CLOSTERIUM MONILIFERUM-EHRENBERGII (CHAROPHYCEAE, CHLOROPHYTA) SPECIES COMPLEX VIEWED FROM THE 1506 GROUP I INTRON AND ITS2 OF NUCLEAR rDNA

To assess phylogenetic relationships and speciation modes in Closterium , we sequenced two noncoding regions of the nuclear ribosomal cistron, the 1506 group I intron in small subunit and the internal transcribed spacer 2, for a total of 58 strains of the Closterium moniliferum-ehrenbergii species complex. These include both homothallic and heterothallic C. moniliferum Erenberg ex Ralfs v. moniliferum , heterothallic C. moniliferum v. submoniliferum (Woronichin) Krieger, and heterothallic C. ehrenbergii Meneghini ex Ralfs that can be divided into several mating groups. We found no or very little sequence divergence within single mating groups of C. ehrenbergii and among all heterothallic strains of C. moniliferum v. moniliferum or C. moniliferum v. submoniliferum. Nevertheless, sequence divergence was much greater between those mating groups of C. ehrenbergii and also among the three traditional taxa . Maximum parsimony and maximum likelihood analyses showed that the taxon C. ehrenbergii was not monophyletic. The two varieties of C. moniliferum appeared as a sister clade to certain mating groups of C. ehrenbergii . Among the clades that were recovered in different trees by maximum parsimony and maximum likelihood analyses, we consistently found two large conspicuous clades: clade I consisted of mating groups A, B, C, H, K, and L of C. ehrenbergii whose zygospores have smooth-walls, and clade II contained the mating groups D, E, I, J, and S whose zygospores are scrobiculate. Phylogenetic incongruences observed are discussed from the viewpoints of the different molecular nature of the group I intron and internal transcribed spacer 2, as well as putative rapid diversification of the mating groups and probable ancient ancestral hybridization.     

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.     

TIME-LAPSE ANALYSES OF SEXUAL ISOLATION BETWEEN TWO CLOSELY RELATED MATING GROUPS OF THE CLOSTERIUM EHRENBERG SPECIES COMPLEX (CHLOROPHYTA)1

Sexual isolation between Groups A and B of Closterium ehrenbergii, two closely related species, was studied by a multiple-choice mating method, as well as the nochoice mating method which has been used in previous work on microalgae. Time lapse photomicrographs and the difference in cell shape and size between the two mating groups allowed identification of a given cell in the mixture as either Group A or B, even when certain morphological changes occurred during the several day culture required for sexual induction. When plus and minus mating types of Group A were mixed with those of Group B (multiplechoice mating), no intergroup hybrid zygospores were formed. However, many intragroup zygospores of either Group A or B were formed. When one plus strain of Group A was mixed with one minus strain of Group B or when one plus strain of Group B was mixed with one minus strain of Group A (no-choice mating), intergroup sexual interactions took place resulting in a small number of hybrid zygospores; however, the process took much longer than intragroup sexual interactions. It was also shown that cell size difference itself hardly affects sexual interactions between haploid and autodiploid strains of Group A. It is suggested that sexual isolation between Groups A and B would be complete in nature, although they may interact sexually in the laboratory.     

Zygospore formation between homothallic and heterothallic strains of Closterium

Zygospore formation in different strains of the Closterium peracerosum-strigosum-littorale complex was examined in this unicellular isogamous charophycean alga to shed light on gametic mating strains in this taxon, which is believed to share a close phylogenetic relationship with land plants. Zygospores typically form as a result of conjugation between mating-type plus (mt⁺) and mating-type minus (mt⁻) cells during sexual reproduction in the heterothallic strain, similar to Chlamydomonas. However, within clonal cells, zygospores are formed within homothallic strains, and the majority of these zygospores originate as a result of conjugation of two recently divided sister gametangial cells derived from one vegetative cell. In this study, we analyzed conjugation of homothallic cells in the presence of phylogenetically closely related heterothallic cells to characterize the reproductive function of homothallic sister gametangial cells. The relative ratio of non-sister zygospores to sister zygospores increased in the presence of heterothallic mt⁺ cells, compared with that in the homothallic strain alone and in a coculture with mt⁻ cells. Heterothallic cells were surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified and confirming the formation of hybrid zygospores between the homothallic cells and heterothallic mt⁺ cells. These results show that at least some of the homothallic gametangial cells possess heterothallic mt⁻-like characters. This finding supports speculation that division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.     

