Mycosphere Doi 10.5943/mycosphere/4/2/6 The nuclear reproductive cycle in the myxomycetes: a review

Clark J1 and Haskins EF2

1Department of Biology, University of Kentucky, Lexington, Kentucky 40506 – [email protected] 2Department of Biology, University of Washington, Seattle, Washington 98195 – [email protected]

Clark J, Haskins EF 2013 – The nuclear reproductive cycle in the myxomycetes: a review. Mycosphere 4(2), 233–248, Doi 10.5943/mycosphere/4/2/6

Our understanding of the , ecology and population biology of myxomycetes has been enhanced by investigations of the nuclear reproductive cycle of taxa in this group. These studies have involved light microscopy, electron microscopy, DNA cytophotometric reports and genetic investigations. Heterothallism with its associated life cycle events of syngamy and meiosis is extensively reviewed as revealed by light microscopy, genetics and DNA spectrophotometric analysis of nuclear ploidy levels. Non-heterothallism, i.e., homothallism and apogamy, specifically automixis and its genetical and evolutionary significance is discussed. Nuclear division, chromosomal number and polyploidy in the myxomycetes is also detailed.

Key words – apogamy – automixis – heterothallism – homothallism – myxomycetes – nuclear- cycle – polyploidy

Article Information Received 11 January 2013 Accepted 30 January 2013 Published online 25 March 2013 Corresponding author: Jim Clark – [email protected]

Introduction and can form large populations; these An understanding of the nuclear amoeboflagellates then form multi-nucleate reproductive cycle, and its variations, can aid vegetative plasmodia which give rise to and inform our understanding of the taxonomy, sporangia to complete the cycle by producing ecology and population biology of myxo- spores. However, while the sequence of stages mycetes. Thus considerable effort has been in the reproductive cycle is consistent from one expended in the study of the occurrence and and isolate to the next, the nuclear location of these nuclear cycle events. While cycle can display considerable variability; that several excellent reviews (Gray & Alexopoulos can be divided into two major categories: 1968, Collins 1979, 1981) have covered parts heterothallism and non-heterothallism (see of this area, they are somewhat limited in review by Clark & Haskins 2010). In the availability and enough new information has heterothallic group, the amoeboflagellates accumulated to warrant an updated review. possess mating types and have a haploid- In the myxomycetes, the reproductive diploid sexual reproductive cycle, whereas the pattern set out by de Bary (1858) is followed non-heterothallic group is not known to by all known species: small generally wind- possess mating types and thus may be either borne spores germinate to release uninucleate sexual (homothallic) or nonsexual (apogamic). amoeboflagellate cells, which are vegetative While a nonsexual organism would not have a 233

Mycosphere Doi 10.5943/mycosphere/4/2/6 nuclear cycle since there would be no change Clastoderma debaryanum: plasmogamy of in ploidy during the reproductive cycle, the paired swarm cells (McManus 1961). occurrence of a haploid/diploid nuclear cycle in Comatricha nigra: a single pre-cleavage a non-heterothallic isolate would indicate that division (Lister 1893); meiosis I & II in it is homothallic. spores (von Stosch 1937). A list of reproductive nuclear cycle Comatricha typhoides: syngamy of paired reports is given in Table I. This list is divided swarm cells (Ross 1957); pre-cleavage into three groups: group A studies where the meiosis I & II (Wilson & Ross 1955). heterothallic/non-heterothallic nature of the Diachea leucopodia: syngamy of paired material was unknown; group B studies where swarm cells (Ross 1957). the isolates were known heterothallics; and Dictydiaethalium plumbeum: syngamy of group C studies where the isolates were known paired swarm cells (Ross 1957). non-heterothallics. The A group, which consist Dictydium cancellatum: no sporangial division mainly of earlier studies, displays rather (Jahn 1901); syngamy of paired swarm variable results and is probably of limited value cells and pre-cleavage meiosis I & II since the lack of background information (Ross 1961). makes it difficult to evaluate the validity and Didymium clavus: meiosis I & II in spores meaning of these results. (Dangeard 1947). Didymium difforme: plasmogamy of multiple Table 1 Reproductive nuclear cycle reports myxamoebae (Cienkowski 1863a, b); plasmogamy of multiple myxamoebae All names according to Martin & Alexopoulos and karyogamy in plasmodium just (1968) prior to sporulation (Skupienski 1926a, b); plasmogamy of paired A. Studies where only cytological swarm cells (Cayley 1929). information was available Didymium iridis (includes all D. nigripes): cinerea: karyogamy in plasmodium, syngamy of paired myxamoebae with and nuclear fusion with meiosis I & II pre-cleavage meiosis I & II (Cadman in spores Kranzlin (1907); synapti- 1931); plasmogamy of paired nemal complexes in young spores myxamoebae and karyogamy in (Aldrich & Mims 1970). plasmodium just prior to sporulation Arcyria incarnata: a single pre-cleavage (Schünemann 1930). division (Lister 1893); synaptinemal Didymium melanospermum: a single pre- complexes in young spores (Aldrich & cleavage division (Harper 1914). Mims 1970). Didymium squamulosum: syngamy of paired Badhamia utricularis: a single pre-cleavage myxamoebae (Ross 1957). division (Lister 1893); a single meiotic Enteridium rozeanum: syngamy of paired pre-cleavage division (Jahn 1933). swarm cells (Ross 1957). Ceratiomyxa fruiticulosa: plasmogamy of Enteridium sp. (Reticularia): a single pre- swarm cells and karogamy in plasmodia cleavage division (Harper 1914). with a single meiotic pre-cleavage Fuligo septica: a single pre-cleavage division division (Jahn 1908); multiple swarm (Harper 1914, Rosen 1893); syngamy cell plasmogamy and plasmodial of paired swarm cells with pre-cleavage karyogamy with meiosis I & II in meiosis I & II (Ross 1961). spores (Gilbert 1935); meiosis I & II in Hemitrichia stipitata: synaptinemal complexes spores (Olive 1907); plasmogamy of in young spores (Aldrich & Mims paired swarm cells (McManus 1958); 1970). meiosis I & II in spores (Wilson & Lamproderma arcyriodes: syngamy of paired Ross 1955); syngamy of swarm cells swarm cells with pre-cleavage meiosis I with meiosis I & II in spores (Sansome & II (Ross 1960). & Sansome 1961, Sansome & Dixon Lamproderma arcyrionema: pre-cleavage 1965). meiosis I & II (Wilson & Ross 1955). 234

