Mycosphere

Reproductive systems in the myxomycetes: a review

Jim Clark1* and Edward F. Haskins2

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. 2010 – Reproductive systems in the myxomycetes: a review. Mycosphere 1(4), 337–353.

A review of reproduction in the myxomycetes reveals that they have a basic one-locus multiple- alleleic heterothallic mating system, which controls syngamy between haploid amoeboflagellates to produce the diploid plasmodium. However, each morphologically defined species contains a number of biological sibling species that can’t interbreed with each other and are centered in different regions of the world. Also, these morphospecies generally contain numerous non- heterothallic strains that can complete the life cycle from a single isolated spore. While there is information that suggests that some of these strains are homothallic, the majority of the evidence supports an apomictic system derived from a blockage of meiosis during spore formation. Thus, these non-heterothallic strains produce diploid amoeboflagellates that can develop directly into plasmodia without the need for crossing.

Key words – apomixis– heterothallism – homothallism – sibling-species

Article Information Received 16 November 2010 Accepted 22 November 2010 Published online 12 December 2010 *Corresponding author: Jim Clark – e-mail – [email protected]

Introduction fusion to produce the diploid plasmodium, with The basic reproductive pattern described meiosis occurring during spore formation. In by deBary (1887) has been found to generally contrast, the non-heterothallic group doesn’t apply to every species of myxomycetes that has display a mating type system. Therefore, the been examined (Martin & Alexopoulos 1969, individuals, in this group, are either homo- Collins 1979, Stephenson & Stempen 1994). thallic (sexual with a ploidal cycle) or apo- Sporocarps are formed from plasmodia and mictic (non-sexual with no ploidal change). produce spores, these spores then germinate The non-heterothallic myxomycetes, following and release uninucleate amoeboflagllates that a long mycological tradition, have often been divide mitotically and produce large clonal automatically labeled homothallic without any populations, which then produce coenocytic evidence as to a change in ploidy; however, plasmodia that grow and fruit to produce more this unsupported practice should not be sporocarps. However, information has accumu- continued. ated over the years, which indicates that there are different nuclear cycles associated with this Heterothallism general pattern, which subdivides this general The first report of heterothallism in the life cycle into two major categories, hetero- mycomycetes, by Pinoy (1908), was an ob- hallism and non-heterothallism. The hetero- vious error, since he concluded that + and – hallic group has a sexual system with mating plasmodia needed to fuse before sporulation types controlling haploid amoeboflagellate could occur. As such the first acceptable

337 reports of heterothallism in a myxomycete apparently only increase the rate of cell fusion were those of Skupienski (1917), working with (a lower rate of fusion occurs without mating an isolate of Didymium diforme, and von typeB heterozygousity), thus providing more Stosch (1935) working with an isolate of opportunities for the matA controlled plasmo- Didymium iridis (identified as Didymium dial transition (Youngman et al. 1981), while nigripes); however, the Skupienski isolate was heterozygousity at the matC locus increases the later determined to be non-heterothallic by pH range in which cell fusions can take place Schünemann (1930). The work of Dee (1960); (Shinnick et al. 1978). Plasmodial color locus using polycephalum and Collins segregation (Collins & Clark 1966) and cyto- (1961, 1962) working with Didymium iridis photometric DNA ploidal level (haploid amoe- and Physarum pusillum marks the generally boflagellates and diploid plasmodia) changes accepted beginnings of current heterothallic (Collins & Therrien 1978) have confirmed that myxomycete studies. In all of these reports, the normal syngamy and meiosis occurs in the mating alleles were of two types (designated + heterothallic isolates of the myxomycetes. and -); however, further studies with additional isolates of D. iridis (Alexopoulos & Zabka Sibling biological species 1962, Collins 1963, Collins & Ling 1964, A study (Henney 1967, Henney & Mukherjee & Zabka 1964, Collins 1965), Henney 1968) of nine isolates identified as Physarum pusillum (Collins et al. 1964), and P. Physarum flavicomum found that all nine isola- polycephalum (Dee 1966), indicated that these tes were heterothallic; however, when the species possessed a one-locus (matA) multiple- mating types of the different isolates were allelic mating system (any two amoebofla- crossed in all possible pair-wise combinations, gellates that differ for the various mating allel- it was found that the isolates formed three es can undergo sexual fusion). Later reports of distinct non-interbreeding mating series. Four a second matB (Youngman et. al 1979) and isolates, which displayed a one-locus multiple- third matC (Kawano et al. 1987) mating locus allelic mating group were then identified as in P. polycephalum have not been found to Physarum rigidum due to slight morphological occur in other species such as D. iridis (Clark differences from the rest of the isolates. The 1983) and the two extra P. polycephalum loci other five isolates formed two genetically iso- are now generally considered to be mating type lated sibling biological species since they could modifier genes (Dee 1978); that control cell not be separated on morphological grounds and fusion rates while the mating locus controls were not interbreeding. One on the sibling nuclear fusion and plasmodial differentiation species, contained three interbreeding isolates, (Bailey et al. 1990). and the other sibling species contained two interbreeding isolates. More extensive studies Syngamy using numerous isolates of the Didymium iridis The amoeboflagellate is a vegetative morphospecies (Collins 1976, Betterley & stage, which also serves as a gamete in the Collins 1983, Clark & Stephenson 1990, Clark heterothallic isolates; therefore, it must under- & Landolt 1993, Clark & Mires 1999, Clark et go a conversion from the vegetative to the al. 2001) have produced similar results. Ten gametic state so that syngamy can take place. separate sibling biological species were report- As the amoeboflagellate population increases ed in the D. iridis morphospecies. However, in density (Shipley & Ross 1978, Pallotta et al. one of them was later assigned to the Didy- 1979), the cells produce a mating factor mium megalosporum morphospecies due to (Youngman et al. 1977, Albert & Therrien sight morphological differences (which over- 1985) which apparently acts as a pheromone lapped with the D. iridis variations). It was also that induces the matA controlled amoebofla- found that the barriers between the different gellate to plasmodial transition (Nader et al. biological species was only partial in that 1984), since it increases the rate of plasmodial certain crosses between some of the biological formation not only in heterothallic crosses; but species sporadically produced plasmodia which also in non-heterothallic cultures. Heterozy- were generally weak and rarely sporulated gousity at the matB locus in P. polycephalum (Clark et al. 1991). Sibling biological species

