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.     

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.     

MORPHOLOGICAL VARIABILITIES OF THREE CLOSELY RELATED MATING GROUPS OF CLOSTERIUM EHRENBERGII MENEGHINI (CHLOROPHYTA)1

The ranges of morphological variabilities in vegetative cells of three closely related mating groups of Closterium ehrenbergii Meneghini were statistically analyzed, having been grown under standard and uniform culture conditions, using Group A clones from Japan and Australia, Group B clones from Japan and Taiwan and Group H clones from Nepal. Significant differences in the morphological characters were not recognized between the two complementary mating types in any of the three groups. It has been shown that cells of Group A are smallest (mean width 50 μm and mean length 250 μm) and cells of Group B are largest (mean width 67 μm and mean length 404 μm), while cells of Group H are intermediate (mean width 57 μm and mean length 333 μm). There are considerable differences in the mean cell size between the three mating groups, although some intergrading clones were recognized. Degrees of the intergrading overlap were shown to be small between the sympatric groups (A and B) and large between the two pairs of allopatric groups (A and H & B and H). It has been shown that cells of Group A are shorter and much more curved than cells of the other two mating groups. Cells of Groups B and H are slender and less curved. It has also been shown that the ranges in cell size of each mating group are smaller than those currently accepted for C. ehrenbergii.     

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.     

DIPLOIDY IN DNA CONTENT IN VEGETATIVE CELLS OF CLOSTERIUM EHRENBERGII (CHLOROPHYTA)1

DNA content of the nucleus in the placoderm desmid, Closterium ehrenbergii Meneghini was measured throughout the life cycle by epifluorescence microspectrophotometry after DNA specific dye [4′,6-diamidino-2-phenylindol (DAPI)] staining. Postulating a mean DNA content of gamete nuclei as 1C, the nucleus of a newly divided vegetative cell was 2C. Most vegetative cells in the stage of exponential growth had a DNA content from 2C to 4C, while most in stationary phase, with the highest frequency of zygote formation, were 2C. They became pre-gametes (2C) upon mixing two heterothallic strains. Four gametes were made by a DNA reduction division of each pre-gamete cell. Therefore, there was a nonmeiotic DNA reduction stage by one half. During germination, the zygote underwent meiosis to produce two gones, each of which contained one surviving nucleus (large nucleus) and one degenerating nucleus (small nucleus). The DNA content of these four nuclei was 1C basically. The DNA of the surviving nucleus duplicated to 2C and further quadruplicated to 4C without cell or nuclear division. These two 4C gones had different cell morphology from ordinary vegetative cells. After the first cell division following meiosis, each gone produced two vegetative cells in which the DNA content became 2C to 4C again.     

PLASMOLYSIS,HECHTIAN STRAND FORMATION,AND LOCALIZED MEMBRANE‐WALL ADHESIONS IN THE DESMID,CLOSTERIUM ACEROSUM (CHLOROPHYTA)1

Closterium acerosum Ehrenberg (Chlorophyta) produced a distinct network of thin cytoplasmic strands, or Hechtian strands, upon controlled plasmolysis in a sucrose solution. The strands persisted for 30 min or longer and could be visualized with both LM and EM. Near the plasma membrane of the polar zones of plasmolyzing protoplasts, the strands formed a “lattice”‐like arrangement with interstrand spacing of 120–130 nm. The strands terminated at the fibrous zone of the inner cell wall stratum. Although actin cables could be found attached to the plasma membrane upon rhodamine phalloidin labeling of membrane ghosts, neither microfilaments nor microtubules were found in Hechtian strands at any stage of development. The formation of strands was not disrupted by centrifugation at 8000 g or by repeated cycles of plasmolysis‐deplasmolysis. Application of microtubule‐ or microfilament‐affecting agents or various proteolytic/polysaccharide‐degrading enzymes did not disrupt the formation of strands. Cold treatment of cells resulted in the formation of Hechtian strands.     

ANALYSIS OF GAMETIC PROTOPLAST RELEASE IN THE CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE COMPLEX (CHLOROPHYTA)1

A biologically active glycoprotein (protoplast-release-inducing protein; PR-IP), which induces the release of gametic protoplasts from mating type minus (mt^-) cells of the Closterium peracerosum-strigosum-littorale complex, was prepared from a medium in which mt^- and mt⁺ cells had been previously incubated together. The process of PR-IP-inducing protoplast release was analyzed. Induction of protoplast release was dependent upon the duration of both PR-IP treatment and preincubation in nitrogen-deficient mating medium before PR-IP treatment. Low cell density in the preculture stage had a significant stimulative effect upon the induction of protoplast release. Light was necessary for protoplast release, especially just before PR-IP treatment. Chloramphenicol and 3-(4-chlorophenyl)-1,1-dimethylurea (CMU) exerted inhibitory effects on protoplast release, especially when they were applied to the preculture stage but not when they were applied to the protoplast-releasing stage after the PR-IP treatment. We suggest that preculture at a low cell density under continuous light conditions that may cause metabolic changes in the chloroplast is a very important stage for gametic protoplast release in this Closterium.     