TIME LAPSE ANALYSES OF SEXUAL REPRODUCTION IN CLOSTERIUM EHRENBERGII (CONJUGATOPHYCEAE)1

The process of sexual differentiation was studied using heterothallic clones of Closterium ehrenbergii Meneghini. The first visible sign of sexual reproduction was agglutination of two or more cells in a group and this was followed by gametangiogenic division and conjugation of gametangial cells. Movements of gametangial cells were carefully studied. Gametangial cells occasionally participated again in gametangiogenesis instead of proceeding directly to the formation of conjugation papilla. The whole process of sexual differentiation from vegetative cell to zygospore was considered to be basically similar in both of the two closely related mating groups, A and B, of C. ehrenbergii. Nevertheless, there were some differences between the two groups in patterns of the sexual differentiation. In Group A, vegetative cell division was completely suppressed by mixing the two complementary mating type clones together into the same medium with high light illumination. This suppression was not caused by the nitrogen depletion in the medium, but by the presence of cells of opposite mating type. In Group B, vegetative cell division and sexual reproduction occurred side by side repeatedly for several days.     

SEXUAL PROCESSES AND PHYLOGENETIC RELATIONSHIPS OF A HOMOTHALLIC STRAIN IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (ZYGNEMATALES,CHAROPHYCEAE)1

Members of the Closterium peracerosum–strigosum–littorale (C. psl.) complex are unicellular charophycean algae in which there are two modes of zygospore formation, heterothallic and homothallic. A homothallic strain of Closterium (designation, kodama20) was isolated from a Japanese rice paddy field. Based on alignment of the 1506 group‐I introns, which interrupt nuclear SSU rDNAs, homothallic kodama20 is most closely related to the heterothallic mating group II‐B, which is partially sexually isolated from group II‐A. Time‐lapse photography of the conjugation process in kodama20 revealed that most of the observed zygospores originated from one vegetative cell. The sexual conjugation process consisted of five stages: (1) cell division resulting in the formation of two sister gametangial cells from one vegetative cell, (2) formation of a sexual pair between the two sister gametangial cells (or between gametangial cells of another adjoined individual), (3) formation of conjugation papillae, (4) release of gametic protoplasts from both members of a pair, and (5) formation of the zygospore by protoplast fusion. For conjugation to progress, the cell density and light condition in the culture was critical. We suggested the presence of a conjugation promotion factor.     

A SEX DETERMINING MECHANISM IN THE CLOSTERIUM EHRENBERGII (CHLOROPHYTA) SPECIES COMPLEX1

Homozygous mt^?/mt^? diploid clones of the Closterium ehrenbergii Menegh. ex Ralfs species complex were obtained by hypertonic treatment from minus vegetative cells, and mating type segregation ratios in the F₁ progeny of “triploid” zygospores between wild type mt⁺ haploid and mt^?/mt^? homozygous diploui were analyzed. The ratio of plus to minus individuals was 1:4.8, and the ratio of the pairs of opposite mating types to those of minus mating type was 1:2.1. The results clearly show that mt^? is dominant to mt⁺ and that the mating type inheritance in these zygospores follows the triploid-like pattern. The validity of our assumption that the two mating types are determined by one genetic factor (mt^? allele dominant) was confirmed in B₁ progeny analyses as well. The results suggest that this sex determining mechanism is working effectively in the C. ehrenbergii species complex, in which several biological species have evolved through polyploidization.     

XYLULOSE,AN ALGAL GROWTH INHIBITOR12

The growth of Chlorococcum echinozygotum was previously known to be inhibited by an autoclaved solution of xylose in Bold's modified Bristol's medium. We now report the isolation of 2 inhibitory compounds derived from xylose during autoclaving, and the identification of one of them as xylulose. Two strains of C. echinozygotum were isolated, one of which had physiologically adapted to xylulose. The growth rates of both strains were studied using varying amounts of xylulose. Cell counting studies indicated that the zoospores of the unadapted strain were most severely affected by xylulose inhibition and that the zygospores were most resistant.     

CULTURE STUDY OF REPRODUCTIVE CYCLES AND SYSTEMATICA IN MOUGEOTIA TRANSEAUI (CHLOROPHYTA)1,2

Asexual and sexual reproductive cyries are described and illustrated for a cultured homothallic strain of Mougeotia transeaui Collins. Reproduction is by aplanospores and zygospores. Aplanospore formation precedes zygospore formation and continues longer with regreening of older cultures occurring regularly from precocious germination of aplanospores. Aplanospores typically form when most of the protoplast moves into the central swollen region of a cell and two new cross walls form to delimit an aplanosporangium. Conjugation is scalariform without the movement and fusion of well-organized gametes. During zygospore maturation, three new cross walls form in the receptive gametangium and conjugation tube to produce a zygosporangium.     

