959.—The 4 nuclear divisions in the ascus of Nectria peziza were studied with the aid of acetoorcein and aceto-carmine techniques. The ascus was found to arise by crozier formation. Synapsis takes place while the chromosomes are still contracted and elongation of chromosomes continues throughout the pachytene phase. The haploid complement was found to consist of 5 chromosomes, the second of which is the nucleolus-organizing chromosome. This chromosome number which had never been reported in the Hypocreales was also found in several other species of this group of fungi. Chromosome numbers in the Hypocreales are discussed with regard to the species concept in the imperfect genus Fusarium.     

Meiosis and ascospore development in Cochliobolus heterostrophus.

Cochliobolus heterostrophus produces eight filiform ascospores per ascus, following meiosis and a postmeiotic mitosis. Early ascus development and nuclear divisions in C. heterostrophus resemble those of the prototypic Pyrenomycete Neurospora crassa. However, the two fungi differ in several important details owing to differences in ascus and ascospore shape, spindle pole body (SPB) behavior during spore delimitation, and ascospore development. In C. heterostrophus, the two spindles at meiosis II, and the four spindles at the postmeiotic mitosis are aligned irregularly, unlike the tandem or ladder rung-like orientation of spindles of N. crassa. Prior to ascospore delimitation, all eight nuclei reorient themselves and their SPB plaques migrate toward the base of the ascus. The SPB plaques facilitate demarcation of the lower end of each incipient ascospore. The filiform ascospores are uninucleate and unsegmented at inception but they become highly multinucleate, multisegmented, and helically coiled when mature. An account of ascus development, nuclear divisions, and ascospore delimitation and maturation is presented here and supported by a series of photomicrographs.     

Meiosis and ascospore development in Cochliobolus heterostrophus

Cochliobolus heterostrophus produces eight filiform ascospores per ascus, following meiosis and a postmeiotic mitosis. Early ascus development and nuclear divisions in C. heterostrophus resemble those of the prototypic Pyrenomycete Neurospora crassa. However, the two fungi differ in several important details owing to differences in ascus and ascospore shape, spindle pole body (SPB) behavior during spore delimitation, and ascospore development. In C. heterostrophus, the two spindles at meiosis II, and the four spindles at the postmeiotic mitosis are aligned irregularly, unlike the tandem or ladder rung-like orientation of spindles of N. crassa. Prior to ascospore delimitation, all eight nuclei reorient themselves and their SPB plaques migrate toward the base of the ascus. The SPB plaques facilitate demarcation of the lower end of each incipient ascospore. The filiform ascospores are uninucleate and unsegmented at inception but they become highly multinucleate, multisegmented, and helically coiled when mature. An account of ascus development, nuclear divisions, and ascospore delimitation and maturation is presented here and supported by a series of photomicrographs.     

Morphogenesis of Ascospores in Saccharomyces cerevisiae

Ultrastructural changes associated with ascospore formation in Saccharomyces cerevisiae were investigated by using freeze-etching and thin-sectioning techniques. The first nuclear division (meiosis I) is indicated by the appearance of spindle fibers within the nucleus. The nucleus subsequently elongates and eventually assumes a barbell shape; the second nuclear division (meiosis II) occurs before nuclear separation. The spindle fibers involved in meiosis II appear to be oriented perpendicular to those observed in meiosis I. A discrete bilaminar structure (forespore wall) progressively delineates each ascospore nucleus and encloses cytoplasmic material including mitochondria and endoplasmic reticulum. The forespores then elongate, close off, and become separated from the ascus cytoplasm by membranes. The ascospores assume a spherical shape as spore coat material is laid down; the latter stages of ascospore formation are characterized by thickening of the ascospore wall and disintegration of the ascus cytoplasm. No structures which could be identified as chromosomes were observed.     