DETECTION AND EVALUATION OF A NOVEL SEXUAL PHEROMONE THAT INDUCES SEXUAL CELL DMSION OF CLOSTERIUM EHRENBERGII (CHLOROPHYTA)1

Mating type-plus (mt⁺; NIES-228) cells of Closterium ehrenbergii undergo a division to form gamete-shaped cells. This cell division is induced by a substance produced by mating type-minus (mt^?; NIES-229) cells. Light and the presence of mt⁺ cells enhanced production of the substance. The active substance is heat labile and has an apparent molecular mass of 20 kDa. From these results, we conclude that the substance is a novel, proteinaceous sexual pheromone involved in reproduction of Closterium ehrenbergii.     

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.     

MONOPHYLY OF THE GENUS CLOSTERIUM AND THE ORDER DESMIDIALES (CHAROPHYCEAE, CHLOROPHYTA) INFERRED FROM NUCLEAR SMALL SUBUNIT rDNA DATA

We newly sequenced the nuclear-encoded small subunit (SSU) rDNA coding region for 21 taxa of the genus Closterium. The new sequences were integrated into an alignment with 13 known sequences of conjugating green algae representing six traditional families (i.e. Zygnemataceae, Mesotaeniaceae, Gonatozygaceae, Peniaceae, Closteriaceae, and Desmidiaceae) and five known charophycean sequences as outgroups. Both maximum likelihood and maximum parsimony analyses supported with high bootstrap values one large clade containing all placoderm desmids (Desmidiales). All the Closterium taxa formed one clade with 100% bootstrap support, indicating their monophyly, but not paraphyly, as suggested earlier. As to the taxa within the genus Closterium , we found two clades of morphologically closely related taxa in both maximum likelihood and maximum parsimony trees. They corresponded to the C. calosporum species complex and the C. moniliferum-ehrenbergii species complex. It is of particular interest that the homothallic entity of C. moniliferum v. moniliferum was distinguished from and ancestral to all other entities of the C. moniliferum-ehrenbergii species complex. Superimposing all 50 charophycean sequences on the higher order SSU rRNA structure model of Closterium , we investigated degrees of nucleotide conservation at a given position in the nucleotide sequence. A characteristic "signature" structure to the genus Closterium was found as an additional helix at the tip of V1 region. In addition, eight base deletions at the tip of helix 10 were found to be characteristic of the C. calosporum species complex, C. gracile , C. incurvum , C. pleurodermatum , and C. pusillum v. maius. These taxa formed one clade with an 82% bootstrap value in maximum parsimony analysis.     

BIOCHEMICAL, PHYSIOLOGICAL, AND MOLECULAR ANALYSIS OF SEXUAL ISOLATION IN THE SPECIES COMPLEX CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE (CHLOROPHYTA)1

Closterium strains obtained from Japan ( NIES-64 and -65 ) and Nepal ( NIES-67 and -68 ) have been classified as the same taxonomic species; however, they are sexually isolated from each other. When NIES-64 and -65 cells were separately incubated in a medium in which both strains had previously been cultured together, release of protoplasts from both strains was observed. We suggest that factors responsible for the release of protoplasts from cells of both NIES-64 and -65 are produced in a mixed culture of these cells and function during conjugation. These factors, however, had no effect on the release of protoplasts from cells of strains NIFS-67 or -68. Alternatively, a protein that is responsible for the release of protoplasts from cells of NIES-68, called the protoplast-release-inducing protein ( PR-IP ), had no effect on the release of protoplasts from cells of strains NIES-64 or -65. When the media obtained from the culture of NIES-64 and -65 cells at various mixing ratios were analyzed by western blotting with antiserum to a 42-kDa subunit of PR-IP, no cross reaction was detected. In Southern hybridization analysis, no hybridizing band was observed when genomic DNAs of NIES-64 and -65 cells were probed with cDNAs encoding the two subunits of PR-IP. We suggest from these results that the factors responsible for the release of protoplasts from NIES-64 and -65 cells are not structurally similar to PR-IP. It is known that the release of PR-IP from NIES-67 cells can be induced by the action of another sex pheromone ( PR-IP inducer ) which is released by NIES-68 cells. In contrast, no protoplast-release-inducing activity was observed from either NIES-64 or -65 in a culture medium conditioned by opposite strains. We suggest that the conjugation systems employed by strains NIES-64/ NIES-65 and strains NIES-67 /NIES-68 differ, and we propose a possible mechanism of sexual isolation between these biological species .     

