Proc. Nat. Acad. Sci. USA Vol. 72, No. 3, pp. 970-973, March 1975

Sexuality in the Cellular Slime Mold Dictyostelium giganteum (meiosis/mating types/self-incompatibility/macrocysts) GREGORY W. ERDOS, KENNETH B. RAPER, AND LINDA K. VOGEN Departments of Bacteriology and Botany, University of Wisconsin, Madison, Wisc. 53706 Contributed by Kenneth B. Raper, December 23, 1974

ABSTRACT By pairing of strains of Dictyostelium MATERIALS AND METHODS giganteum in various combinations this species was shown to be heterothallic. Four mating types were iden- Most of the cultures of Dictyostelium giganteum were obtained tified. Some strains could not be assigned a mating type from Mr. John Sutherland, formerly of this laboratory, and and others showed no mating reaction. No self-compatible were isolated by him from various prairie habitats in Wiscon- strains were found. Mutations were introduced in several strains and genetic crosses were performed. The results of sin. Others were obtained from the collection of cellular slime these crosses show that mating and macrocyst formation molds assembled and maintained in this laboratory. Stock are controlled by a single locus-multiple allele incom- cultures were maintained on phosphate-buffered 0.1% lactose/ patibility system. The results also support the view that 0.1% peptone agar, pH 6 (7) in association with the food the myxamoebae that emerge upon germination of the Forty-two different macrocysts are the products of meiosis. bacterium, Escherichia coli strain B/r. isolates were tested for their mating type by pairing them in In the cellular slime molds the macrocyst is a developmental various combinations according to the procedure described in alternative to the sorocarp. Macrocyst formation begins with earlier studies (4). Three isolates were selected for further the development of a large cell that increases in size as it genetic analysis: WS-588, WS-589, WS-606. Mutations were ingests surrounding myxamoebae (1, 2). Prior to their in- induced in these strains by means of N-methyl-N'-nitro-N- gestion the associated myxamoebae have formed the loose, nitrosoguanidine according to the method described by Weber fibrillar primary wall. The ingested myxamoebae are con- and Raper (8). After exposure to the mutagen myxamoebae tained in vacuoles of the enlarging macrocyst protoplast. were resuspended in liquid medium (9) and allowed to grow When ingestion is complete, the single large cell forms a rigid to a concentration of 106 cells per ml. They were then spread secondary wall and a more flexible, trilaminar tertiary wall on solid media containing 250 /Ag/ml of cycloheximide at (2). As the macrocyst matures the ingested amoebae are concentrations of 103 to 106 cells per plate. Resistant strains fragmented into smaller and smaller bodies until they are arose as plaques in the lawn of concurrently inoculated completely digested, giving the cyst a homogeneous ap- bacteria. Cells were also spread on nonselective media at 10 to pearance. After an appropriate period of maturation, the time 20 cells per plate and clonal isolates were tested for tempera- varying with the species, the protoplast of the macrocyst ture sensitivity at 270. cleaves into myxamoebae which escape when the walls break Mutant strains were crossed among themselves and with (3). wild-type strains. Crosses were made by techniques previously described for macrocyst formation in D. discoideum (4). s adgeinth celllar sim mldomeirom a ultrastructural Germination of the macrocysts was attempted after 5-6 weeks studyofsuch cyn oetI,where late of incubation. The macrocysts were removed from the plates in development meiotic chromosomes were observed (2). It and washed three times in a solution containing 250 ,g/ml of was also shown that the large cell of the nascent macrocyst was streptomycin sulfate. They were then resuspended in sodium at first binucleate but became uninucleate by the time in- dodecyl sulfate (0.0125%) and shaken for 2 hr, after which the gestion of the adjacent myxamoebae began. These facts in- cysts were again washed three times in the streptomycin solu- dicated that the macrocyst protoplast arose from the fusion of tion followed by 2 hr shaking in 0.7% EDTA. After three two cells followed by nuclear fusion and subsequent meiosis,\ more washes in streptomycin solution the macrocysts were thus making the giant cell a zygote. \\ spread on nonnutrient agar containing 250 ,/g/ml of