338 Mycosphere have also been found within the morphospecies modial formation does not occur. Also, the of cinerea (Clark et al. 2002), Badha- very small size of myxomycete chromosomes mia gracilis (Clark et al. 2002), Didymium makes accurate chromosome counts and the ovoideum (Clark 2004), Didymium annellus identification of meioitic figures very difficult (Clark & Landolt 2001), Didymium squamulo- and not very definitive (see Collins 1979). sum (ElHage et al. 2000), minu- Haskins (1976) had to use high voltage elec- tum (Clark & Haskins 1998), Physarum globu- tron microscopy to count the 124 very tiny liferum (Henney 1968), and Physarum pusillum chromosomes in an apomictic isolate of Echi- (Clark &Landolt 2001). The sporangial mor- nostelium minutum. Synaptonemal complexes phology of the D. ovoideum biological species (EM evidence of meiotic chromosome pairing) was also found to overlap with the D. iridis also does not prove that a sexual cycle is morphology (isolated originally identified as D. present, since isolates of Echinostelium minu- iridis were later found to belong to the D. tum, Didymium iridis and Stemonitis flavo- ovoideum biological species). genita in which the complexes were found (Carroll & Dykstra 1966, Aldrich & Carroll Non-heterothallism 1971, Haskins et al. 1971, Gaither & Collins The first report of non-heterothallism in 1984) where later proven by cytophotometric the myxomycetes was by Jahn (1911), who DNA studies (Therrien & Yemma 1974, used an isolate of Didymium squamulosum. Haskins & Therrien 1978, Therrien & Haskins This was followed by reports by Cayley (1929) 1981, Collins et al. 1983) to lack a haploid- on isolates of Didymium diforme and D. iridis diploid ploidal cycle. Apparently, these isolates (two isolates, one identified as D. nigripes), are automictic, with the chromosomes pairing and by Schünemann (1939) on isolates of Phy- during first division meiosis, but then the first sarum leucopus, D. diforme, and D. iridis division products fuse to reform the diploid (identified as D. nigripes). Von Stosch (1935) state. Thus, the only method that can determine also found that isolates of Didymium iridis, D. whether an isolate is homothallic or apogamic difforme, D. squamulosum and Physarum cine- is via means of amoebofagellate and plasmo- reum were non-heterothallic and suggested that dial photospectrometric DNA ploidy determi- they were apogamic on the basis of chromo- nations. However, this is fairly difficult and has some studies. The report by Kerr and Sussman been done for only a handful of species and (1958) and Kerr (1961) that the amoebofla- isolates. Of the 12 non-heterothallic isolates gellates of a non-heterothallic strain of D. iridis examined by cytophotometry to date, the fol- (identified by them as D. nigripes) underwent lowing results have been obtained: one isolate cell fusion (which they believed to be gamete of Echinostelium minutum (Haskins & Therrien fusion) and was thus homothallic, was later 1978, Therrien & Haskins 1981), four isolates contradicted by von Stosch et al. (1964), who of Stemonitis flavogenita (Collins et al. 1983), concluded that the same strain was apogamic and three isolates of Didymium iridis (Therrien on the basis of chromosome studies. Kerr’s & Yemma 1974, Therrien et al. 1977) were later report (1967) also found that plasmodia apogamic and four isolates of D. iridis (Ther- formation in this strain could occur without rien et al. 1974) were considered to be homo- amoeboflagellate fusion. A number of reports, thallic. However, one of the homothallic D. illustrated by the extensive studies on meiosis, iridis isolates was later found to be an apomict by Wilson and Ross (1955), and syngamy, by that had partially converted to heterothallism (Ross 1957), on a number of species and iso- (Collins 1980, Collins & Gong 1985). Both lates of myxomycetes could not settle the haploid and diploid amoeboflagellates were homothallic versus apomictic question. Thus, present in the culture, but apparently only the while amoeboflagellate cell fusion, is sugges- cells having haploid nucleic were considered to tive of syngamy, it is not a reliable criterion of be amoeboflagellates. Thus, the occurrence of sexual reproduction, since it has been shown polyploid amoeboflagellates and plasmodia (Bailey et al. 1990) that amoeboflagellate cells (Mulleavy & Collins 1979), chromosome eli- homoallelic for mating type can fuse and their mination (Collins et al. 1978, and mixoploid nuclei can also fuse to form a diploid, but plas- plasmodia (containing nuclei of several diffe-

339 rent ploidal levels) (Kubbies et al. 1986) makes order to form plasmodia. Thus, while apomictic even DNA level determinates of apogamic or clones are produced, they are only temporary, homogamic cycles difficult in the non-hetero- and a mutation to block meiosis would be thallic isolates. required to produce permanent apomictic lines. Since apomictic myxomcyetes do not undergo Apomictic-heterothallic conversion meiosis, they also do not need to have identical Three isolates, that were at first desig- chromosome pairs. Thus, chromosome aberra- nated as non-heterothallic on the basis of plas- tions, including partial polyploidy, could be a modial formation by single spore derived mechanism to block meiosis that would pro- amoeboflagellates, were later found, after a duce permanent apomictic lines. period in culture, to also produce amoebofla- gellate clones that could form plasmodia only Reproductive variations after crossing with a heterothallic clone dis- While most myxomycete isolates have playing a different mating type. In the Phi-1 either heterothallic or non-heterothallic repro- (Yemma et al.1980) and Pan-4 (Collins 1980) ductive cycles, there are a number of variations isolates of D. iridis, and the CR-1 isolate of that can depart from these common systems. Stemonitis flavogenita (Collins et al. 1983), stable plasmodial producing non-heterothallic Haploid amoeboflagellate selfing lines and stable mating type lines were esta- Heterothallic amoeboflagellate clones, blished. Cytophotometric DNA measurements, having a single mating type, can occasionally in both of the D. iridis isolates (Yemma et al. produce plasmodia without crossing to a dif- 1980, Collins & Gong 1985) and the Stemonitis ferent clone. While these plasmodia are often flavogenita (Collins et al. 1983) isolate, found weak and have sporulation problems they can that the stable non-heterothallic lines were sometime complete the entire life cycle in diploid apomicts, and the crossing lines were culture. This clonal haploid selfing is generally haploids that produced diploid plasmodia when sporadic and variable and has been shown to crossed. These apomictic-heterothallic conver- occur in D. iridis (Collins 1961, Collins & Ling sions would seem to indicate that some apo- 1968), P. flavicomum (Henney 1967), P. poly- mictic isolated from nature, and therefore cephalum (Dee 1960, Collins 1975, Adler & probably most non-heterothallic isolates, are Holt 1975), and several additional species created by a meiotic blockage that produces (Collins. unpublished, but cited in Collins diploid spores containing both mating types. 1979). The available evidence from genetic and cytophotometric studies on D. iridis (Collins & Polyploidy and apomixes Ling 1968, Yemma & Therrien 1972, Therrien The mechanism for the formation of & Yemma 1975) indicates that the resulting apomictic lines is not known; however, it is plasmodia are haploid and were thus produced likely that polyploidy is somehow involved. apogamously. It has been postulated (Yemma Polyploid amoeboflagellates of the myxomy- et al. 1974) that selfing is controlled in D. iridis cetes occur and can function normally as a by a cytoplasmic factor which acts on the vegetative cell and also undergo sexual fusion mating type gene to induce plasmodial forma- if it is a heterothallic line with mating type tion, and that some mating types are more alleles (Collins et al. 1978). Mulleavy and susceptible to this induction. Wheals (1970), Collins (1979, 1981) have shown that when Cooke & Dee (1974), and Anderson et al. two compatible diploid clones are crossed, a (1976) have reported a homothallic clone of P. tetraploid plasmodium is produced that then polycephalum, which is actually a haploid produces diploid spores when it sporulates. clone that is not only capable of crossing with Half of these diploid spores produce amoebo- other mating type clones; but is also capable of flagellate clones that are heterozygous for producing plasmodia 100% of the time when it mating type and are also capable of producing is not crossed. Generally, these plasmodia are plasmodia without crossing. However, when produced by the formation of a haploid binu- these diploid plasmodia sporulate they produce cleate by mitosis followed by their fu- normal haploid lines that must be crossed in sion to form a diploid nucleus and plasmodial

340 Mycosphere formation (Anderson et al. 1976). However, it matA-h locus behave like mtA-2 alleles may also occur by the crossing of two amoe- (Anderson 1978). How the other mutant types boflagellate cells, each carrying the mutant interact in regard to controlling plasmodial matA-h allele, since Wheals (1970) was able to formation is still unknown, as is the exact show mating type and plasmodial fusion gene mechanism of how plasmodial formation is recombination and segregation. Collins & Tang controlled. (1988) studied a D. iridis heterothallic isolate which had selfed in nature, since it displayed Sterility only one mating type and could also produce It was noticed in the early myxomycete plasmodia without crossing. Thus, rare haploid reproductive studies (Collins 1961) that some facultative apomictic lines can occur in nature. amoeboflagellate clones did not form plasmo- A series of papers (Therrien & Collins 1976, dia either by themselves (in non-heterothallic Collins et al. 1978, Ritch & Therrien 1987, isolates) or when crossed (in heterothallic iso- 1988) investigated the possibility that a poly- lates), and while some of these failures were no ploid clone could induce the selfing of a doubt due to unknown cultural problems such haploid strain since it appeared that only as pH tolerance (Collins & Tang 1973), many haploid plasmodia were produced when crosses of them were likely due to sterility derived were attempted between the two clones. How- from a mutation or a developmental abnorma- ever, closer examination indicated that sexual lity. Similarly, plasmodia produced in culture fusion and plasmodial formation had taken or isolated from nature are often found to not place, but that the polyploidy chromosomes sporulate in culture (Clark & Collins 1976, were rapidly eliminated from the plasmodium. Stephenson et al. 2004). Again many of these cases are due to unknown sporulation require- Mating type mutation ments (Collins 1979), but apparently some of While Collins (1965) reported that he had them are also due to genetic mutations or de- recovered a mating type mutant in D. iridis, the velopmental abnormalities. Thus sterile amoe- major examination of mating type and plasmo- boflagellate clones and plasmodia lines appear dial formation mutations has occurred in P. to be produced fairly often in the myxomycetes. polycephalum. The extensive studies (Adler et This sterility and the fact that both amoebo- al. 1975, Anderson et al. 1976, Davidow & flagellates and plasmodia can produce resistant Holt 1977, Anderson 1979, Anderson & structures during poor growth conditions (see Youngman 1985, Anderson et al. 1989) on mu- Collins 1976) and that amoeboflagellates and tants that effect plasmodium development in P. plasmodial are potentially immortal (Clark polycephalum have found that most of these 1984, Clark 1992) would seem to suggest that mutations are located on the mating type chro- it is quite likely that sterile amoeboflagellate mosome and fall into three groups. These are and plasmodial lines exist in nature. (1) the amoebal-plasmodium transition (apt) mutants that block plasmodial formation in Occurrence clones carrying the Colonia mtA-h allele that All of the known reproductive system allows plasmodial formation without crossing, reports for myxomycete species are shown in (2) non-plasmodial forming mutants (npfA, Table 1 except for a few papers with ambi- npfB, npfC) that also block plasmodial forma- guous results and those dealing with the details tion in mtA-h lines, and (3) greater asexual of plasmodial developmental regulation using development mutants (gad) that increase plas- mutations (see Anderson et al. 1989). Some modial formation without crossing in all reproductive system reports that have been mating types. The interaction between these published for certain species are not recognized mutants is complex, but it appears that the gad as valid, either because the identification was mutants permit higher levels of selfing by in error or the evidence indicates that the pre- regulating the npfB locus (Anderson et al. 1989) sumed species is a variant of another species. and are thus like the mtA-h allele. Therefore, The numerous references to Didymium nigripes the mtA-h allele is probably a gad mutant of a by Kerr (for example 1961, 1967, 1968) have mtA-2 allele, since npfB mutants with the been incorporated into the D. iridis species