REPRODUCTIVE ISOLATION BY SEX PHEROMONES IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (ZYGNEMATALES,CHAROPHYCEAE)1

The Closterium peracerosum–strigosum–littorale (C. psl.) complex consists of unicellular algae and is known to be composed of several reproductively isolated mating groups of heterothallic strains. Group I‐E is completely isolated from mating groups II‐A and II‐B, groups II‐A and II‐B are partially isolated from each other, and only mating‐type plus (mt⁺) cells of group II‐A and mating‐type minus (mt^?) cells of group II‐B form zygotes. Based on the alignment of 1506 group I introns, significant phylogenetic relationships were observed among mating groups II‐A and II‐B, while mating group I‐E was distant from groups II‐A and II‐B. Sexual cell division in both mating‐type cells of group II‐A was stimulated in conditioned media in which cells of group II‐B had been cultured. When mt^? cells of group II‐B were stimulated in conditioned medium derived from group II‐A, mt⁺ cells of group II‐B did not respond to the conditioned medium. Conditioned media derived from group I‐E did not exhibit sexual cell division (SCD)–inducing activity against any strain except those within its own group. From the alignment of deduced amino acid sequences from orthologous protoplast‐release‐inducing protein (PR‐IP) Inducer genes, we detected a significant similarity among groups II‐A and II‐B, and mating group I‐E had low similarity to other mating groups. The existing degree of reproductive isolation can be partially explained by differences in molecular structures and physiological activities of sex pheromones of these heterothallic mating groups.     

A POLYPLOID SPECIES COMPLEX OF SPIROGYRA COMMUNIS (CHLOROPHYTA) OCCURRING IN NATURE 1

Investigations were conducted to determine whether ploidal changes found in laboratory cultures of Spirogyra also occur in nature. In an earlier study filament types identifiable as three different species (Spirogyra singu-laris Nordstedt, S. communis (Hassall)Kütz., S. fragilis Jao) arose from an original clonal culture through vegetative growth and sexual reproduction. These three “species” or filament groups differed in filament width, chloroplast number, zygospore size, and chromosome number. The differences in chromosome number represented a polyploid series of diploid (S. communis), triploid (S. fragilis), and tetraploid (S. singularis) forms in which width increased with ploidal level. The three width groups constituted a “species complex.” Five years after isolation of the original strain in this species complex, filaments corresponding to two of the width groups (S. singularis and S. communis) were found at the original collection site in the Santa Catalina Mountains in southern Arizona. The two field-collected groups were indistinguishable from the laboratory species complex in morphology and chromosome number. Homothallic conjugation within the two field width groups yielded progeny similar to those from homothallic conjugation of groups in the laboratory species complex. Filament widths of progeny were generally within the width limits of respective parental groups. The four possible intergroup crosses between the two laboratory and two field width groups yielded progeny similar to the wider parent (S. singularis) or the parent of intermediate width (S. fragilis). Progeny characteristics were determined by the width groups of parents, regardless of whether parents came from the laboratory or field. The similarities in morphology, chromosome numbers, and reproductive behavior of laboratory and field width groups imply that the laboratory species complex of S. communis has a natural counterpart in the field.     

Morphological and cytogenetic characteristics of intergroup hybrids between closely related mating groups of the Closterium ehrenbergii species complex (Desmidiales, Chlorophyta)

Five F₁ hybrid strains were established from rare survivors in intergroup crosses between three closely related mating groups (A, B and H) of the Closterium ehrenbergii Meneghini ex Ralfs species complex. Cell sizes of these five strains studied under our standard culture conditions were compared to those of their parental stains and also to the total range of cell-size variation in each mating group. All five F₁ strains were larger in mean cell width than their parental strains. In cell length, three of them were larger than, one was the same as, and the other was intermediate between their parental strains. Their cell sizes were always larger than the range of their respective smaller parental mating group and three of them were larger than the range of their respective larger parental mating group.     

OBSERVATIONS ON FILAMENT FORMATION IN MICRASTERIAS FOLIACEA (DESMIDIACEAE,CHLOROPHYTA)1

Filaments of Micrasterias foliacea Bailey were isolated from a sample of a Texas lake. Cultures were established and examined by light and scanning electron microscopy (SEM). Enzymatic removal of mucilage proved necessary to obtain well preserved cells for SEM investigation. The development of the overlapping polar lobes and the formation of the interlocking apical teeth are described. The importance of these, of mucilage and the primary wall for filament formation is discussed.     