Mycosphere Doi 10.5943/mycosphere/4/2/6 : syngamy of paired Stemonitis flavogenita: plasmogamy of swarm cells (Ross 1957); a single pre- myxamoebae and or swarm cells cleavage division (Harper 1914); pre- (Koevenig 1961, 1964). cleavage meiosis I & II (Wilson & Ross Stemonitis fusca: plasmogamy between a 1955). myxamoeba and a swarm cell Lycogala exiguum: a single pre-cleavage (McManus 1961, Benedict 1962); a division (Conrad 1910). single pre-cleavage division (Bisby Metatrichia vesparium (Hemitrichia 1914); pre-cleavage meiosis I & II vesparium): syngamy of paired swarm (Koevenig 1964); syngamy of paired cells (Ross 1957); pre-cleavage swarm cells (Ross 1957). meiosis I & II (Wilson & Ross 1955). Stemonitis herbatica: syngamy of paired Mucilago crustacean: pre-cleavage meiosis I & swarm cells (Ross 1957); synaptinemal II (Schure 1949). complexes in young spores (Aldrich & Perichaena vermicularis: pre-cleavage meiosis Mims 1970). I & II or in spores depending upon Stemonitis pallida: pre-cleavage meiosis I & II sporulation conditions (Ross 1967b). (Wilson & Ross 1955). Physarella oblonga: a single pre-cleavage Stemonitis nigrescens: syngamy of paired division (Bisby 1914); syngamy of swarm cells (Ross 1957). paired myxamoebae with pre-cleavage botrytis (fragilis): a single pre-cleavage meiosis I & II (Ross 1961). division (Lister 1893). Physarum bogoriense: synaptinemal com- Trichia persimilis: nuclear fusion in plexes in young spores (Aldrich & sporangium with meiosis I & II at spore Mims 1970). germination (Kranzlin 1907); syngamy Physarum cinereum: synaptinemal complexes of paired swarm cells (Ross 1957); pre- in young spores (Aldrich & Mims cleavage meiosis I & II (Wilson & Ross 1970). 1955). Physarum didermoides: syngamy of paired Trichia decipiens (fallax): a single pre- myxamoebae with one possible pre- cleavage division (Strasburger 1884); cleavage meiotic division (Jahn 1911); nuclear fusion in sporangium with one pre-cleavage division (Schure meiosis I & II at spore germination 1949). (Kranzlin 1907); a single pre cleavage Physarum gyrosum: syngamy of myxamoebae division (Lister 1893). and or swarm cells with pre-cleavage Trichia varia: a single pre-cleavage division division figures (Koevenig 1964). (Jahn 1933). Physarum leucophaeum: a single pre-cleavage Tubifera ferruginosa: synaptinemal complexes division (Lister 1893). in young spores (Aldrich & Mims Physarum oblatum: syngamy of paired 1970). myxamoebae cells (Ross 1957). Tubifera microsperma: pre-cleavage meiosis I Physarum polycephalum: syngamy of swarm & II (Ross 1961). cells with a single pre-cleavage division (Howard 1931); a single pre- B. Studies involving known heterothallic cleavage division (Dalleux 1940); isolates syngamy of swarm cells with pre- Didymium iridis (including all D. nigripes): cleavage meiosis I & II (Ross 1961). syngamy of paired swarm cells with Physarum psittacinum: karyogamy in meiosis I & II in spores (von Stosch plasmodium (Prowazek 1904). 1935); microspectrophotometry Reticularia lycoperdon: syngamy of paired showing pre-cleavage meiosis I & II swarm cells with pre-cleavage meiosis I (Therrien 1966); syngamy of & II (Wilson & Cadman 1928). myxamoebae (Ross 1967a). Stemonitis axifera: syngamy of paired swarm CR 5 strain: microspectrophotometry of DNA cells (Ross 1957). levels in amoeboflagellates and