341 Table 1 Heterothallic and non-heterothallic reproductive system reports.

Ceratiomyxales (Ceratiomyxaceae) fructiculosa (Müll.) T. Macbr. 1 non-heterothallic isolate: Costa Ricaa

Echinosteliales (Echinosteliaceae) Echinostelium coelocephalum T.E. Brooks & H.W. Keller 17 heterothallic isolates: 17 isolates (1 Arizona USb, 16 California USb) with a minimum of 9 mating types in one biological species Echinostelium minutum deBary 30 non-heterothallic isolates: 30 isolates (various locations in the USc) 7 heterothallic isolates: A1 biological species: 6 isolates (6 California USd) with 4 mating types A2 biological species: 1 isolate (Arizona USd) with 2 mating types Semimorula liquescens Haskins, McGuinness & Berry 1 non-heterothallic isolate: Washington USa

Liceales (Liceaceae) Licea biforis Morgan 1 non-heterothallic isolate: Washington USa

Physales (Physaraceae) afinis Rostaf. 1 non-heterothallic isolate: Texas USe Badhamia apiculospora (Härk.) Eliasson & Lundq. 2 non-heterothallic isolate: 1 isolate as Physarum apiculosporum (Finlandf), 1 isolate as Badhamia semiannulata (Florida USg) Badhamia follicola A. Lister 1 non-heterothallic isolate: California USh Badhamia gracilis (T. Macbr.) T. Macbr. (also recognized as B. melanospora Speg.) 61 non-heterothallic isolates: 2 isolates (Canary Islands Spaini), 12 isolate (Mexicoi), 27 isolates (Puerto Ricoi), 1 isolate (Taiwanj), 9 isolates (Arizona USi), 2 isolates (California USi), 3 isolates (New Mexico USi), 5 isolates (Texas USi) 5 heterothallic isolates: A1 biological species: 3 isolates (Arizona USi) with 4 mating types A2 biological species: 2 isolates (New Mexico, USi) with 4 mating types (Bull) Berk. 2 heterothallic isolate: 1 isolate (Great Britiank), 1 isolate (California USh), could not be tested for multiple allelism. obovatum Peck 1 non-heterothallic isolate: as Badhamia curtissi (unknown locationl) cinerea (Schwein.) Morgan 1 non-heterothallic isolate: Tahitim (L.) F.H. Wigg. 6 non-heterothallic isolates: 1 isolate (Alaska USn), 3 isolates (Texas USo), 1 isolate (West Virginia USn), 1 isolate (Wyoming USn) 4 heterothallic isolates: A1 biological species: 4 isolates (Texas USo) with 4 mating types Physarella oblonga (Berk. & M.A. Curtis) Morgan 2 non-heterothallic isolate: 1 isolate (Venezuelan), 1 isolate (unknown locationp) Physarum cinerum (Batsch) Pers. 7 non-heterothallic isolates: 2 isolates (Curacaoq), 1 isolate (Great Britain s), 2 isolates (Indonesiaq), 1 isolate (Taiwanr), 1 isolate (Venezuelaq) 2 heterothallic isolate: 1 isolate (Germanyt) with two mating types, 1 isolate (Venezuelah) with 2 mating types; the two isolates could not be tested for multiple alleles or biological species Physarum compressum Alb. & Schwein. 48 non-heterothallic isolates: 6 isolates (Australiau), 18 isolates (Costa Ricah,u,v), 1 isolate (Columbiau), 8 isolates (Ecuadoru,v), 3 isolate (Guatemalau,v), 1 isolate (Guyanau), 2 isolates (Hondurash,v), 1 isolate (Panamah), 4 isolates (Puerto Ricou,v), 2 isolates (Thailandv), 1 isolate (Tennessee USu), 1 isolate (West Virginia USu)

342 Mycosphere

Table 1 (Continued) Heterothallic and non-heterothallic reproductive system reports.

2 heterothallic isolates: A1 biological species: 2 isolates (1 Canary Islands Spainu, 1 Costa Ricov) with 4 mating types Physarum didermoides (Pers.) Rostaf. 1 non-heterothallic isolate: unknown locationw 2 heterothallic isolates: A1 biological species: 2 isolates (1 Costa Ricaa, 1Texas USa) with 4 mating Physarum flavicomum Berk. 5 heterothallic isolates: A1 biological species: 3 isolates (Texas USx,y) with 6 mating types A2 biological species: 2 isolates (1 Philippinesx,y, 1 Texas USx,y) with 4 mating types Physarum gyrosum Rostaf. 5 non-heterothallic isolates: 1 isolate (Brazilt), 2 isolates (Guyanat), 1 isolate (Puerto Ricoa), 1 isolate (unknown locationh) Physarum globuliferum (Bull.) Pers. 3 heterothallic isolates: A1 biological species: 1 isolate (Texas USz) with two mating types A2 biological species: 1 isolate (Texas USz) with two mating types A3 biological species: 1 isolates as P. bilgramii (Texas USz) with two mating types Physarum leucopus Link 1 non-heterothallic isolate: Germanyaa Physarum melleum (Berk. & Broome) Massee 21 non-heterothallic isolates: 1 isolate (Costa Ricabb), 1 isolate (Mexicou), 18 isolates (Puerto Ricobb), 1 isolate (South Carolina USu) Physarum nicaraguense T. Macbr. 1 non-heterothallic isolate: from UScc (probably should be included in P. compressum) Physarum nudum T. Macbr. in Peck and Gilbert 1 non-heterothallic isolate: Great Britaink Schwein. 7 heterothallic isolates: A1 biological species: 7 isolate (1Russiadd, 1 Indiana USee, 1 Iowa USff, 2 Japangg, 1 North Carolina USee, 2 Wisconsin UShh,ii, 3 unknown USA originee,ii) with at least 15 mating types Physarum pusillum (Berk. & M. A. Curtis) G. Lister 11 non-heterothallic isolates: 1 isolate (Australiaa), 6 isolates (Costa Ricaa,q), 1 isolate (Puerto Ricoq), 1 isolate (Trinidada), 1 isolate (Venezuelah), 1 isolate (Michigan USh) 3 heterothallic isolates: A1 biological species: 1 isolate (Arizona USjj) with 2 mating types A2 biological species: 1 isolate (Arizona USjj) with 2 mating types A3 biological species: 1 isolate (Mexicojj) with 2 mating types Physarum rigidium (G. Lister) G. Lister 4 heterothallic isolates: A1 biological species: 4 isolates (1 Costa Ricax,y, 1 Philippinesw,x, 2 Texas USx,y) with 8 mating types Physarum roseum Berk. & Broome 1 non-heterothallic isolate: California USn Protophysarum phloiogenum M. Blackw. & Alexop. 1 heterothallic isolate: A1 biological species: 1 isolate (Colorado USjj) with 2 mating types Wilkommlangea reticulate (Alb. & Schwein.) Kuntze 1 non-heterothallic isolate: Pennsylvania USll as Cienkowski reticulata