strepto- It has been shown that there ar heterothaliic as ie 1 as mycin sulfate. This method was devised (10) to eliminate homothallic forms. This was first demonstrated in Dictyo- contamination of the germination plates by bacteria, fungi, stelium discoideum (4), where three intercompatible mating and extraneous slime mold spores and myxamoebae that may types were identified. Later, other species were also shown to have remained. Plates were examined daily for contamination have heterothallic forms (5). The heterothallic nature of by myxamoebae that were not the product of germination. If Dictyostelium giganteum Singh (6) is the subject of this report. such contamination was found the plates were discarded. This species is a member of the Dictyostelium mucoroides com- After 3 days in a lighted incubator at 250 the macrocysts plex and is characterized by strongly phototropic sorocarps began to germinate. that have long sinuous stalks bearing large, white sori con- Progeny were recovered by using a sterile needle to pick sisting of propagative spores. The mating system of this up individual sorocarps that had arisen from single macro- species as well as some preliminary genetic analyses are cysts. (Each germinated macrocyst generally yields one small presented here. sorocarp.) The source of each sorocarp was confirmed micro- 970 Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 72 (1976) Sexuality in Dictyodelium 971

TABLE 1. Macrocyst production from pairings of TABLE 2. Results of progeny analysis in crosses between single the four different mating types mutant strains and wild-type strains*

WS-589 WS-606 WS-607 WS-588 Al cycD X A2 cycD+ Al tsgA + X AS tsgA Al AS AS A4 + WS-598 cycD cycD+ Total tsgA tsgA Total Al _ + + + Al 33 27 60 Al 26 19 45 WS-606 AS 21 28 49 AS 27 27 54 AS + - + + Total 54 55 109 Total 53 46 99 WS-607 x2 = 2.66, P = 0.5 (3 df) x2 = 1.8, P = 0.65 (3 df) AS + + - + for a 1:1:1:1 segregation. fo. a 1:1:1:1 segregation. WS-588 A4 + + + AS cycC+ X A4 cycC AS tsgA X A4 tsgA+ cycC+ cycC Total tsgA tsgA + Total scopically. The spores from each sorocarp were suspended in A2 32 39 71 AS 28 33 61 0.6 ml of a heavy bacterial suspension in distilled water. The A4 32 34 66 A4 29 25 54 spore suspension was divided equally among five tubes, each Total 64 73 137 Total 57 58 115 containing 2.5 ml of molten (450) growth medium with 0.7% XI = 0.95, P = 0.8 (3 df) x2 = 1.24, P 0.75 (3 df) agar. These were quickly agitated and poured over plates of for a :1:1:1 segregation. for a 1:1:1:1 segregation. solid growth media. Clonal isolates appeared after 3 days at 250 as clear areas in the soft agar where they had consumed * Numbers presented in table represent macrocysts analyzed. the bacteria. These isolates were then subcultured and tested df = degrees of freedom. for mating type and mutant characteristics. In later experi- ments (see Results) spores from sorocarps were subcultured clones that emerged from any one macrocyst were genetically directly without clonal isolation. alike but that among the total macrocyst progeny all possible RESULTS combinations of markers could be observed in each cross. For this reason clonal isolation was later abandoned and spores Of the 42 isolates tested, four mating types (Al, A2, AS, and from sorocarps were subcultured directly in order to determine A4) could be clearly identified among 17 of them. The mating the identity of a given macrocyst. In the accompanying tables types are self-incompatible and each one is cross-compatible each number represents macrocysts analyzed, their identity with any of the other three types (Table 1). Seven strains being established by either clonal isolation or direct subculture showed inconsistent or aberrant patterns in their mating re- methods. In all, some 1400 macrocysts have been germinated actions and could not be assigned to any of the four mating and analyzed; a portion of this analysis is the subject of this types. Neither did the results justify designation of additional report. mating groups to accommodate them. Sixteen strains showed The results of the crosses performed are presented in Tables no mating reaction under any of the circumstances investi- 2-4. In those crosses where only one of the parents carries a gated, nor were any self-compatible (homothallic) strains mutant marker (Table 2), random segregation of the mating of D. giganteum observed. type and the mutant gene is observed among the