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Mycosphere Doi 10.5943/mycosphere/4/2/6 plasmodium indicated a haploid/diploid spores (von Stosch et al. 1964); cycle (Collins & Therrien 1976). synaptinemal complexes in young CR 25 strain: microspectrophotometry of DNA spores (Aldrich 1967); light micro- levels in amoeboflagellates and scopy, electron microscopy and plasmodium indicated a haploid/diploid microphotospectrometry determinations cycle with polyploidy in one found a basic pattern of meiosis in amoeboflagellate clone (Collins & young spores with all but one resulting Therrien 1976). nucleus disintegrating, but there Hon 1 strain: pre-cleavage synaptinemal were considerable variation in the complexes (Carroll & Dykstra 1966); details including some pre-cleavage synaptinemal complexes in young meiosis (Laane & Haugli 1976). spores (Aldrich & Carroll 1991) with Physarum pusillum microphotospectrometry Carroll and Dykstra’s report probably showing pre-cleavage meiosis I & II due to abnormal cleavage (examined (Therrien 1966). old blocks); microspectrophotometry of DNA levels in amoeboflagellates and C. Studies involving known non- plasmodium indicated a haploid/diploid heterothallic isolates cycle (Collins & Therrien 1976). Didymium difforme: pre-cleavage meiosis I Ky 1 strain: microspectrophotometry of DNA with meiosis II at spore germination levels in amoeboflagellates and (Cayley 1929). plasmodium indicated a haploid/diploid Didymium iridis (including all D. nigripes): cycle (Collins & Therrien 1976). possible pre-cleavage meiosis I & II Pan 1 strain: microspectrophotometry of DNA figures (Schünemann 1930) and pre- levels in amoeboflagellates and cleavage figures in same isolate (von plasmodium indicated a haploid/diploid Stosch 1935). cycle (Collins & Therrien 1976). Kerr’s strain: plasmogamy of paired swarm Pan 2 strain: microspectrophotometry of DNA cells (Kerr 1961); micro- levels in amoeboflagellates and spectrophotometry indicated haploid plasmodium indicated a haploid/diploid amoebae and diploid zygotes (Therrien cycle (Collins & Therrien 1976). 1966); presumed pre-cleavage meiotic Pan 3 strain: microspectrophotometry of DNA figures with no synaptinemal com- levels in amoeboflagellates and plexes (Schuster 1964). plasmodium indicated a haploid/diploid Cal 1 strain: microspectrophotometry of DNA cycle with polyploidy in one levels in amoeboflagellates and amoeboflagellate clone (Collins & plasmodium indicating a haploid- Therrien 1976). diploid cycle (Collins & Therrien Physarum flavicomum: meiosis I & II with 1976). synaptinemal complexes in young Ga 1 strain: microspectrophotometry of DNA spores with some disintegrating levels in amoeboflagellates and nuclei (Aldrich 1976); microspectro- plasmodium indicating a diploid/diploid photometry showing pre-cleavage cycle (Collins & Therrien 1976). meiosis I & II (Therrien 1966). Ha 1 strain: microspectrophotometry of DNA Physarum globuliferum: synaptinemal levels in amoeboflagellates and complexes in young spores (Aldrich plasmodium indicating a diploid/diploid 1976). cycle with polyploidy (Collins & Physarum polycephalum: plasmogamy and Therrien 1976). karyogamy occurs between any two Mo 1 strain: microspectrophotometry of DNA myxamoebae but plasmodial levels in amoeboflagellates and differentiation only occurs when they plasmodium indicating a haploid- are heteroallelic for mating type (Bailey diploid cycle (Collins & Therrien et al. 1990); one meiotic pre-cleavage 1976); synaptinemal complexes in division (Guttes et al. 1961); meiosis in young spores (Sherman & Mims 1985). 236