Didymiaceae Didymium annelus Morgan 7 non-heterothallic isolates: 5 isolates (Peruq), 2 isolates (Puerto Ricoq) 2 heterothallic isolates: A1 biological species: 1 isolate (Puerto Ricoq) with 2 mating types A2 biological species: 1 isolate (Puerto Ricoq) with 2 mating types Didymium annulisporum H. W. Keller & Schokn. 1 non-heterothallic isolate: from unknown locationmm Didymium atrichum Henney & Alexop. 1 non-heterothallic isolate: Texas USnn

343 Table 1 (Continued) Heterothallic and non-heterothallic reproductive system reports.

Didymium difforme (Pers.) Gray 10 non-heterothallic isolates: 4 isolates (Germany.n,s,aa,), 1 isolate (Great Britainr), 2 isolates (California USf), 1 isolate (Minnesota USf), 1 isolate (Michigan USf), 1 isolate (Ohio USf) Didymium dubium Rosaf. 2 non-heterothallic isolates: 1 isolate (Indonesiaa), 1 isolate (West Virginia USn) Didyium laxifillum G. Lister & J. Ross 2 non-heterothallic isolates: 1 isolate (Curacaoq), 1 isolate (California USf) Didymium megalosporum Berk. & M.A. Curtis 2 non-heterothallic isolates: 1 isolate (Costa Ricaoo), 1 isolate (Thailandoo) 9 heterothallic isolates: A1 biological species: 9 isolates with some early reports as D. iridis (1 Curacaooo, 3 Mexicooo,pp, 2 Thailand00, 1 Georgia USpp, 2 Hawaii USoo) with 11 mating types Didymium minus (G. Lister) Morgan 1 non-heterothallic isolate: West Virginia USn 1 heterothallic isolate: A1 biological species: 1 isolate (Louisiana USh) with 2 mating types Didymium iridis (Ditmar) Fr. 68 non-heterothallic isolates: 7 isolates (Costa Ricaf,oo,qq,ss), 1 isolate (Dominicass), 3 isolates (Ecuadoroo), 2 isolate (Francef,oo,ss,tt) with the Kerr D. nigripes isolate being one of them, 4 isolates (Germanyrs,t,aa), 1 isolate (Guatemalapp), 2 isolates (Indonesiaoo), 1 isolate (Japanpp), 4 isolates (Panamaf,ss), 1 isolate (Philippinesf,ss), 2 isolates (Puerto Ricaoo), 2 isolates (South Africaf,pp), 3 isolates (Thailandoo),1 isolate (Trinidadf), 3 isolates (Tanzanian), 9 isolates (California USf,pp,rr,ss), 1 isolate (Georgia USf,rr,ss), 3 isolates (Hawaii USoo,ss), 1 isolate (Kansas USf), 6 isolates (Minnesota USf,rr), 1 isolate (Missouri USf,rr,ss), 1 isolate (North Carolina USpp), 3 isolates (South Carolina USoo), 2 isolates (Texas USf,oo,ss), 2 isolates (Washington USa,f,rr) 36 heterothallic isolates: A1 biological species: 20 isolates (10 Costa Ricaf,oo,rr,ss,uu, 1 Guatemalaqp, 3 Hondurasf,m,rr,ss,ww, 4 Panamaf,pp,rr,ss,ww, 1 New York USxx, 1 Iowa USf,rr,ss) with 18 mating types A2 biological species: 3 isolates (1 Kentucky USf,h,rr,ss, 1 Virginia USuu, 1 West Virginia USoo) with 5 mating types A3 biological species: 1 isolate (Washington USrr,ss) with 2 mating types A4 biological species: 1 isolate (Panamayy) with 2 mating types A5 biological species: the A5 biological species was found to be D. megalosporum A6 biological species:1 isolate (Florida USzz) with 2 mating types A7 biological species: 1 isolate (Great Britainzz) with 2 mating types A8 biological species: 1 isolate (Hawaii USoo) with 2 mating types A9 biological species: 2 isolates (2 Costa Ricaoo) with 4 mating types A10 biological species: 1 isolate (Mexicooo) with 2 mating types Undetermined biological species (not tested to the other biological species): 3 isolates (1 Australiaaaa,1 Hondurasaaa,bbb, 1 Hawaiiaaa) with uncertain connections to the A1 biological species have 8 mating types and are in 2 biological species; 2 isolates (Germanyt, Georgia USf,rr) with no known connections to any of the known biological species each with two mating types Didymium ovoideum Nann.-Bremek. 9 non-heterothallic isolates: 3 isolates (Costa Ricaoo,), 5 isolates (Indonesiaoo), 1 isolate (Tennessee USoo) 16 heterothallic isolates: A1 biological species: 15 isolates (1 Tennessee USoo, 10 Virginia USccc, 4 West Virginia USccc) with 2 mating types A2 biological species: 1 isolate (South Carolina USa) with 2 mating types Didymium saturnus H. W. Keller 3 non-heterothallic isolates: 2 isolates (Peruq), 1 isolate (Iowa USmm) Didymium squamulosum (Alb. & Schwein.) Fr. 45 non-heterothallic isolates: 4 isolates (Australiau), 26 isolates (Costa Ricah,u,ddd), 4 isolates (Germanyh,t,eee), 2 isolates (Indonesiaddd), 3 isolates (Puerto Ricoddd), 1 isolate (Thailandu),1 isolate (Hawaii USu), 1 isolate (Michigan USh), 1 isolate (Oregon USu), 1 isolate (Tennessee USu), 1 isolate (unknown locationfff) 19 heterothallic isolates: A1 biological species: 1 isolate (Costa Ricaddd) with 2 mating types A2 biological species: 4 isolates (4 Costa Ricaddd) with 6 mating types A3 biological species: 1 isolate (Puerto Ricoddd) with 2 mating types A4 biological species: 3 isolates (3 Costa Ricou) with 6 mating types A5 biological species: 1 isolate (Mexicou) with 2 mating types A6 biological species: 9 isolates (6 Australiau, 2 New Zealandu, 1 Thailandu) with 13 mating types

344 Mycosphere

Table 1 (Continued) Heterothallic and non-heterothallic reproductive system reports.

Didymium vaccinum (Durieu & Mont.) Buchet 1 non-heterothallic isolate: Mexicoa

Stemonitales (Stemonitaceae) Comatricha laxa Rostaf. 4 heterothallic isolates: A1 biological species: 4 isolates (Arizona USggg) with 2 mating types Comatricha lurida A. Lister 1 heterothallic isolate: A1 biological species: 1 isolate Arizona UShhh with 2 mating types Lamproderma arcyrionema Rostaf. 1 non-heterothallic isolate: Costa Ricaggg Stemonitis flavogenita E. Jahn 7 non-heterothallic isolates: 1 isolate (Costa Ricah), 1 isolate (Ecuadorh), 1 isolate Hondurash), 1 isolate (Indonesian), 1 isolate (California USh), 1 isolate (Iowa USh), 1 isolate (Washington USh) 1 heterothallic isolate: A1 biological species: 1 isolate (Costa Ricah,iii,jjj) with 2 mating types Stemonitis fusca Roth 1 heterothallic isolate: A1 biological species: 1 isolate (Utah USkkk) with 2 mating types Stemonitis herbatica Peck 1 non-heterothallic isolate: Indiakkk

Trichiales (Trichiaceae) Arcyria cinerea (Bull.) Pers. 10 non-heterothallic isolates: 8 isolates (Costa Ricalll), 1 isolate (Ecuadorlll), 1 isolate (Mexicolll) 3 heterothallic isolates: A1 biological species: 1 isolate (Ecuadorlll) with 2 mating types A2 biological species: 2 isolates (Ecuadorlll) with 2 mating types Perichaena vermicularis (Schwein.) Rostaf. 1 non-heterothallic isolate: Indonesiaa