CYTOLOGY OF HELMINTHOSPORIUM TURCICUM AND ITS ASCIGEROUS STAGE,TRICHOMETASPHAERIA TURCICA

Knox- Davies , P. S., and J. G. Dickson . (U. Wisconsin, Madison.) Cytology of Helmintho sporium turcicum and its ascigerous stage, Trichometasphaeria turcica . Amer. Jour. Bot. 47(5) : 328—339. Illus. 1960.–The cells of the vegetative hyphae were generally multinucleate. Interphase nuclei resembled those of higher organisms, with a matrix of thread-like chromatin material surrounding a spherical nucleolus. “Beaked” nuclei frequently associated with anastomosing hyphae were interpreted as migrating nuclei. Nuclear division in the vegetative hyphae was rapid. Various division stages were distinguished but it was difficult to make accurate chromosome counts. The nucleoli were discarded at prophase or prometaphase and were reorganized in daughter nuclei at telophase. An outstanding feature of nuclear division was that all the nuclei in a cell divided simultaneously. Conidiophores and conidia were occasionally joined by wide cytoplasmic connections. They were multinucleate throughout their development. Mechanisms therefore exist for the perpetuation of heterokaryons through the conidium. Ascus development was studied in a hybrid between a dark and an albino isolate. Crozier formation was typical and nuclear fusion occurred in the young ascus. Four nuclear divisions were completed in the ascus before there was evidence of ascospore delimitation. Further nuclear division took place in the ascospores whose cells were multinucleate. The occurrence of less than 8 ascospores in an ascus appeared to follow degeneration of nuclei rather than the incorporation of a number of division-Ill nuclei in a single ascopore. Chromosome counts and irregularities in the appearance and behavior of nuclei and chromosomes in the asci indicate that aneuploidy occurs in Trichometasphaeria turcica. It is suggested that aneuploidy is a common phenomenon in the conidial stage of the fungus H. turcicum, and possibly also in other imperfect fungi.     

Cytological and genetic studies of the life cycle of Saccharomycopsis lipolytica

Summary In the alkane yeast Saccharomycopsis lipolytica (formerly: Candida lipolytica) the variability in the ascospore number is caused by the absence of a correlation between the meiotic divisions and spore wall formation. In four spored yeasts, after meiosis II, a spore wall is formed around each of the four nuclei produced by meiosis II. However, in the most frequently occurring two spored asci of S. lipolytica, the two nuclei are already enveloped by the spore wall after meiosis I due to a delay of meiosis II. This division takes place within the spore during the maturation of the ascus. In this case germination of the binucleate ascospore is not preceded by a mitosis. It follows that the cells of the new haploid clones are mononucleate. In the three spored asci, which occur rarely, only one nucleus is surrounded by a spore wall after meiosis I; the other nucleus undergoes meosis II before the onset of spore wall formation. The result is one binucleate and two mononucleate spores. In the one spored asci the two meiotic divisions occur within the young ascospore, i.e. spore wall formation starts immediately after development of the ascus. These cytological observations were substantiated by genetic data, which in addition confirmed the prediction that binucleate spores may be heterokaryotic. This occurs when there is a postreduction of at least one of the genes by which the parents of the cross differ. This also explains the high frequency of prototrophs in the progeny on non-allelic auxotrophs since random spore isolates are made without distinguishing between mono-and binucleate spores. The possibility of analysing offspring of binucleate spores by tetrad analysis is discussed. These findings enable us to understand the life cycle of S. lipolytica in detail and we are now in a position to start concerted breeding for strain improvement especially with respect to single cell protein production.     

Diverse programs of ascus development in pseudohomothallic species of Neurospora,Gelasinospora, and Podospora

Meiosis and ascospore development in the four-spored pseudohomothallic ascomycetes Neurospora tetrasperma, Gelasinospora tetrasperma, Podospora anserina, and P. fefraspora have been reexamined, highlighting differences that reflect independent origins of the four-spored condition in the different genera. In these species, as in the heterothallic eight-spored N. crassa, fusion of haploid nuclei is followed directly by meiosis and a postmeiotic mitosis. These divisions take place within a single unpartitioned giant cell, the ascus, which attains a length of >0.1 mm before nuclei are enclosed by ascospore walls. Two basically different modes underlie the delivery of opposite mating type nuclei into each of the four ascospores in the different genera. In N. tefrasperma on the one hand, the mating type locus is closely centromere-linked. Mating types therefore segregate at the first meiotic division. The second division spindles of N. tefrasperma overlap and are usually parallel to one another, in contrast to the their tandem arrangement in N. crassa. As a result, nonsister nuclei of opposite mating type are placed close together in each half-ascus and a pair is enclosed in each ascospore. In the Podospora and Gelasinospora species on the other hand, the second-division spindles are in tandem, with sister nuclei of opposite mating type associated as a pair in each half-ascus. It is established for P. anserina and inferred for P. fetraspora and G. fefrasperma that a single reciprocal crossing over almost always occurs in the mating type-centromere interval, ensuring that mating types segregate at the second meiotic division and that nuclei of opposite mating type are enclosed in each ascospore. Other differences are also seen that are less fundamental. Neurospora tetrasperma differs from the other species in the orientation of chromosomes and spindle pole body plaques at interphase (I.) Third-division spindles are oriented parallel to the ascus wall in Gelasinospora but across the ascus in Podospora and Neurospora. The two Podospora species differ from one another in nuclear behavior following mitosis in the young ascospores. In P. tefraspora, two of the four nuclei migrate into the tail cell, which degenerates, leaving one functional nucleus of each mating type. In P. anserina, by contrast, only one of the four nuclei moves into the tail cell, leaving the germinating ascospore with two functional nuclei of one mating type and one of the other. The pseudohomothallic condition with its heterokaryotic vegetative phase has significant consequences for both the individual organism and the breeding system. Genetic controls of development and recombination are complex. Inbreeding is not obligatory. © 1994 WiIey-Liss, Inc.     