Mycosphere Doi 10.5943/mycosphere/4/2/6 Pan 1 strain: microspectrophotomertry of DNA cytophotometric studies of this strain, found heterothallic mating type which normally displayed a diploid- amoebal clones and diploid diploid amoeboflagellate plasmodium plasmodial forming lines (Yemma et al. cycle, found haploid amoeboid clones 1983). (Collins & Tang 1988) which were Pan 4 strain: microspectrophotometry of DNA produced during vegetative growth, that levels in amoeboflagellates and had mating types and could fuse to plasmodium indicating a haploid- form diploid plasmodia (Collins et al. diploid cycle (Collins & Therrien 1976) 1983a). however (Collins 1980, Collins et al. 1983, Collins & Gong 1985) found that Heterothallism this strain contained both plasmodial Heterothallic myxomycete isolates have forming diploid amoeboflagellates and a sexual cycle in which the haploid haploid amoeboflagellates with two amoeboflagellate stage undergoes syngamy to different mating types (an apparent produce a diploid zygotic cell that develops conversion from the diploid strain into a diploid plasmodium. Functional during long term clonal culture). syngamy with the production of a diploid Pan 5 strain: microspectrophotometry of DNA plasmodium requires that the two amoebo- levels in amoeboflagellates and flagellate cells involved, each carry a different plasmodium indicating a haploid- allele of the multiple allelic system (Collins diploid cycle (Collins & Therrien 1963). During sporulation the diploid nuclei 1976). undergo meiosis in the young developing Ph-1 strain: microspectrophotometry of DNA spores and each haploid nucleus receives one levels in amoeboflagellates and of the mating type alleles (Collins 1961). plasmodium indicating a diploid/diploid cycle (Therrien & Yemma 1974); Syngamy haploid sexual clones isolated from the Syngamy, in the myxomycetes, consists apogamic strain (Yemma et al. 1980); of two related events, plasmogamy (the fusion synaptinemal complexes in young of the gametic cells prior to nuclear fusion) and spores (Aldrich & Carroll 1971). karyogamy (the fusion of the haploid nuclei to Didymium squamulosum: pre-cleavage division form the diploid zygotic nucleus), and was (von Stosch 1937). reported to occur between two amoebo- Echinostelium minutum: microspectro- flagellates in several very early studies. photometry of DNA levels in amoebo- However, considerable controversy occurred in flagellates and plasmodium indicating a regards to the number of amoeboflagellates diploid/diploid cycle with polyploidy in involved and their state (myxamoebae or one line (Haskins & Therrien 1978); flagellate swarm cells) at the time of syngamy. synaptinemal complexes in young Several reports indicated that plasmogamy spores (Haskins et al. 1971); micro- involved the possible coalescence of a number spectrophotometry of a division within of myxamoebae prior to plasmodial formation the developing spore did not show the (Cienkowski 1863a, b, Skupienski 1926a, b, reduction in DNA levels expected Wilson & Cadman 1928, Gilbert 1935, during meiosis (Therrien & Haskins Wollman 1966), while others (Jahn 1911, 1981). Cayley 1929, Schünemann 1930, McManus Physarum cinereum: a single pre-cleavage 1961) reported only paired fusions. There division (von Stosch 1935). were also differing reports of plasmogamy Physarum pusillum: synaptinemal complexes occurring only between myxamoebae (Jahn in young spores (Aldrich & Mims 1911), only between swarm cells (Wilson & 1970). Cadman 1928, Howard 1931, von Stosch 1935, Stemonitis flavogenita: synaptinemal com- McManus 1961) or between either (Ross 1957, plexes in young spores of the CR-1 Koevenig 1961, 1964, Kerr 1961). These early strain (Gaither & Collins 1984), studies also generally reported that karyogamy 237

Mycosphere Doi 10.5943/mycosphere/4/2/6 occurred shortly after plamogamy (Jahn 1911, examined in Didymium iridis by Clark (1984) Wilson & Cadman 1928, Cadman 1931, using three clones carrying different Howard 1931, von Stosch 1935, Gilbert 1935, plasmodial incompatibility genes (see review Ross 1957, Koevenig 1961), although others by Clark & Haskins 2011). These clones were reported that karyogamy was delayed and selected so that a plasmodium resulting from a occurred in the plasmodium (Prowazek 1904, multiple fusion event would display a Olive 1907, Kranzlin 1907, Jahn 1908, particular plasmodial fusion phenotype only if Skupienski 1926, 1927, Schünemann 1930). genetic information from all three clones were However, by the end of this early period, a present. The resulting plasmodia often carried general consensus, relying heavily on careful this phenotype, but over a period of time in studies by Ross and Koevenig, considered that culture the plasmodia generally changed to a sexual myxomycetes underwent plasmogamy different phenotype (apparently the polypoid between myxamoebae or swarm cells in pairs nuclei shed some chromosomes). with some species favoring one method, but Time-lapse micro-cinematography of most employing either according to live immunofluorescence stained amoebo- circumstances, and that karyogamy occurred flagellates of Physarum polycephalum (Bailet shortly after plasmogamy. et al. 1990) showed that the mating locus The discovery of sexual heterothallism controlled plasmodial differentiation and not in Didymium iridis (Collins 1961) and cell or nuclear fusion. Apparently any two Physarum polycephalum (Dee 1960) opened up competent amoeboflagellates can undergo a new avenue for syngamy research in the plasmogamy and karygamy, but plasmodial myxomycetes since it allowed researchers to form only in the mating type heteroallelic make controlled crosses of known sexual pairings, while the fused cells and nuclei of the strains. Ross (1967) using a heterothallic strain homoallelic pairings apparently separate or of Didymium iridis confirmed the general degenerate without forming plasmodia. consensus arrived at by the earlier studies; and However, there may be rare cases of with others (Ross & Cummings 1970, Ross & illegitimate plasmodial formation involving Shipley 1973, Albert & Therrien 1985) went homoallelic pairings since clonal mating-type on to show that amoeboflagellates must first isolates will occasionally produce plasmodia become competent before they can fuse and without crossing (Collins & Ling 1968, that competency required that the cells reach a Anderson & Truitt 1983). Termed selfing, minimum population density. Both Didymium these plasmodia are haploid (Yemma & iridis and Pysarum polycephalum mating type Therrien 1972) and grow poorly, but will amoeboflagellate clones were grown separately sometimes sporulate to produce amoebo- (Shipley & Holt 1982) to the competency flagellates having only the original mating type density before mixing, to demonstrate that both (thus they are not due to some type of mating types must be competent before accidental contamination mating). The syngamy would occur and that competency Colonia isolate of Physarum polycephalum is involved the production of an extracellular an extreme example of this selfing behavior; in inducer material released by the growing this isolate clonal amoeboflagellate populations amoeboflagellates (Youngman et al. 1977, have mating types and cross (Poulter & Honey Pallotta et al. 1979, Nader et al. 1984, Albert & 1977) to produce diploid plasmodia, but they Therrien 1985). Ross & Cummings (1970) can also produce haploid plasmodia when they also observed that the mixing of mating type have no mating partner (Cooke & Dee 1974, clones after they had reached maximum Anderson et al. 1976, Mohberg 1977). During competency, produced a rapid fusion of this process, some haploid cells undergo an multiple amoeboflagellates that formed extended cell cycle during which they become polyploid nuclei in large synctia. This committed and gain plasmodial characteristics behavior, under very artificial conditions, (Bailey 1995). Therefore, the Colonia isolate is would explain the occasional observations of heterothallic and faculatively apomictic and not multiple cell fusions observed in earlier homothallic as it is often designated. Thus, studies. The fate of these polyploid nuclei was syngamy in the myxomycetes, as presently 238