progeny in Three strains were selected for further genetic analysis: each case, and thus recombination between mating type and WS-589 (Al), WS-606 (A2), WS-588 (A4). With mutants mutant gene was established. These data are consistent with derived from these strains crosses were performed. Mutants the expectation that a typical meiotic process is operating. cycC and cycD are resistant to cycloheximide at 250 /Ag/ml in There is no evidence that mating type is closely linked to any solid media and are otherwise developmentally normal. The of the mutant loci studied. When these same mutant strains tsgA mutant is temperature sensitive in failing to grow at 270, are crossed with each other (Tables 3 and 4), mating type but it develops normally at 22.50. Wild-type will develop segregates in a random fashion but recovery of the mutant normally at 300. markers is not random. In the cross summarized in Table 3 Under the conditions used for germination a small sorocarp the wild-type class for the mutant markers is significantly normally emerges directly from the broken cyst wall. The inflated relative to the other groups and the double mutant absence of food on the germination plates prevents the class shows the lowest representation. In the cross shown in myxamoebae from undergoing further vegetative growth and Table 4 the double mutant class is severely depressed with re- as a result they immediately aggregate and fruit in situ. In gard to the other groups, which are equally represented among this way the product of each individual cyst could be easily identified and isolated. This also helped to insure that the TABLE 3. Results of progeny analysis in the cross sorocarps were not contaminated by amoebae from other Al cycD tsgA+ X A2 cycD+ tsgA* germinating cysts. By this method a cyst-by-cyst analysis could be conducted. In the crosses analyzed the percentage of cycD cycD+ cycD cycD + germination varied with the cross-and was between 1% and tsgA + tsgA tsgA tsgA + Total 4%. The spores of the sorocarps arising from the germinated Al 12 5 3 21 41 cysts were of the haploid size (about 7.5 X 4.1%,um). AS 8 7 4 24 43 In the early analyses the spores from each sorocarp were Total 20 12 7 45 84 clonally isolated. An average of about 20 clones could be re- covered from a single sorocarp but on rare occasions as few as -9 -j 7A-%-_ two or as many as 100 were recoverable. It was found that all * Numbers presented in table represent macrocysts analyzed. Downloaded by guest on October 1, 2021 972 Genetics: Erdos et al. Proc. Nat. Acad. Sci. USA 72 (1975)

TABLE 4. Results of progeny analysis in the c708 cross involving cycC and tsgA (Table 4). To a lesser extent it is A2 cycC+ tsgA X A4 cycC tsgA +* also true of the cross involving cycD and tegA- (Table 3), where the double mutant class is significantly depressed in cycC+ cycC cycC cycC+ regard to the other classes. It may also be that these strains tegA tegA + tegA tegA + Total carry undetected genetic lesions, linked to the known markers, AS 11 5 4 9 29 that when acting in concert, reduce viability or affect some A4 7 13 1 10 31 com- 19 60 step in the germination process in cysts carrying certain Total 18 18 5 binatibns of these mutations. This type of interaction could well account for the uneven ratios in these crosses (Tables 3 x2 = 14.91,P 0.04 (7df) foraa 1:1:1:1: 1: 1 segregation. the low rate of macrocyst germination makes * Numbers presented in table represent macrocysts analyzed. and 4). However, definitive interpretation of these results difficult. the progeny. There is no indication of linkage in either of these Those progeny that show anomalous mating reactions are crosses. Crosses between strains containing cycC and cycD thought to be the result of some form of aneuploidy. The produced no viable macrocysts. breakdown of the "double maters" after several transfers sug- In most cases a small number of anomalous progeny (not gests that they are disomic and that the extra chromosome is shown in the tables) arise. Between 0 and 4.1% of the progeny eventually lost. Their instability tends to rule out their being do not mate with either of the parental types nor with any of the result of recombination within the mating-type locus. the other known mating types. This nonmating characteristic The non-maters may result from the loss of the chromosome, has been maintained through several clonal passages. A second or part of the chromosome, that carries the