Morphology,development and cytology of Taphrina maculans Butler

Morphology, development and nuclear behavior of the ascogenous stroma and asci in the infection spots have been described inTaphrina maculans Butler. The fungus forms subcuticular and intercellular mycelium in the leaf tissues and the ascogenous layers originate through division of the subcuticular hyphal cells in the infection sites. Germination of ascogenous cells starts with their elongation in the uppermost layer forming asci and ascospores without formation of stalk cells. Meiosis of the fusion (diploid) nucleus occurs in the young ascus as in otherTaphrina species devoid of stalk cells. The haploid chromosome complement in this species consists of 3 chromosomes (n=3). All the cells in the stromatic layer are potential ascogenous cells and ascus formation continues, until all of them are exhausted in the infection spot. Eight ascospores are normally formed in each ascus, but multi-plication of ascospores may occurin situ later. Three morphologically distinct types of ascus opening are encountered, which are apparently not correlated with prevalent environment. Multiplication of ascospores after their discharge from mature asci occurs by budding proceded by a mitotic division of the spore nucleus. Blastospores (budded cells) germinate into short hyphae and binucleate condition of cells originates by mitotic division of the nucleus. Occurrence of giant cells containing 2 nuclei is often observed. Possible origin of Uredinales fromTaphrina-like ancestors has been indicated due to their close resemblance.     

Metschnikowia lunata sp. nov.

Ascosporulation in the yeast strain designated asSelenotila intestinalis Krassilnikov was achieved. On the basis of mode of ascus formation and ascospore morphology it is included in the genusMetschnikowia Kamienski as a new species,M. lunata.     

Biphasic diurnal cycle of ascus development of Taphrina maculans Butler

Taphrina maculans Butler inciting the brown leaf spot disease of turmeric (Curcuma longa L.) produces cuboid ascogenous cells several layers in depth in the subcuticular interspaces of the epidermis. The ascogenous cells germinate to produce asci centrifugally maturing toward the periphery of the stroma, expelling octosporous microcolonies of asco-blastospores on the leaf surface inciting secondary infection in favorable environment. Occurrence of a rhythmic cycle of ascus development and ascospore discharge giving 2 peaks of ascospore discharge each day has been demonstrated in Taphrina maculans. The cycle is directly affected by atmospheric temperature, availability of free moisture on the leaf surface and sunlight. Free moisture on a leaf surface appropriately soaks the infection spots (ascogenous cells) and induces ascus elongation and ascospore discharge, when a suitable atmospheric temperature is reached. Sunlight may adversely affect the cycle by increasing the temperature and lowering humidity in the atmosphere.     

Ultrastructure of ascospore delimitation in freeze substituted samples ofAscodesmis nigricans (Pezizales)

Summary Freeze substitution proved to be a valuable technique for studying the early stages of ascosporogenesis inAscodesmis nigricans. Our observations indicate that the ascus vesicle originated from the ascus plasma membrane. Invaginations of the plasma membrane produced ascus vesicle initials consisting of two closely spaced unit membranes. The appearance of the outer leaflet of each of these membranes was identical to that of the inner leaflet of the ascus plasma membrane. Apparent points of continuity between ascus vesicle initials and the plasma membrane were observed. Ascus vesicle initials accumulated in the ascus cytoplasm near the plasma membrane and then coalesced to form the ascus vesicle, a peripheral, cylinder-like structure consisting of two closely spaced unit membranes that extended from the ascus apex to the ascus base. The ascus vesicle then became invaginated in a number of regions and subsequently gave rise to eight sheet-like segments, or ascosporedelimiting membranes, that encircled uninucleate segments of cytoplasm forming ascospore initials. Like the ascus vesicle, each ascospore-delimiting membrane consisted of two closely spaced unit membranes, the inner of which became the ascospore plasma membrane. The ascospore wall then developed between the spore plasma membrane and the outer membrane. Many details of ascospore maturation were clearly visible in freeze substituted samples.     