Table references: aClark et al. 2004, bHaskins et al. 2000, cHaskins personal communication, dClark & Haskins 1998,eBlackwell personal communication, fBetterley 1981, gRaub et al. 1979, hClark & Collins 1976, iClark et al. 2003, jLiu 1990, kCarlile 1974a,b,lRoss 1964, mCollins 1961, nClark 1995, oAlexopoulos & Henney personal communication, pGehenio & Luyet 1950, qClark & Landolt 2001,rShen 1964, sCayley 1929, tvon Stosch 1935, uClark & Stephenson 2003, vIrawan et al. 2000, wPolanowski 1973, xHenney 1967, yHenney & Henney 1968, zHenney 1968, aaSchünemann 1930, bbClark & Stephenson 2000, ccSolis 1962, ddCollins & Tang 1977, eeDee 1966, ffCollins 1975, ggKawano & Kuroiwa 1989, hhDee 1960, iiKirouac-Brunet et al. 1981, jjCollins et al. 1964, kkBlackwell 1974, llTherrien personal communication, mmKeller personal communication, nnHenney et al. 1980, ooClark et al. 2001, ppClark & Landolt 1993, qqCollins 1965a,b,rrBetterley & Collins 1983, ssCollins 1976, ttKerr & Sussman 1958, uuClark & Stephenson 1990, vvCollins & Ling 1964, wwCollins 1965, xxCollins & Tang 1989, yyCollins & Gong 1985, zzClark et al. 1991,aaaMukherjee & Zabka 1964, bbbAlexopoulos & Zabka 1962, cccClark & Stephenson 1994,dddElHage et al. 2000, eeeJahn 1911,fffGustafson & Thurston 1974, gggClark & Haskins 2002, hhhMcGuinness & Haskins 1985, iiiCollins et al. 1983, jjjCollins & Tang 1988, kkkClark1997, lllClark et al. 2002. ______

(Collins 1976), based on a reappraisal of its , three in the Echinosteliales, and six morphology. While D. nigripes is considered to in the Stemonitiales. This distribution of be a valid species, these authors do not believe reports, in regards to taxonomic order, is that it has so far been successfully cultured. probably due to a number of factors, including On the other hand, P. bilgramii, is treated as a the number of species per order, and the variant of P. globuliferum due to cultural particular interests of individual researchers. studies (Henney 1968). Of the 51 species in However, the overriding factors are probably the table, 38 are in the , one in the the abundance, distribution and culturability of Ceratomyxales, one in the , two in the a particular species, with the “weedy” species

345 being much more likely to be cultured (Clark Clark 2000, Clark 2004). The recent review 2004). article by Haskins and Wrigley de Basanta (2008) on agar culture methods for the myxo- Heterothallism and non-heterothallism mycetes should be the starting point for anyone Fourteen of the 51 species in the table wishing to culture these organisms. Once an have both heterothallic and non-heterothallic isolate is in culture, a fresh well formed reports, eight have only heterothallic reports, sporangium should be used as a source for and 29 have only non-heterothallic reports. single spore amoeboflagellate isolations. Using However, only five of the eight species with sterile techniques, a spore suspension is pro- only heterothallic reports have five or more duced and diluted to a level that provides a few studied isolates, and only four of the 27 species widely spaced spores when spread on agar. with only non-heterothallic reports have five or Single spores can be picked up with a needle more studied isolates. Therefore, it is likely using a dissecting microspore with 60X magni- that investigations involving more isolates, fication and sub-stage lighting (Collins 1963), would show that many of these species display and then placed in a new plate with the both reproductive systems. However, it is pos- appropriate food organism. The spore will then sible that some species, such as Echinostelium germinate and grow into a clonal population. coelocephalum, are totally or mostly hetero- Alternatively, the dilute spore suspension can thallic, and others, such as Physarum melleum, be spread on a plate with a lawn of food are totally or mostly non-heterothallic. organisms and the germinating spores will form plaques, that can be transferred to new Multiple alleles plates (McGuinness and Haskins 1985), or the Thirteen of the sixteen species, having spore suspension, mixed with a food organism, more than two heterothallic isolates in the same can be spotted on the plates and the ger- sibling species, displayed multiple alleles at the minating spores will then form a population in mating locus. Only the Didymium ovoideum the spots from which they can be isolated. In A2 sibling species, in which sixteen isolates all of these procedures, getting a proper dilute were examined, the Arcyria cinerea A2 sibling is the critical part, since it is necessary to insure species, in which two isolates were examined, that only a single spore is present in each case. and Comatricha laxa, in which 4 isolates were Once isolated, the amoeboflagellate clones can examined, displayed only two mating alleles. be maintained by serial transfers to new plates Thus, multiple alleles, at the mating locus, or slant tubes. Non-heterothallic isolates will appear to be nearly universal in the myxomy- produce plasmodia in these clones, while cetes. heterothallic isolates, require crossing by mix- ing two different clones together (Collins 1979). Sibling species For any particular heterothallic isolate half of Fourteen of the sixteen species (which the amoeboflagellate clones will be of one could be tested) having two or more hetero- mating type and the other half will have a thallic isolates, displayed sibling biological different mating type. You need approximately species. Only Physarum polycephalum (7 isola- 15 clones to insure that you can detect hetero- tes) and Didymium megalosporum (9 isolates) thallism, and a second generation of clones, seem to lack sibling species. Thus, sibling derived from a first generation cross, should be biological species seems to be the norm in the produced for both heterothallic and non- myxomycetes. heterothallic isolates to insure that the system is stable. Methods The determination of homothallism or The determination of heterothallism and apomictic development is very difficult since non-heterothallism in the myxomyctes is normal cytological procedures are not adequate straightforward once an isolate is in culture, in the myxomycetes. Amoeboflagellate fusion however while many species are relatively easy is not proof of syngamy since homoallelic to culture, others have been resistant to all amoeboflagellates can fuse and form diploid attempts to date (see Clark & Collins 1976, nuclei; however, this produces a polyploidy

346 Mycosphere amoeboflagellate and not a plasmodium variability in the ease of culture for those (Bailey et al. 1990). These polyploidy amoeo- species which can be easily obtained for study. laellates, if heterothallic, can fuse to produce a While the cosmopolitan and common Didy- plasmodium that then undergoes chromosome mium iridis is easy to culture, the equally elimination so that it can have the same amount common and cosmopolitan epiden- of chromosomes and DNA as the amoebo- drum has never been cultured despite numerous lagellates (Ritch & Therrien 1988). Also, attempts. Except for Cribariaceae, at least one diploid apomictic lines, after a period in culture, species has been cultured from every family can revert some of the amoeboflagellates back (Clark 1995), although a number of families to haploid cells with mating types, therefore, are poorly represented in culture and four the detection of haploid nuclei in a non- (Cribariaceae, Reticulariacae, Dianemaceae, heterothallic culture is support, but not proof Clastodermataceae) have no mating system for a determination of homothallism (Collins & reports. Thus the reproductive systems studied Tang 1988). Because of these problems, the are biased toward common cosmopolitan easily only sure way of separating the two possibi- grown species, with a few rare specialized ities (homothallism and apogamy) is by genetic species present due to the particular interests of recombination studies (this would occur at high a number of investigators. The moist chamber frequency only in homothallism), or microcine- method (small substrate samples are placed in a atography of the amoeboflagellate transition to Petri dish with water and monitored for plasmodia coupled with cytophotometric deter- plasmodial formation and sporulation) which is minations of DNA levels in the amoebofla- commonly used to produce collections for gellates and plasmodium. However, recombi- study is also likely to bias the sample. Since nation studies require genetic markers which non-heterothallic isolates require only a single are rarely available, while microcinemato- spore to produce a collection, they would pro- graphy and cytophotometry requires special- duce plasmodia and sporangia more frequently ized equipment and procedures (see Collins & in moist chambers even if the number of Therrien 1976, Bailey et al. 1990) combined heterothallic and non-heterothallic spores in the with careful cultural studies (need to start with region were identical. a freshly re-cloned culture, to insure that the Based on these reproductive system non-heterothallic behavior examined is not due studies, myxomycetes essentially have a hete- to a mixed population of apomictic and rever- rothallic one-locus multiple-allelic mating sys- tant heterothallic amoeboflagellates). Thus, it is tem with a number of derived variations. seldom useful to go beyond a non-heterothallic During sporulation, meiosis occurs to produce determination for an isolate, unless there is a spores which germinate to produce haploid compelling research need for the information. amoeboflagellate cells that divide mitotically to produce a clonal population of identical cells. Conclusions As the cell density increases, the amoebo- Of the 900 or so recognized species of flagellates become competent to act as gametes myxomycetes, 51of them have had one or more and fuse with another cell having a different isolates investigated as to their reproductive mating type allele. This fusion creates a zygote system. While this would seem to be a which then grows into the coenocytic plas- reasonable sample size, it is not a random modium that completes the lifecycle by sample, since reproductive systems can only be sporulating. This basic system has been fre- determined with cultured species. However, quently modified by the development of non- whether or not a myxomycete species has been heterothallic systems. The homothallic system, cultured is dependent upon its abundance in which is probably rare if it is actually present, nature and its specific requirements. Approxi- retains the sexual reproduction system with mately 30% of the recognized myxomycete haploid amoeboflagellate cells and diploid species (Schnittler & Mitchell 2000) are known plasmodia, but has lost the mating types, so from only a single or a few collections, and that any two amoeboflagellates can form a until recently, the culture of these collections zygote. On the other hand, the apomictic was seldom attempted. There is also a great system, which is probably the most common