Quantitative nuclear DNA changes inEleusine (Gramineae)

2C nuclear DNA amounts were determined in 30 collections belonging to 10 species ofEleusine. About a 2.5-fold variation in genome size is evident in the genus. The 2C DNA amount in the diploid species ranged from 2.50 pg inE. verticillata to 3.35 pg inE. intermedia. In contrast, the tetraploid species showed a range from 4.95 pg inE. africana to 6.13 pg inE. floccifolia. At intraspecific level 10 collections ofE. coracana, 6 ofE. indica, 4 ofE. africana, 2 ofE. tristachya, and 2 ofE. kigeziensis did not show any significant variation. However, 2 collections ofE. floccifolia, connected with polyploidy, displayed about 90% variation. Polyploid species showed approximately double the genome size of that of their corresponding diploids. An evolutionary increase in DNA amount is evident inE. coracana during the course of its origin and domestication fromE. africana.     

The distribution of the transposable elementBari-1 in theDrosophila melanogaster andDrosophila simulans genomes

The distribution of the transposable elementBari-1 inD. melanogaster andD. simulans was examined by Southern blot analysis and byin situ hybridization in a large number of strains of different geographical origins and established at different times.Bari-1 copies mostly homogeneous in size and physical map are detected in all strains tested. Both inD. melanogaster and inD. simulans a relatively high level of intraspecific insertion site polymorphism is detectable, suggesting that in both speciesBari-1 is or has been actively transposing. The main difference between the two sibling species is the presence of a large tadem array of the element in a well-defined heterochromatic location of theD. melanogaster genome, whereas such a cluster is absent inD. simulans. The presence ofBari-1 elements with apparently identical physical maps in allD. melanogaster andD. simulans strains examined suggests thatBari-1 is not a recent introduction in the genome of themelanogaster complex. Structural analysis reveals unusual features that distinguish it from other inverted repeat transposons, whereas many aspects are similar to the widely distributedTc1 element ofC. elegans.     

Bipolar heterothallism,a principal mating system of<Emphasis Type="Italic">Cordyceps militaris in vitro</Emphasis>

Interest inin vitro study of entomopathogenic fungi, includingCordyceps species, has been increasing due to their valuable bioactive compounds and biocontrol effects. AmongCordyceps species,in vitro stromata ofC. militaris has been successfully produced and cultivated for industrial purposes. However, genetic study onin vitro stromata formation ofC. militaris has not been carried out yet. Here, relationship between mating system and perithecial stromata formation ofC. militaris is reported. Mating system was determined by observing perithecial stromata formation from mono-ascospore cultures and their pair-wise combinations. Certain combinations of mono-ascospore strains produced perithecial club-shaped stromata, whereas other combinations produced either no stromata or only abnormal non-perithecial stromata. Similarly, monoascospore cultures without combination produced either no stromata or only abnormal nonperithecial stromata. Despite obvious heterothallism, self-fertility was occasionally observed in few strains ofC. militaris. These observations indicated thatC. militaris behaves as a bipolar heterothallic fungus and requires two mating compatible strains in order to produce regular clubshaped perithecial stromata, a fundamental requirement for its industrial cultivation.     

Nectar flow and pollinators' efficiency in two co-occurring species ofCapparis (Capparaceae) in Israel

Two closely related species ofCapparis, C. ovata andC. spinosa, which are exposed to the same pollination environment were studied. The nectar volume and concentration ofC. ovata are higher than inC. spinosa. Both species have similar pollination efficiency, but the relative contribution of each pollinator is different.C. ovata is pollinated mainly hy hawkmoths while the principal pollinators inC. spinosa are bees.     

多形小克银汉霉、暗孢小克银汉霉和褐孢小克银汉霉应否被接受为独立的种?