Mycosphere Doi 10.5943/mycosphere/4/2/6 understood, occurs between pairs of competent in the cleaved spores, or meiosis occurs in the amoeboflagellates with karyogamy following mature spores just prior to germination. plasmogamy, however plasmodial different- However, since only Kranzlin (1907) and to a iation, in general, only occurs when the certain extent Cayley (1929) supported the amoeboflallates are heteroallelic for the mating mature spore model, the major disagreement locus. While this sexual fusion usually results involves whether meiosis occurs before or after in a diploid plasmodial nucleus, multiple cell spore cleavage. Light microscopic cytological fusions can occur under some unusual studies supported both the pre-cleavage (Jahn circumstances to produce generally temporary 1908, 1933, Wilson & Cadman 1925, polyploidy nuclei. While the many reports of Schünemann 1930, Cadman 1931, Schure plamogamy and karyogamy (Table I) may 1949, Wilson & Ross 1955, Kovenig 1964, serve to indicate a possible sexual cycle in Ross 1967a) and post-cleavage spore models those species and isolates, by themselves, they (Olive 1907, Gilbert 1935, von Stosch 1935, cannot serve as a definitive proof of sexuality, 1937, Gilbert 1947, Wilson & Ross 1955, since this requires evidence of a syngamy Sansome & Sansome 1961, Sansome & Dixon which produces a diploid nucleus that 1963, von Stosch et al. 1967). However, Ross, undergoes genetic segregation. a major proponent of the pre-cleavage model, also reported that he could shift the site of Meiosis meiosis from pre to post-cleavage in Meiosis consists of two connected Perichaena vermicularis by changing the nuclear divisions that reduce the chromosome sporulation conditions (Ross 1967b). number by one half. In meiosis I the DNA spectrophotometric studies, in chromosomes replicate, pair, undergo crossing known heterothallic isolates, supports a haploid over and then separate into separate nuclei; the ameoboflagellate diploid plasmodium nuclear replicated chromosomes then divide during cycle in Didymium iridis (Collins & Therrien meiosis II. Unfortunately, in the myxo- 1979), Physarum pussillum (Therrien 1966), P. mycetes, the nuclei and chromosomes are flavicomum (Therrien 1966), P. polycephalum extremely minute and adequate cytological (Cooke & Dee 1974, Laane & Haughi 1976), preparations and chromosome counts are and Stemonitis flavogenita (Collins & Tang difficult to obtain. Thus early investigators had 1988). Therrien (1966) also found DNA to rely on estimated chromosome counts and spectrophotometric evidence for pre-cleavage the occurrence of two nuclear divisions where meiosis in the Didymium iridis, Physarum apparent chromosome bridges or rings occur in flavicomum and P. pussilum isolates. the first division. Later workers were able to However, all electron microscopic studies on utilize several additional techniques such as known heterothallic isolates have found electron microscopy and DNA spectro- synaptinemal complexes and other evidence of photomectrics. Synaptinemal complexes, seen meiosis, only in the developing spores: in electron micrographs, are generally found Physarum flavicomum (Aldrich 1967) using the only with paired chromosomes and are thus same isolate studied by Therrien, P. gyrosum evidence for the occurrence of division I of (Aldrich 1967), P. polcephalum (Aldrich meiosis (Moses & Coleman 1964). Also, 1967), and Didymium iridis (Aldrich & Carroll careful comparative DNA level studies of the 1991). An earlier study on Didymium iridis by nuclei of different life cycle stages, determined Carroll & Dykstra (1966) which reported a pre- by spectrophotometrics, can be used to replace cleavage meiosis, was re-investigated by or supplement chromosome counts. Aldrich & Carroll (1991) and was found to be While almost all researchers agree due to an abnormal cleavage in the material (Table I) that meiosis takes place during used. In a study using coordinated light and sporulation, there is considerable disagreement electron microscopy, Laane & Haugli (1976) among workers on whether it precedes or reported that Physarum polycephalum follows spore cleavage. The three conclusions displayed a basic system with meiosis in the of the different researchers are that meiosis young spores with all but one of the resulting occurs prior to spore cleavage, meiosis occurs nuclei degenerating; however, many minor 239