347 non-heterothallic form, has dispensed with sex Apparently many, if not most, of the morpho- altogether and produces diploid amoebofla- logically defined species consists of a number gellate cells, by blocking meiosis, that are then of sibling biological species that are regionally capable of producing plasmodia without fusion. centered, and also of a large number of geneti- Also, a rare selfing (facultative haploid apo- cally isolated non-heterothallic clonal lines. mixes of heterothallic amoeboflagellates) sys- The local biological species can evolve into tem is present in a few isolates, where haploid minor variants of the widely dispersed morpho- amoeboflagellate cells produce haploid plas- logical species, and the clonal non-heterothallic modia apomictically. There are probably also a lines could also evolve into a variety of in- number of sterile reproductive systems in tegrating morphological forms. This can be nature, where amoeboflagellate populations seen in the cluster of recognized morphological (that can’t produce plasmodia) and plasmodia species (D. nigripes, D. megalosporum, D. (which can’t sporulate) are present and func- ovoideum, D. bahiense, D. verrucosporum, D. tional. eximium) that centers on Didymium iridis. The driving forces behind the evolution Culture and genetic studies have shown that a of multiple alleles at the mating locus and non- minimum of 20% of the heterothallic isolates heterothallic lines are probably identical. The identified as D. nigripes on morphological resistant wind borne spores provide the myxo- grounds are actually D. iridis, and a similar mycetes with a very efficient dispersal mecha- percentages of D. megalosporum and D. ovoi- nism, which could very easily produce a situa- eum isolates are identified as D. iridis. These tion where an amoeboflagellate population studies also show that the morphological iden- derived from a single heterothallic spore would ified heterothallic isolates of D. bahiense and D, not have a mating partner. This problem could verrucosporum are common variants that are be partially overcome by multiple alleles, since found in the D. iridis biological species. Didy- the greater the number of mating types in the ium eximum, like D. nigripes (all reported population at large, the greater the probability cultures were actually D. iridis) has never been that amoeboflagellates derived from two ran- cultured (or attempted), and in the opinion of dom spores deposited on the same substrate the senior author, it is the same as D. ovoideum would be compatible. And in the case of the (D. ovoideum is the newer name and therefore non-heterothallic lines this problem is com- this species should probably be called D. exi- pletely eliminated. The formation of biological um). In terms of the non-heterothallic isolates, sibling species is also probably related to dis- they all blend together, and except for D. ovoi- persal. While the spores are apparently capable eum isolates which has a yellow instead of of maintaining a widespread morphological brown plasmodium, only a few morphological species, the great majority of individuals within distinct forms can be separated from D. iridis a particular region would tend to be genetically (Clark & Mires 1999). A similar cluster of related, since most spores would not travel recognized morphological species is centered great distances. This relatedness would allow on Diymium squamulosum, this cluster is pro- the local populations to adapt to the local ably even more complex, since it appears that conditions and could also allow the develop- there are a large number of recognized morpho- ment of reproductive barriers between distance ogical species that are rare and are separated sexual populations, which would produce the from D. squamulosum by one or at most a few sibling species. Also, since the non-heterothal- fairly minor traits. It is quite likely that some lic lines are completely isolated from the of these rare species are local non-heterothallic sexual population and from each other, they lines that have evolved a distinct character trait. would be free to adapt to very specific local Other examples of similar, but smaller, species conditions, and also to fix minor morphological clusters can be seen centered on Physarum variations. flavicomum and Physarum compressum, and others are probably quite common.

Taxonomy Acknowledgments This complex reproductive system has This review is distilled from the work of taxonomy implications for the myxomycetes. many people who conducted research on

348 Mycosphere myxoycete reproduction. We hope that we Betterley DA, Collins OR. 1983 – Reproduc- have done justice to everyone’s contributions, tive systems, morphology, and genetical and have produced a useful summary for future diversity in Didymium iridis (Myxomy- investigators. cetes). Mycologia 75, 1044–1063. Blackwell M. 1974 – A study of sporophore References development in the myxomycete Proto- physarum phloiogenum. Archive of Adler PN, Holt CE. 1975 – Mating type and Microbiology 99, 331–334. the differentiated state in Physarum poly- Carlile MJ. 1974a – The myxomycete Physa- ephalum. Developmental Biology 43, rum nudum: life cycle and pure culture of 240–253. plasmodium. Transactions of the British Albert AM, Therrien CD. 1985 – Cytophoto- Mycological Society 62, 213–215. metric evidence of mating fusion compe- Carlile MJ. 1974b – Incompatibility in the tence and its induction in Didymium myxomycete Badhamis utricularis. Tran- iridis. Cytobios 44, 189–204. sactions of the British Mycological Aldrich HC, Carroll GC. 1971 – Synaptonemal Society 62, 401–402. complexes and meiosis in Didymium Carroll G, Dykstra R. 1966 – Synaptinemal iridis: a reinvestigation. Mycologia 63, compexes in Didymium iridis. Mycologia 308–316. 58,166–169. Alexopoulos CJ, Zabka GG. 1962 – Production Cayley DM. 1929 – Some observations on of hybrids between physiological races of of the genus Didymium. the true Didymium iridis. Transactions of the British Mycological Nature 193, 598–599. Society 14, 227–248. Anderson RW. 1979 – Complementation of Clark J. 1983 – The Didymium iridis mating amoebal-plasmodial transition mutants in system: no evidence for a matB locus. Physarum polycephalum. Genetics 91, Mycologia 75, 318–320. 409–419. Clark J. 1984 – Life spans and senescence in Anderson RW, Youngman PJ. 1985 – Comple- six slime molds. Mycologia 76, 366–369. mentation of npf mutations in diploid Clark J. 1992 – The myxamoebae of Didymium amoebae of Physarum polycephlaum: the iridis and Physarum polycephalum are basis for a general method of comple- immortal. Mycologia 84, 116–118. mentation analysis at the amoebal stage. Clark J. 1995 – Myxomycete reproductive sys- Genetic Research 45, 21–35. tems: additional information. Mycologia Anderson RW, Cooke DJ, Dee J. 1976 – 87, 779–786. Apogamic development of plasmodia in Clark J. 1997 – Myxomycete reproductive Physarum polycephalum. Protoplasma 89, systems: Stemonitis species. Mycologia 29–40. 89, 241–243. Anderson RW, Hutchins G, Gray A, Price J, Clark J. 2000 – The species problem in the Anderson SE. 1989 – Regulation of myxomycetes. Stapfia 73, 39–53. development by the matA complex locus Clark J. 2004 – Reproductive systems and in Physarum polycephalum. Journal of taxonomy in the Myxomycetes. Systema- General Microbiology 135, 1347–1359. tics and Geography of Plants 74, 209– Bailey J, Anderson RW, Dee J. 1990 – Cellular 216. events during sexual development from Clark J, Collins OR. 1976 – Studies on the amoeba to plasmodium in the slime mating systems of eleven species of mould Physarum polycephalum. Journal myxomycetes. American Journal of of General Microbiology 136, 739–751. Botany 63, 783–789. Betterley DA. 1981 – Reproductive systems, Clark J, Collins OR, Tang H-C. 1991 – enzymes, enzyme polymorphism, and Didymium iridis mating systems: partial speciation in the myxomycete Didymium compatibility between mating series. iridis. Ph.D. Dissertation, University of Mycologia 83, 210–213. California, Berkleley.