A POLYPLOID SPECIES COMPLEX IN SPIROGYRA MAXIMA (CHLOROPHYTA,ZYGNEMATACEAE), A SPECIES WITH LARGE CHROMOSOMES1

A species complex in Spirogyra consists of the series of filament morphotypes of various ploidal levels arising from an original morphotype within a clonal culture or in nature. A clonal culture of filaments identified as Spirogyra maxima (Hassall) Kützing produced several morphotypes, i.e. filament types of distinctly different widths and ploidal levels. Banding patterns and satellites were visible on chromosomes stained at mitotic prophase and metaphase. The original culture of S. maxima contained filaments averaging 127 μ wide. Vegetative cells of the original culture contained six large chromosomes (>4 μ long), identifiable as three distinct pairs based on banding patterns and presence of satellites: (1) one pair of short chromosomes (ca. 5.0 μ); (2) one pair of long chromosomes (ca. 8.0 μm); and (3) a second pair of long chromosomes (ca. 9.0 μm) including a nucleolar organizing region and satellite. A larger morphotype averaging 175 μm in width contained 12 chromosomes, with two pairs of short chromosomes and four pairs of long chromosomes (satellites were usually indistinct). Aneuploid chromosome numbers ranging from 5 to 13 were observed in a few cells. Binucleate and trinucleate cells were also observed. A twobanded chromosome fragment was observed in a few cells with 6 chromosomes and a few cells with 12 chromosomes. The variety of morphotypes derived in this study could be identified as four different species of Spirogyra by conventional taxonomic criteria. The banding patterns and satellites on chromosomes suggest that three pairs of homologous chromosomes are present in filaments of the original clonal culture and that these filaments are themselves autopolyploid (diploid) descendants of ancestral form with a base chromosome number of x = 3.     

COMPOSITION AND SYNTHESIS OF THE PECTIN AND PROTEIN COMPONENTS OF THE CELL WALL OF CLOSTERIUM ACEROSUM (CHLOROPHYTA)

Closterium acerosum Ehrenberg (Chlorophyta) possesses a trilayered cell wall consisting of an outer tri-laminate stratum, a fibrous middle layer, and a thick inner fibrous layer. The outermost layer has a series of external parallel ridges and valleys. At the bases of the valleys are the wall pores, the site of mucilage release. Pure fractions of cell walls were isolated and inclusive pectin and wall protein fractions were extracted and characterized. Two pectin-like fractions were isolated: a CDTA-extracted polymer consisting of 60.1% galacturonic acid and a Na₂CO₃-extracted fraction consisting of 39.9% galacturonic acid. Two major protein fractions, one with a molecular mass of 23.5 kDa and one with a molecular mass of 28.5 kDa, were isolated by preparative gel electrophoresis. The former was glycine-rich, whereas the latter contained both significant amounts of glycine and hydroxyproline. Antibodies were raised to both the pectin fractions and the 23.5-kDa wall protein fraction. Immunocytochemical labeling of whole cells and wall fragments using antibodies raised against CDTA and Na₂CO₃ extracts showed that these pectin-like components were found throughout the wall strata and were more concentrated at the polar tips, the site of new wall synthesis in growing semicells. Immunogold labeling showed that their production was focused on the trans- Golgi network of the Golgi apparatus. Immunolabeling with an antibody raised against the 23.5-kDa glycine-rich wall protein showed close association of the protein with the wall pores. Similarly, immunogold labeling revealed that the protein was processed throughout the entire Golgi body even when large mucilage-containing vesicles were being processed. The roles of the secretory apparatus and putative spitzenkorper-like regions of the cell are discussed.     

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 SIMPLIFIED,RAPID METHOD FOR IDENTIFYING MATING TYPE IN ALGAE: RESULTS WITH THE PANDORINA MORUM (CHLOROPHYCEAE) SPECIES COMPLEX1

As a collection expands, increasing numbers of test crosses are required to identify new isolates in algal species containing numerous pairs of mating types. A short-cut is described that utilizes a reduced number of preliminary test crosses. The method was used successfully with the colonial green flagellate, Pandorina morum Bory de St. Vincent, to identify a new pair of milling types (syngen) from Japan and five new isolates from China of a previously known syngen. When tested on the 20 previously described syngens, it revealed one unexpected multi-clone effect on mating. The method should be valuable for identifying newly collected clones, examining potential, interactions among incompatible mating types, and for screening other highly specific inter-organism interactions such as host-parasite infections.