Mycosphere Doi 10.5943/mycosphere/4/2/6 variations occurred, including some pre- Collins & Gong 1985, Collins & Tang 1988, cleavage meiosis and delayed nuclei Yemma et al. 1980) which added to the puzzle. degeneration. Since Aldrich (1967) was However, these convertants also provided a unable, in his studies, to modify the site of solution to the puzzle, since the most meiosis, by manipulating the environmental reasonable explanation of the reversion to conditions during sporulations, a controversy heterothallism is that the original isolates were still remains as to the exact site of meiosis. automictic. Since there are no generally However, the preponderance of evidence accepted definitions of automixis and related would seem to indicate that meiosis occurs terms (Maynard Smith 1978, Mogie 1986), we during the development of the young spore and will use the following description in regards to that three of the products of the meiosis the myxomycetes; in automixis the diploid degenerate to leave the spore uninucleate and nucleus in the spores undergo division I of haploid (Aldrich 1967, Laane & Haughi 1970), meiosis (synaptinemal complexes present), but although later mitotic divisions may produce the second division is suppressed to produce multinucleate spores. two diploid nuclei, or division II occurs with the immediate fusion of sister pro-nuclei to Non-heterothallism regenerate diploid nuclei. While there is no Cytophotometric DNA studies and direct evidence, in myxomycetes, for the electron microscopy seemed to have solved the occurrence of sister pro-nuclei fusion, there is puzzle concerning homothallism or apogamy in good evidence for the suppression of division the non-heterothallic isolates of myxomycetes: II in Echinostelium minutum (Therrien & some appeared to be homothallic and others Haskins 1981). Although either process would apogamic. Cytophotometric studies of seven retain some heterozygous loci during a Didymium iridis isolates (Therrien & Yemma particular division, the line would soon become 1974, Therrien et al. 1977) reported a haploid homozygous after a few generations due to amoeboflagellate diploid plasmodial cycle in inbreeding. Thus, the line would produce four isolates (Cal 1, Mo 1, Pan 4, Pan 5) and a genetically identical diploid nuclei in all of its diploid/diploid cycle in three other isolates (Ga spores, which then directly produces identical 1, Ha 1, Ph 1). Diploid/diploid cycles have diploid plasmodia. In morphologically com- also been demonstrated in Stemonitis plex organisms (most animals) with separate flavogenita strain CR 1 (Collins et al. 1983a) sexes, anisogametes, and a diploid only life and Echinostelium minutum (Haskins & cycle, automixis has serious difficulties, since Therrien 1978). Also an electron microscope it must occur by parthenogenesis in the female study (Sherman & Mims 1985) of the Mo 1 egg (sperm cannot differentiate) and the isolate of Didymium iridis, determined that resulting initial homozygousity often produces synaptinemal complexes occurred in this a homozygous recessive lethal condition (the isolate during spore formation. Thus, there recessive lethal alleles are normally protected appeared to be reasonable data that confirmed by heterozygousity). However, the myxo- that homothallism occurred in some non- mycetes with isogametes and an alteration of heterothalic isolates (Therrien et al. 1977, haploid and diploid vegetative stages, avoid Sherman & Mims 1985). However, many of the problems of automixis; recessive synaptinemal complexes were also un- lethal alleles are generally selected against in expectedly found in the presumably apogamic the haploid phase and any diploid spore is Didymium iridis Ph 1 isolate (Aldrich & capable of developing into the next stage Carroll 1971), the Stemonitis flavogenita CR 1 The apparent homothallism, in some isolate (Gaither & Collins 1984), the isolates having haploid DNA levels in their Echinostelium mimutum isolate (Haskins et al. amoeboflagellates (Therrien et al. 1977) could 1971) and a Physarum pusillum isolate be explained as revertant cultures. All of the (Aldrich & Mims 1970). Also, a number of non-heterothallic isolates, showing a haploid- non-heterothallic isolates were discovered that diploid cycle, that were studied were long term reverted to heterothallism (Collins & Therrien cultures, and unfortunately were not re-cloned 1976, Collins 1980, Collins et al. 1983a, b, from single amoebae just prior to DNA 240