多形小克银汉霉(Cunninghamella polymorpha Pisnek)、暗孢小克银汉霉(Cunninghamella phaeospora Boedijn)和褐孢小克银汉霉(Cunninghamella brunnea J.N. Rai,S.C. Agarwal & J.P. Tewari)自分别在1929, 1958, 1968年发表以来,一直存在着不同意见上的争论,或承认或不承认,或合并到这个种或合并到那个种等等.我们在研究中国的小克银汉霉属的过程中,分离得到了九株多形小克银汉霉的菌株。为了确定这个种的分类地位以及澄清它与其他一些相关种的关系,我们将这些菌株与从国外交换获得的一些相关种的模式后代作了比较研究,所得结果表明多形小克银汉霉、暗孢小克银汉霉及褐孢小克银汉霉三种既不能相互合并亦不能合并到其他任何一种中去,而应作为独立的种被人们所接受。在我们的工作之前,这三个种的有性型均未有报道。我们在国内尚未发现暗孢小克银汉霉和褐孢小克银汉霉,只找到了多形小克银汉霉,但同时也找到了它的有性型.因此,多形小克银汉霉为我国的新记录种,其有性型的发现则为全世界的首次报道。多形小克银汉霉的主要区别性特征为孢囊梗主枝形成顶生泡囊或不形成而在顶部叉分为二至数个分枝,同一孢囊梗上的分枝长短不齐,接合孢子囊不正球形,配囊柄细长可达80微米等等.暗孢小克银汉霉的主要区别性特征为孢囊梗分枝很少或不分枝,在同一孢囊梗上分枝相距很远且都很短,泡囊形状多不规则且略呈多角形。褐孢小克银汉霉更易与其他种相区别,它的孢囊梗分枝很多,分枝成堆且指向不同的方向,泡囊典型地呈多角形,内外璧不重合。这三个种首次发表时以及后来经过不同作者重新研究后发表的描述及绘图均过于简单而未能充分显示出它们的区别性特征,因此我们在本文中再次作了描述并再次提供了详尽的线条图和显微照相。     

A CYTOCHEMICAL STUDY OF ACID PHOSPHATASE AND NUCLEASE ACTIVITY IN THE PLASTID OF CLOSTERIUM ACEROSUM1

Acid phosphatase activity in Closterium acerosum has been studied using the Gomori and the azo dye procedures. A modification of the Gomori method was used for detecting the distribution of acid nucleases. The plastid is the major site of acid phosphatase activity luhich may be primarily within pyrenoids, between pyrenoids, or throughout the plastid. The Gomori procedure showed activity within the pyrenoids or in the central core of the plastid, whereas the azo dye method showed activity throughout with an occasional tendency to be localized near the ends. No other cytoplasmic activity was ob-served but evidence for occasional activity in the nucleus is presented. Alkaline phosphatase could not be detected. Acid nuclease activity, which results in the degradation of DNA, RNA, and RNA-Core, has been found in both the nucleus and the plastid. Plastid activity is heat labile, whereas nuclear activity is only slightly diminished after 5 min at 100 C. The results arc interpreted as indicating at least 2 acid phosphatases and 2 nucleases in C. acerosum. The findings are discussed with respect to the distribution of similar enzymes in other organisms.     

Revision of the genus Absidia (Mucorales, Zygomycetes) based on physiological, phylogenetic, and morphological characters; thermotolerant Absidia spp. form a coherent group, Mycocladiaceae fam. nov.

The genus Absidia comprises ubiquitously distributed soil fungi inhabiting different growth temperature optima ranging from 20–42 °C. Some of the mesophilic species are important biotechnologically in the biotransformation of steroids or as producers of rennin-like components, whereas species with higher growth temperature optima are of clinical relevance as opportunistic human pathogens. The aim of this study was to investigate the phylogenetic relationships between these species and to establish a revision of their systematics. For this purpose single and combined genealogies based on distance, MP, ML, and Bayesian analyses of aligned nucleotide sequences of the nuclear-encoded genes for actin (act) and for the 5.8S ribosomal RNA flanked by the ITS regions 1 and 2 (comprising 807 and 828 characters, respectively) of 16 Absidia species were reconstructed. The phylogenetic reconstructions suggest a trichotomy of the Absidia genus consisting of a mesophilic, a fast-growing thermotolerant, and a slowly-growing mycoparasitic Absidia group. The trichotomous phylogenetic grouping is concordant with the morphology of the zygospores, which are zygotes resulting from sexual conjugation between two compatible mating partners. Whereas the mesophilic group comprises the majority of absidiaceaeous species forming sterile hair-like, mycelial appendages on the suspensors of their zygospores, the thermotolerant group is characterised by the formation of smooth-walled zygospores, and the mycoparasitic group, namely Absidia parricida and A. zychae, by Mucor-like rough-walled zygospores. Based on the phylogenetic coherence of mesophilic and thermotolerant Absidia species, we propose that the two groups are separated into two distinct genera, Absidia for the mesophilic Absidia species resembling the Absidiaceae and Mycocladus for the thermotolerant species A. corymbifera, A. blakesleeana and A. hyalospora. Because Mycocladus is physiologically, phylogenetically, and morphologically distinct from the Absidiaceae s. str. we suggest that they are classified as a separate family, Mycocladiaceae fam. nov., which comprises the three species M. corymbifer, M. blakesleeanus and M. hyalospora.     