Cytology of recessive sexual-phase mutants from wild strains of Neurospora crassa.

Wild-collected strains of Neurospora crassa harbor recessive mutations that are expressed in the sexual phase when homozygous. Thirty-two representative mutants that produced barren perithecia were examined cytologically. Six of these mutants failed to form asci. Of the remaining 26, chromosome pairing was disturbed in 12 and meiosis was disturbed at pachytene or diplotene in 5. Seven mutants showed normal meiosis I but then diverged from the normal sequence, and two showed perithecial beak abnormalities. In many mutants, ascus development and nuclear divisions continued after the initial defect, albeit abnormally. Nuclear divisions were often delayed, essentially uncoupling them from other ascus events such as the formation of enlarged spindle pole body plaques, ascospore wall membranes, and spore delimitation. All 32 mutants were recessive and none showed obvious morphological abnormalities during vegetative growth. This phenotype contrasts sharply with that of numerous laboratory-induced ascus mutants, which are frequently expressed pleiotropically in the vegetative phase and several are dominant in the sexual phase.     

Programmed ascospore death in the homothallic ascomycete Coniochaeta tetraspora

Immature asci of Coniochaeta tetraspora originally contain eight uninucleate ascospores. Two ascospore pairs in each ascus survive and mature, and two die and degenerate. Arrangement of the two ascospore types in individual linear asci is what would be expected if death is controlled by a chromosomal gene segregating at the second meiotic division in about 50% of asci. Cultures originating from single homokaryotic ascospores or from single uninucleate conidia are self-fertile, again producing eight-spored asci in which four spores disintegrate, generation after generation. These observations indicate that differentiation of two nuclear types occurs de novo in each sexual generation, that it involves alteration of a specific chromosome locus, and that the change occurs early in the sexual phase. One, and only one, of the two haploid nuclei entering each functional zygote must carry the altered element, which is segregated into two of the four meiotic products and is eliminated when ascospores that contain it disintegrate. Fusion of nuclei cannot be random-a recognition mechanism must exist. More study will be needed to determine whether the change that is responsible for ascospore death is genetic or epigenetic, whether it occurs just before the formation of each ascus or originates only once in the ascogonium prior to proliferation of ascogenous hyphae, and whether it reflects developmentally triggered alteration at a locus other than mating type or the activation of a silent mating-type gene that has pleiotropic effects. Similar considerations apply to species such as Sclerotinia trifoliorum and Chromocrea spinulosa, in which all ascospores survive but half the spores in each ascus are small and self-sterile. Unlike C. tetraspora, another four-spored species, Coniochaetidium savoryi, is pseudohomothallic, with ascus development resembling that of Podospora anserina.     

Nuclear division in the red algae Chondria nidifica and Chondria tenuissima

Cytological investigations are reported for two Chondria species, the Pacific species Chondria nidifica Harvey and Chondria tenuissima (Goodenough et Woodward) C. A. Agardh from the shore of the Marmara Sea in Istanbul. Nuclear division during mitosis and meiosis has been followed in somatic cells and in tetrasporangial mother cells respectively of diploid tetrasporic plants. The spherical interphase nucleus stains densely, showing many chromatin granules. Mitotic nuclei in the apical groove show a large number of chromosomes at metaphase; the chromosome number has been estimated at diakinesis to be 40 in both C. nidifica and C. tenuissima. The meiotic nuclei of tetraspore mother cells in prophase contain several relatively large nucleolar-derivatives in both species. The nucleolar derivatives disappear completely before the chromosomes begin to differentiate. In meiotic prophase the tetraspore mother cell enlarges from its original diameter. The period of the second meiotic anaphase seems to be extremely short in comparison with other nuclear phases. When the chromosomes reach the poles, they spread and subsequently form a relatively compact mass at telophase. The spindle has not been observed in C. tenuissima. Photographs are presented of nucleoli and nucleolar-derivatives in mitotic and meiotic divisions.     