Mycosphere Doi 10.5943/mycosphere/4/2/6 determinations, thus the possibility of mixed interfere with their reversion to heterothallism revertant amoeboflagellate cultures cannot be (Collins et al. 1983). dismissed. In a mixed culture the haploid form would be more prevalent since they grow Nuclear division, chromosome number and faster and in the large clonal populations polyploidy studied only a few diploids would be needed to As far as it is known, mitotic nuclear produce the non-heterothallic results, or the division in the uninucleate myxamoebae stage haploids could have functional mating types is astral in type with a centriole at each pole and cross to produce plasmodia. Therefore to and the nuclear membrane breaking down in demonstrate homothallism, in the myxo- late prophase (Koevenig 1964, Kerr 1967, Ross mycetes, one would need to find haploid 1967, Aldrich 1969, Hinchee & Haskins amoeboflagellates and diploid plasmodia in an 1980a). On the other hand, the synchronous isolate recently isolated from a single amoeba. mitotic divisions in the multinucleate Betterley & Collins’ (1983) studied 32 plasmodial stage lack centrioles and the nuclear non-heterothallic isolates of Didymium iridis, membrane does not break down during mitosis and found: that they were all genetically (Kerr 1976, Guttes et al. 1968, Hinchee & different except for several isolates that were Haskins 1980b). Meiotic nuclear divisions are collected in the same area. In addition, all of also intra-membranous and lack centrioles the spores from an isolate were genetically (Aldrich 1969). identical, and all attempts to inbreed these The presumed haploid chromosome isolated among themselves or with numbers reported for over 25 different species heterothallic isolates failed, also supported the of myxomycetes (see review by Collins 1979) automictic model of non-heterothallism. vary from 4 to 90 and they can vary widely for While it appears that automixis is the major the same species: 4 to 81 for Didymium iridis, cause of non-heterothallism, the occurrence of 6 to 87 for Fuligo septica, and 8 to 90 for strict apomixes (as seen in the haploid selfing Physarum polycephalum. Therefore, these of heterothallic clones) or homothallism cannot light microscope determinations are unreliable be ruled out as a minor cause of non- due to the small sizes of the nuclei and the heterothallism. Non-heterothallism could also chromosomes which can cause very difficult occur due to the inclusion of several meiotic technical problems (Mohberg 1977). This products carrying different mating types in the difficulty is well illustrated by Haskins (1976) same spore. However, while multinucleate who required the use of high voltage EM of spores, carrying one, two or four nuclei have serial sections to determine that there were 124 been reported to occur in many species of chromosomes in the plasmodial nuclei of myxomycetes (see review in Gray & Echinostelium minutum. Thus, reported Alexopoulos 1968), the expected mix of self- chromosome numbers are generally more of an fertile and self-sterile spores has not been approximation that an actual count. reported (Clark & Haskins 2010). Therefore, Early reports of polyploidy in the the most likely cause of these multinucleate myxomycetes were also based on light spores would be mitotic nuclear division, or in microscope chromosome counts (von Stosch the case of automixis both of the two identical 1935, Wilson & Ross 1955), which were rather diploid nuclei surviving in the spore. While difficult and unreliable due to their small size. automixis involves an abbreviated meiosis, no However, more recent studies have utilized haploid gametic stage is produced, and thus spectrophotometric measurements of DNA this form of reproduction, using a broad levels and genetic studies as well as the more definition, can be termed apogamic. Although classic light microscopic techniques. These non-heterothallic automicts are certainly studies have confirmed that polyploidy occurs derived from normal heterothallic isolates, and in Physarum polycephalum (Alder & Holt while some of them can revert back to 1975, Mohberg 1977), Didymium iridis heterothallism, it is highly likely that other (Collins & Therrien 1976, Mulleavy & Collins automicts have accumulated genetic and 1979) and Echinostelium mimutum (Haskins chromosomal abnormalities which would 1976) and that it is fairly common. The 241

Mycosphere Doi 10.5943/mycosphere/4/2/6 occurrence of these polyploids is, no doubt, chromosome and DNA level (derived from part of the reason for the variable chromosome crossing two 2N clones) were followed from counts that have been reported in the literature plasmodial formation to senescence, it could be for the same species. Polyploidy is especially shown that there was a general lost of DNA evident in myxomycetes that have been over time till the 2N level was reached, but that cultured for any extended period of time; for during senescence there was a new increase in example, high-resolution flow cytometry polyploidy levels (Clark 1989). This increase (Kubbies et al. 1986) on various cultured in polyploidy starting just prior to senescence strains of Physarum polycephalum found a is probably a reflection of a general loss of high degree of genomic size variations between plasmodial controls at this time (Clark & strains and mixoploidy (nuclei of multiple Hakim 1980, McCullough et al. 1973). ploidy levels) within the strains. Although polyploidy seems to be rather Mulleavy (1979, Mulleavy & Collins common in the myxomycetes, there appears to 1981), using myxamoebal size as in indicator, be mechanisms which control or alleviate the was able to isolate naturally occurring and problems produced by these extra chemically induced N, 2N, 4N and 8N chromosomes. amoeboflagellate clones from Didymium iridis mating type cultures. These polyploidy clonal Summary lines retained the ability to mate and produce The small sizes of the nuclei and plasmodia when crossed to the appropriate chromosomes, and the frequent occurrence of mating type. Crosses of a diploid myxamoebae polyploidy has made cytology rather difficult clone carrying two A1 mating types with one in the myxomyetes. However, much progress carrying two A5 mating types produced has been made and we now have the tetraploid plasmodia which displayed the information needed to understand their nuclear expected tetraploid ratio of 1 A1A1 : 4 A1A5 : 1 reproductive cycle. The cycle in the A5A5 after sporulation. The A1A1 and A5A5 heterothallic myxomycetes appears myxamoebae clones were self-sterile and complicated, due to the alternation of the crossed with other appropriate mating type amoeboflagellate and plasmodial vegetative clones. The A1A5 clones, on the other hand, stages, but, in general, it is a standard cycle produced plasmodia without the need for with a number of generally minor variations. crossing. Thus, they behaved like non- Amoeboflagellates become competent to heterothallics, however, this was only function as gametes when they reach a critical temporary since these diploid plasmodia density and produce an extracellular inducer produced normal haploid clones after factor. Apparently, any amoeboflagellate that sporulation (Mulleavy & Collins 1979). is competent can undergo plasmogamy and However, not all crosses produced plasmodia karyogamy with any other competent that segregated normally. When a particular amoeboflagellate, but true syngamy only polyploid clone (CR 2-25) was crossed to occurs when the two amoeboflagellates are haploid clones the resulting plasmodium heteroallelic for mating types; since these produced polyploid plasmodia which shed heteroallelic fusion develop into diploid many of its chromosomes and these ended up plasmodia, while homoallelic pairings and their with different DNA levels ranging from fused nuclei either separate or undergo haploid to the maximum expected polyploidy degeneration. In the heterothallic myxo- level of the cross (Therrien & Collins 1976, mycetes, meiosis occurs in the young Collins et al. 1978). Polyploidy, with the developing spores, although there appears to be uncovering of lethal alleles during segregation some possible variation in the timing such that could also explain the occurrence of skewed pre-cleavage meiosis may also occur. In any mating type segregation ratios reported in a case, degeneration of three of the meiotic number of other Didymium iridis isolates nuclear products apparently occurs to leave the (Clark & Landolt 1993) and in Comatricha spore uninucleate and haploid (later mitotic lurida (McGuinness & Haskins 1985). When divisions may produce multinucleate spores). plasmodia having an original tetraploid 242