349 Clark J, Haskins EF. 1998 – Heterothallic Collins OR. 1963 – Multiple alleles at the mating systems in the Echinostelium incompatibility locus in the myxomycete minutum complex. Mycologia 90, 382– Didymium iridis. American Journal of 388. Botany 50, 477–480. Clark J, Haskins EF. 2002 – Reproductive Collins OR. 1965a – Evidence for a mutation at systems of the Myxomycetes Comatricha the incompatibility locus in the slime laxa and Lamproderma arcyrionema. mold, Didymium iridis. Mycologia 57, Nova Hedwigia 75, 237–240. 314–315. Clark J, Landolt JC. 1993 – Didymium iridis Collins OR. 1965b – Homothallic behavior in reproductive systems: additions and two Costa Rican isolates of the slime meiotic drive. Mycologia 85, 764–768. mold Didymium iridis. American Journal Clark J. Landolt JC. 2001 – Myxomycete of Botany 52(abstracts), 634. biosystematics: various Didymium and Collins OR. 1975 – Mating types in five Physarum species. Nova Hedwigia 73, isolates of Physarum polycephalum. 437–444. Mycologia 67, 98–107. Clark J, Mires A. 1999 – Biosystematics of Collins OR. 1976 – Heterothallism and homo- Didymium: the non-calcareous, long- thallism: a study of 27 isolates of Didy- stalked species. Mycotaxon 71, 369–382. mium iridis, a true slime mold. American Clark J, Stephenson, SL. 1990 – Didymium Journal of Botany 63, 138-143. iridis reproductive systems: new Collins OR. 1979 – Myxomycete biosystema- additions. Mycologia 82, 274–276. tics: some recent developments and Clark J, Stephenson SL. 1994 – Didymium future research opportunities. Botanical ovoideum culture and mating system. Review 45, 145–201. Mycologia 86, 392–396. Collins OR 1980 – Apomictic-heterothallic Clark J, Stephenson SL. 2000 – Biosystematics conversion in a myxomycete, Didymium of the myxomycete Physarum melleum. iridis. Mycologia 72, 1109–1116. Nova Hedwigia 71, 161–164. Collins OR, Clark J. 1966 – Inheritance of the Clark J, Stephenson SL. 2003 – Biosystematics brown plasmodial pigment in Didymium of the myxomycetes Didymium squa- iridis. Mycologia 58, 743–751. mulosum, Physarum compressum, and Collins OR, Gong T. 1985 – Genetic Physarum melleum: additional isolates. relatedness of a former apomicts and a Mycotaxon 85, 85–89. heterothallic isolate in Didymium iridis Clark J, Haskins EF, Stephenson SL. 2003 – (Myxomycetes). Mycologia 77, 300–307. Biosystematics of the myxomycete Bad- Collins OR, Ling H. 1964 – Further studies in hamia gracilis. Mycologia 95, 104–108 multiple alleomorph heterothallism in the Clark J, Haskins EF, Stephenson SL. 2004 – myxomycete Didymium iridis. American Culture and reproductive systems of 11 Journal of Botany 51, 315–317. species of mycetozoans. Mycologia 96, Collins OR, Ling H. 1968 – Clonally-produced 36–40. plasmodia in heterothallic isolates of Clark J, Schnittler M, Stephenson SL. 2002 – Didymium iridis. Mycologia 60, 858–868. Biosystematics of the myxomycete Arcy- Collins OR, Tang H-C. 1973 – Physarum ria cinerea. Mycotaxon 82, 343–346. polycephalum: pH and plasmodiaium Clark J, Stephenson SL, Landolt JC. 2001 – formation. Mycologia 65, 232–236. Biosystematics of the Didymium iridis Collins OR, Tang H-C. 1977 – New mating super species complex: additional types in Physarum polycephalum. Myco- isolates. Myxotaxon 79, 447–454. logia 69, 421–423. Collins OR. 1961 – Heterothallism and homo- Collins OR, Tang H-C. 1988 – Further studies thallism in two myxomycetes. American on conversion from non-heterothallism to Journal of Botany 48, 674–683. heterothallism in Stemonitis flavogenita Collins OR. 1962 – Mating types in the slime (Myxomycetes, Stemonitales). Mycolo- mold Physarum pusillum. American gia 80, 405-407. Journal of Botany 49 (abstracts), 659.

350 Mycosphere Collins OR, Tang H-C. 1989 – Reproductive Gehenio PM, Luyet BJ. 1950 – Complete de- behavior of a new isolate of Didymium velopment of mycetozoan from a single iridis (Myxomycetes). Mycologia 81, spore or a single amoeba. Biodynamica 7, 149–150. 11–23. Collins OR, Therrien CD. 1976 – Cytophoto- Gustafson RA, Thurtson EL. 1974 – Calcium metric measurement of nuclear DNA in deposition in the myxomycete Didymium seven heterothallic isolates of Didymium squamulosum. Mycologia 66, 397–412. iridis, a myxomycete. American Journal Haskins EF. 1976 – High voltage electron of Botany 63, 457–462. microscopical analysis of chromosomal Collins OR, Alexopoulos CJ, Henney M. 1964 number in the slime mold Echinostelium – Heterothallism in three isolates of minutum de Bary. Chromosoma 56, 95– Physarum pusillum. American Journal of 100. Botany 51(abstracts), 679. Haskins EF, Hinchee AA, Cloney RA. 1971 – Collins OR, Gong T, Clark J, Tang H-C. 1983 The occurrence of synaptonemal com- – Apomixis and heterothallism in pexes in the slime mold Echinostelium Stemonitis flavogenita (Myxomycetes, minutum de Bary. Journal of Cell Stemonitales). Mycologia 75, 614–622. Biology 51, 898–903. Collins OR, Therrien CD, Betterley DA. 1978 Haskins EF, Therrien CD. 1978 – The nuclear – Genetical and cytological evidence for cycle of the myxomycete Echinistelium chromosome elimination in a true slime minutum. I. Cytophotometric analysis of mold, Didymium iridis. American Journal nuclear DNA content of amoebal and of Botany 65, 660–670. plasmodial phases. Experimental Myco- Cooke DJ, Dee J. 1974 – Plasmodial formation logy 2, 32–40. without change in nuclear DNA content Haskins EF, McGuinness MD, Clark J. 2000 – in Physarum polycephalum. Genetic Heterothallic mating systems in the Research. 23, 307–317. Echinosteliales II. Echinostelium oeloce- de Bary A. 1887 – Comparative Morphology phalum. Mycologia 92, 1080–1084. and Biology of the Fungi, Mycetozoa and Haskins EF, Wrigley de Busanta D. 2008 – Bacteria. Clareton Press, London. Methods of agar culture of Myxomycetes: Davidow LS, Holt CE. 1877 – Mutants with an overview. Revista Mexicana de Mico- decreased differentiation to plasmodia in logia 27, 1–7. Physarum polycephalum. Molecular and Henney MR. 1967 – The mating type system of General Genetics 155, 291–300. the myxomycete Physarum flavicomum. Dee J. 1960 – A mating type locus in an Mycologia 59, 637–652. acellular slime-mould. Nature 185, 780– Henney MR. 1968 – The mating type systems 781. in the myxomyctes Physarum globu- Dee J. 1966 – Multiple alleles and other factors liferum and Physarum bilgramii. Ameri- affecting plasmodial formation in the true can Journal of Botany 55(abstract), 720. slime mould Physarum polycephalum. Henney MR, Alexopoulos CJ, Scheetz RS. Journal of Protozoology 13, 610–616. 1980 – Didymium atrichum, a new Dee J. 1978 – A gene unlinked to mating type myxomycete from south-central Texas. affecting crossing between strains of Mycotaxon 75, 150–164. Physarum polycephalum. Genetic Re- Henney MR, Henney HR. 1968 – The mating search 31, 85–92. type systems of the myxomycetes Phy- ElHage N, Little C, Clark J, Stephenson SL. sarum rigidum and Physarum flavico- 2000 – Biosystematics of the Didymium mum. Journal of General Microbiology squamulosum complex. Mycologia 92, 53, 321–332. 54–64. Irawan B, Clark J, Stephenson SL. 2000 – Gaither T, Collins OR. 1984 – Synaptonemal Biosystematics of the Physarum com- complexes in an apomictic line of pressum morphospecies. Mycologia 92, Stemonitis flavogenita (Myxomyctes, 884-893. Stemonitales). Mycologia 76, 1123–1125.