Division spindle and centrosomal plaques during mitosis and meiosis in some Ascomycetes

The behaviour of the division spindle and centrosomal plaques is described in four species of Ascomycetes (Ascobolus immersus, Ascobolus stercorarius, Podospora anserina and Podospora setosa) studied by light and electron microscopy. Two unique features of the kinetical apparatus were observed: presence of centrosomal plaques and intranuclear location of the spindle. In all types of mitoses (mycelium, crosier and postmeiotical mitosis) the apparatus is structurally identical to that found in meiosis. The centrosomal plaques, present in all divisions, are always contiguous with the nuclear envelope and never show centrioles similar to those commonly found in Metazoa and Protozoa. During metaphase and anaphase the plaque is constituted of two zones situated on each side of the nuclear envelope: an electron opaque outer zone and inner one less opaque in which most of the microtubules end. In Podospora the outer zone appears in sections as consisting of two dark layers separated by a clear one. Two dispositions of plaques are possible: either they are entirely contiguous with the nuclear envelope (Ascobolus) or only partially so, the remainder being perpendicular to the nuclear envelope (Podospora). — The localisation of the plaques in the ascus was determined by light and electron microscopy. The nuclear envelope was shown to remain intact during division. It was possible to observe that the sporal wall of each spore originated from the same unique double membrane formed in the ascus during the meiotic second division and postmeiotical mitosis. This fact is of genetical interest for the study of morphological and physiological characters of the spores.     

THE DISTRIBUTION OF SELECTED DIAGNOSTIC CHARACTERS IN THE LECANORALES

Traditional classification concepts in lichenology are often, but not always, supported by molecular results. Molecular data should be compared and correlated with micro-morphological and ultrastructural information before systematic rearrangements are undertaken. Visualization of the distribution of morphological and other characters in specified groups is considered as a desirable resultper se, but it is also important to discover whether correlating characters are dependent on each other or not; and if not, whether their distribution in a group might support existing classification concepts. A data set for lecanoralean and other lichenized and lichenicolous genera, comprising 90—mostly multi-state—characters was used to store morphological, chemical and ecological data, and to test character correlations. Several examples of such analyses are presented. The following pairs of characters show some degree of dependence: ascospore septation and number per ascus, ascospore wall type and pigmentation, ascospore and epihymenium pigmentation. Several authors postulated that ascus types are good phylogenetic markers. Ascus types have been widely used for classification concepts of the Lecanorales. Two-dimensional correlation queries of ascus types with the following morphologcal characters were made: substratum preference, thallus growth form and ascospore septation. These correlations supply characteristic profiles for the various ascus types, which have to be compared with forthcoming phylogenetic hypotheses based on molecular data.     

Ascospore aggregation and oxylipin distribution in the yeast Dipodascopsis tothii

Upon cultivation of the yeast Dipodascopsis tothii in its sexual stage, small ascospores are released individually from the ascus tip, which then assemble in sheathed cluster balls. In contrast to Dipodascopsis uninucleata, this yeast produced smooth bean shaped ascospores with sheath-like appendages that assemble in a disordered sheathed ball of ascospores outside the ascus. Strikingly, upon release, the ascus tip contained 3-hydroxy oxylipins, while the released ascospore clusters contained little or no 3-hydroxy oxylipins as indicated by immunofluorescence microscopy. In D. uninucleata, these oxylipins are concentrated on the spore surface and interspore matrix, but not on the ascus tip.     

DNA CONTENT OF SEVEN SPECIES OF ASTEREAE AND ITS SIGNIFICANCE TO THEORIES OF CHROMOSOME EVOLUTION IN THE TRIBE

Relative amounts of nuclear DNA were determined in root tip cells of seven species of Astereae: Aster hydrophilus Greene, A. oblongifolius Nutt., A. riparius H.B.K., Machaeranthera boltoniae (Greene) Turner and Home, M. brevilingulata (Sch-Bip.) Turner and Home, M. parviflora Gray, and M. tenuis (S. Wats.) Turner and Home. The results show that A. hydrophilus and M. brevilingulata, with a chromosome number of n = 9, have less nuclear DNA than other closely related species which are either n = 4 or n = 5. Cytological analyses of meiosis in the intergeneric hybrid M. parviflora X A. hydrophilus showed cells with two or more small chromosomes of the latter species pairing with single large chromosomes of the former. Pachytene cells of the hybrids M. parviflora X A. hydrophilus, M. parviflora X A. riparius, and M. boltoniae X M. tenuis showed some unpaired chromosome segments. The significance of these results to chromosome evolution in the tribe Astereae is discussed.