Mycosphere Doi 10.5943/mycosphere/4/2/6 On the other hand, non-heterothallic adapted to find and quickly exploit scattered reproduction seemed quite confusing since, and ephemeral habitats, while the sexual without knowledge of the nuclear cycle during isolates maintain a long term ability to exploit reproduction, it was impossible to determine if a variety of habitats. An ability to switch the isolates were homothallic or apogamic. between modes would seem to provide the However, enough information has now species with some advantage; however, this accumulated to provide a basic hypothesis. ability seems to be easily lost due to polyploidy Most, if not all, non-heterothallics are and other aberrations that can occur during a automicts in which meiosis I occur in the number of generations without meiosis (Collins developing spore (or possibly pre-cleavage), et al. 1983). However, the high level of genetic but meiosis II does not take place, thus leaving diversity found between different apogamic the spore diploid and uninucleate after one of isolates (Betterley & Collins 1983) seems to the nuclei degenerates (multinucleate spores indicate that either apogamic lines are could occur due to both nuclei surviving or by continually produced at high frequencies from mitotic division in the mature spore), or sister heterothallic lines, and or that there is some pro-nuclei fuse during meiosis II to produce type of low level recombination occurring in diploid spores. Since, automixis is a form of the apogamic lines, similar to that seen in apogamy (broadly defined), it appears that higher plants (Thompson & Ritland 2006, van most, if not all, non-heterothallic isolates are Baarlen et al. 2000). apogamic. Acknowledgments Discussion This review is derived from the Since, both sexual heterothallism and research of many investigators of the nuclear automictic reproduction occurs in different reproductive cycle in the myxomycetes. We isolates of the same species, it raises the can only hope that we have done justice to question; how is this situation maintained. everyone’s contribution to this field and have Although, all isolates of Echinostelium produced a useful summary for future minutum collected in temperate environments researchers. have been found to be non-heterothallic, and all of those from arid deserts heterothallic (Clark References & Haskins 1998), in other species (Clark et al. 2001) the two reproductive modes are found in Albert M, Therrien CD. 1985 – Cytophoto- near proximity in the same habitat. Apparently metric evidence of mating fusion there are forces which maintain this dual competence and its induction in occurrence even when there is no obvious Didymium iridis. Cytobios 44, 189– selection for different habitats. Of course, the 204. sexual isolates undergo genetic recombination Adler PN, Holt CE. 1975 – Mating type and and can thus adapt to a variety of the differentiated state in Physarum environmental conditions, but the patchy, and polycephalum. Developmental Biology often ephemeral, nature of suitable habitats 43, 240–253. could also require a high dispersal ability and Aldrich HC. 1967 – Ultrastructure of meiosis rapid growth. It has been shown, that the in three species of Physarum. successful colonization of a habitat, by a sexual Mycologia 59, 127–148. isolate, would require a spore rain 2.4 times Aldrich HC. 1969 – The ultrastructure of that of an asexual isolate (Schnittler & Tesmer meiosis in myxamoebae and plasmodia 2008). It has also been shown (Betterley & of Physarum flavicomum. American Collins 1983) that apogamic isolates of Journal of Botany 56, 290–299. Didymium iridis are generally quicker to form Aldrich HC, Carroll G. 1971 – Synaptinemal plasmodia and undergo sporulation, and that complexes and meiosis in Didymium plasmodial of this species undergo competition iridis: a reinvestigation. Mycologia 63, under crowded conditions (Clark 1980a, b). 308–316. Thus, the apogamic strains appear to be Aldrich HC, Mims CW. 1970 – Synaptinemal 243

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