351 Jahn E. 1911 – Myxomycetenstudien. 8. Der lines of a myxomycete, Didymium iridis. Sexualakt. Berichte der Deutschen American Journal of Botany 66, 1067– Botanischen Gesellschaft 29, 231–247. 1073. Kawano S, Kuroiwa T. 1989 – Transmission Mulleavy P, Collins OR. 1981 – CIPC-induced pattern of mitochondrial DNA during and spontaneously produced diploid plasmodial formation in Physarum poly- myxamoebae in a myxomycete, Didy- cephalum. Journal of General Microbio- mium iridis: a study of mating type logy 135, 1559–1566. heterozygousity. Mycologia 73, 62–77. Kawano S, Kuroiwa T, Anderson RW, 1987 – Nader WF, Shipley GL, Huettermann A, Holt A third multialleic mating-type locus in CE. 1984 – Analysis of an inducer of the Physarum polycephalum. Journal of amoebal-plasmodial transition in the General Microbiology 133, 2539–2546. myxomyctes Didymium iridis and Kerr NS. 1961 – A study of plasmodial Physarum polycephalum. Developmental formation by the true slime mold Biology 103, 504–510. Didymium nigripes. Experimental Cell Pallotta DJ, Youngman PJ, Shinnick TM, Holt Research 23, 603–611. CE. 1979 – Kenetics of mating in Kerr NS. 1967 – Plasmodial formation by a Physarum polycephalum. Mycologia 71, minute mutant of the true slime mold, 68–84. Didymium nigripes. Experimental Cell Pinoy E. 1908 – Sur l’existence d’um dimor- Research 45, 646–655. phisme sexuel chez un Myxomycete. Kerr NS, Sussman M. 1958 – Clonal Comptes Rendus des Séances de la development of the true slime mold, Société de Biologie 64, 630–631. Didymium nigripes. Journal of General Polanowski FP. 1973 – Quantitative cyto- Microbiology 19, 173–177. chemical analysis of normal and selfing Kerr NS. 1968 – Ploidy level in the true slime strains of three species of myxomycetes. mold Didymium nigripes. Journal of Ph.D. Dissertation, Pennsylvania State General Microbiology 53, 9–15. University, University Park. Kirouac-Brunet J, Masson S, Pallota D. 1981 – Raub TJ, Keller HW, Gaither TW. 1979 – A Multiple allelism at the matB locus in new species of Badhamia with smooth Physarum polycephalum. Canadian spores. Mycologia 71, 119–126. Journal of Genetics and Cytology 23, 9– Ritch DL, Therrien CD. 1987 – An investiga- 16. tion of the apomictic induction hypothe- Kubbies M, Wick R, Hildebrandt A, Sauer HW. sis in Didymium iridis. Cytobios 51, 171– 1986 – Flow cytometry reveals a high 181. degree of genomic size variations and Ritch DL, Therrien CD. 1988 – A cytophoto- mixoploidy in various strains of the mertric investigation of haploid x poly- acellular slime mold Physarum poly- ploidy matings in the acellular slime cephalum. Cytometry 7, 481–486. mold Didymium ridis. Caryologia 41, Liu C-H. 1990 – Myxomycetes of Taiwan. VI. 169–179. Badhamia gracilis. Taiwania 35, 57–63. Ross IK. 1957 – Syngamy and plasmodial Martin GW, Alexopoulos CJ. 1969. Mono- formation in the myxogasres. American graph of the Myxomycetes. University of Journal of Botany 44, 843–850. Iowa Press, Iowa City, Iowa. Ross IK. 1964 – Pure culture of some McGuinness MD, Haskins EF. 1985 – Genetic myxomycetes. Bulletin of the Torrey analysis of the reproductive system of the Botanical Club 91, 24–228. true slime mold Comatricha lurida. Schnittler M, Mitchell DW. 2000 – Species Mycologia 77, 646–653. diversity in myxomycetes based on the Mukherjee KL, Zabka GG. 1964 – Studies in morphological species concept – a the myxomycete Didymium iridis. Cana- critical examination. Stapfia 73, 55–62. dian Journal of Botany 42, 1459-1466. Schünemann E. 1930 – Untersuchungen über Mulleavy P, Collins OR. 1979 – Development die Sexualität der Myxomyceten. Planta of apogamic amoebae from heterothallic 9, 645–672.

352 Mycosphere ShenYF. 1964 – A study of the life cycle of iridis. American Journal of Botany 64, grown in culture. 286–291. Taiwania 10, 63–72. von Stosch HA. 1935 – Untersuchungen über Shinnick TM, Pallotta DJ, Jones-Brown YR, die Entwicklungsgeschichte der Myxo- Youngman PJ, Holt CE. 1978 – A gene, myceten, Sexualität und Apogamie bei imz, affecting the pH sensitivity of zygote Didymiacean. Planta 23, 623–656. formation in Physarum polycephalum. von Stosch HA, Van Zul-Pischinger M, Current Microbiology 1, 163–166. Deresch G. 1964 – Nuclear phase alter- Shipley GL, Ross IK. 1978 – The effect of nance in the myxomycete Physarum proteases on plasmodial formation in polycephalum. Tenth International Bota- Didymium iridis. Cell Differentiation 7, nical Congress Abstracts 481–482. 21–32. Wheals AE. 1970 – A homothallic strain of the Skupienski FX. 1917 – Sur la sexualité de myxomycete Physarum polycephalum. Champignons Myxomycètes. Comptes Genetics 66, 623–633. Rendus del’Academie des Sciences Wilson M, Ross IK. 1955 – Meiosis in the (Paris) 165, 118–121. myxomycetes. American Journal of Solis BC. 1962 – Studies on the morphology of Botany 42, 743–749. Physarum nicaragense Macbr. Master of Yemma JJ, Therrien CD. 1972 – Quantitative Science Thesis, University of Iowa, Iowa microspectrophotometry of nuclear DNA City. in selfing strains of the myxomycete Stephenson SL, Clark J, Landolt JC. 2004. – Didymium iridis. American Journal of Myxomyctes occurring as single genetic Botany 59, 828–835. strains in forest soils. Systematics and Yemma JJ. Therrien CD, Jakupein GF. 1980 – Geography of Plants 74, 287–295. Isolation of haploid clones bearing a Stephenson SL, Stempen H. 1994. Myxo- mating allele from a diploid apomictic mycetes: a Handbook of Slime Molds. isolate of Didymium iridis. Cytobios 29, Timber Press Portland, Oregon. 145–157. Therrien CD, Collins OR. 1978 – Apogamic Yemma JJ, Therrien CD, Ventura S. 1974 – induction of haploid plasmodia in a Cytoplasmic inheritance of the selfing myxomycete Didymium iridis. Develop- factor in the myxomycete Didymium mental Biology 49, 283–287. iridis. Heredity 32, 231–239. Therrien CD, Haskins EF. 1981 – The nuclear Youngman PJ, Adler PN, Shinnick TM, Holt cycle of the myxomycete Echinostelium CE. 1977 – An extracellular inducer of minutum. II. Cytophotometric analysis of asexual plasmodial formation in the sporangial phase. Experimental Physarum polycephalum. Proceedings of Mycology 5, 229–235. the National Academy of Science 74, Therrien CD, Yemma JJ. 1974 – Comparative 1120–1124. measurements of nuclear DNA in a Youngman PJ, Anderson RW, Holt CE. 1981 – heterothallic and a self-fertile isolate of a Two multiallelic mating compatibility myxomycete, Didymium iridis. American loci separately regulate zygote formation Journal of Botany 61, 400–404. and zygote differentiation in the Therrien CD, Yemma JJ. 1975 – Nuclear DNA myxomycete Physarum polycephalum. content and ploidy values in clonally- Genetics 97, 513–530. developed plasmodia of the myxomycete Youngman PJ, Pallotta DJ, Hosler B, Struhl G, Didymium iridis. Caryologia 28, 313–320. Holt CE. 1979 – A new mating Therrien CD, Bell WR, Collins OR. 1977 – compatibility locus in Phy sarum Nuclear DNA content of myxamoebae polycephalum. Genetics 91, 683–693. and plasmodia in six non-heterothallic isolates of a myxomycete, Didymium

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