Molecular relationships between closely related strains and species of nematodes

Summary Electrophoretic comparisons have been made for 24 enzymes in theBergerac andBristol strains ofCaenorhabditis elegans and the related species,Caenorhabditis briggsae. No variation was detected between the two strains ofC. elegans. In contrast, the two species,C. elegans andC. briggsae exhibited electrophoretic differences in 22 of 24 enzymes. A consensus 5S rRNA sequence was determined forC. elegans and found to be identical to that fromC. briggsae. By analogy with other species with relatively well established fossil records it can be inferred that the time of divergence between the two nematode species is probably in the tens of millions of years.The limited anatomical evolution during a time period in which proteins undergo extensive changes supports the hypothesis that anatomical evolution is not dependent on overall protein changes.     

Chromosomenverhältnisse,konstitutives Heterochromatin und Geschlechtsbestimmung bei einigen Arten der GattungChrysomya (Calliphoridae,Diptera)

Compared with the strongly monogenic blowfliesChrysomya albiceps andC. rufifacies investigated previously, the closely related speciesC. chloropyga, C. putoria andC. varipes turned out to be amphogenic, i.e. each female of these species produces both sexes in nearly equal frequencies.C. chloropyga, C. putoria andC. varipes possess similar karyotypes consisting of five pairs of long identifiable autosomes and one pair of smaller sex chromosomes, which are homomorphic (XX) in the female and heteromorphic (XY) in the male. In all species the Y is shorter than the X. Among the cytologically known amphogenicChrysomya speciesC. varipes has the smallest heterochromosomes. Male meiosis inC. chloropyga, C. putoria andC. varipes is achiasmatic. During mitosis the autosomes show a high degree of somatic pairing but heterochromosomes, which form a heteropyknotic mass in the interphase nuclei, usually separate in the course of early mitotic prophase. A comparative study of C-banding patterns in mitotic and meiotic chromosomes ofC. chloropyga, C. putoria, C. varipes andC. rufifacies revealed that constitutive heterochromatin is present in almost all chromosomes. In the five pairs of large chromosomes it is confined to short regions adjacent to the kinetochores. An interstitial C-band has been observed only in the shorter arm of autosome no. 3 inC. putoria. With the exception of the tiny Y chromosome ofC. varipes, which seems to be completely euchromatic, all the other sex chromosomes of the amphogenicChrysomya species as well as the small chromosome no. 6 ofC. rufifacies possess longer or shorter C-segments. The karyotype ofC. varipes resembles closely that of the monogenic speciesC. albiceps andC. rufifacies thus indicating that the monogenic species could have evolved from an ancestral species closely related toC. varipes. The occurrence of two exceptional XO females and one exceptional XXY male among normal XX and XY animals in a wild stock ofC. chloropyga demonstrates that the Y chromosome of C. chloropyga and most probably of all amphogenicChrysomya species bears an epistatic male determining factor.

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Herrn Professor Dr. G. Krause zum 70. Geburtstag gewidmet     

Karyosystematic studies on threeCrepis species (Asteraceae) endemic to Greece

This work examines the cytogeographical distribution, the morphological characters, and the karyotypes of threeCrepis species endemic to Greece (C. sibthorpiana, C. incana, andC. heldreichiana). C. sibthorpiana is diploid (2n = 2x = 8),C. incana is diploid (2n = 2x = 8) and tetraploid (2n = 4x = 16, 17), andC. heldreichiana is always dekaploid (2n = 10x = 40). The Giemsa positive bands, usually pairs of dots, are mainly centromeric inC. incana, while they are terminal inC. sibthorpiana (on the short arm of all chromosomes) and inC. heldreichiana (on both arms of all chromosomes). Intercalary C-bands are scarce and usually variable within karyotypes, individuals, and species. The most variable karyotype both in Feulgen and Giemsa preparations is that ofC. incana, in which also supernumerary chromosomes were observed, which are polysomic to standard set members. On the basis of morphological and karyological data the evolutionary relationships among the threeCrepis taxa are discussed.     

THE ZYGOSPORIC STAGE OF THE GENUS PIRELLA (MUCORACEAE)

Hesseltine , C. W. (Northern Utilization Research and Development Division, Peoria, Ill.) The zygosporic stage of the genus Pirella (Mucoraceae). Amer. Jour. Bot. 47(3) : 225—230. Illus. 1960.–Pirella circinans Bainier has been found to be heterothallic. The zygospores, which have never before been reported, are formed when + and – strains are mated at 15°C. on such media as tomato paste-oatmeal or potato dextrose agars. Zygospores are formed in a diffuse manner in the aerial mycelium and are similar to those found in the genus Mucor. The single species, P. circinans, is described on a synthetic medium and its synonyms are listed. It is believed to represent a genus between Mucor and the family Thamnidiaceae.