incompatibility ssmall group (0.9-5.5%) will mate with both parent types. locus. The presence of undetected sterility factors that inter- When clonal isolates are made of these types, three groups are fere with mating might also account for nonmaters among the recovered: those that will mate with one or the other parent, progeny, as well as for natural isolates that do not mate at all but not both, and those that will mate with both parents. or do not mate as expected. When those that maintained the double mating type were re- In a recent report on the homothallic species Dictyostelium grown and re-cloned, the three types emerged again. After mucoroides, evidence for meiosis via the macrocyst has been repetition of this process several times the "double maters" presented (14). Although the data are inconclusive, the as- were eventually lost and only those having a single mating sumption is probably correct. There is a strong indication that, type could be recovered. in this species, several duplications of the diploid nucleus occur, followed by multiple meiotic events. Additionally, from DISCUSSION the ratios presented by MacInnes and Francis, there is a From the data showing that new genetic combinations are distinct indication of mitotic selection following the meioses. possible via the macrocyst, several steps in the formation and These complications in the process make accurate interpreta- development of that structure become clear. There must be a tions of the results difficult. The system in D. giganteum fusion of cells of opposite mating type followed by the fusion seems to be free of both these complications, indicating that of their respective nuclei in order to obtain the results ob- this species might be more amenable to genetic analysis. served. Additionally, there must be some method for the The accumulation of evidence concerning the macrocyst of haploidization of the diploid nucleus. The results presented in the cellular slime molds now leaves little doubt that it is the Table 2 are consistent with a meiotic pattern of segregation, site of a fairly typical sexual process, barring some bizarre and it can therefore be assumed that meiosis is the system of situation heretofore unknown. We can conclude that mating haploidization. The fact that only one type of progeny can be in D. giganteum (and probably in other heterothallic species recovered from any one cyst, while all types are present in the as well) is controlled by a single locus-multiple allele system, total progeny, indicates that three of the four meiotic nuclei four alleles having been identified. Furthermore, such mating are lost in a random fashion. This same situation can be found results in the formation of a true zygote which undergoes in the fungus Rhizopus stolonifer (11), in the soil amoeba meiosis yielding haploid progeny of new genetic associa- Sappinia (12), and in a species of the plasmodial slime mold tions. Physarum (13), where it has been shown that three of the meiotic nuclei are destroyed in autophagic vacuoles. This pat- We are grateful to Dr. Lindsay S. Olive and Dr. Thomas J. in Leonard for critical reading of the manuscript. This work was tern also parallels the formation of polar bodies higher supported by National Science Foundation Grant GB-40545 animals and the degeneration of three of the four megaspores and National Institutes of Health, U.S. Public Health Service in the ovules of seed plants. We suggest that the lost meiotic Grant AI-04915. products may degenerate in a fashion similar to that found in Physarum, although cytological evidence in support of this 1. Filosa, M. F. & Dengler, R. E. (1972) "Ultrastructure of notion is lacking. macrocyst formation in the cellular slime mold, Dictyo- in the crosses stelium mucoroides: Extensive phagocytosis of amoebae by a The nonrandom segregation of the markers specialized cell," Develop. Biol. 29, 1-16. containing two mutations (Tables 3 and 4) is likely due to the 2. Erdos, G. W., Nickerson, A. W. & Raper, K. B. (1972) nature of these mutations and their combined effects on the 'The fine structure of macrocysts in Polysphondylium progeny rather than to some complexity of segregation, since violaceum," Cytobiologie 6, 351-366. singly the markers segregate in a random fashion and each 3. Erdos, G. W., Nickerson, A. W. & Raper, K. B. (1973) "The fine structure of macrocyst germination in Dicdyo- appears to represent a mutation at a single locus. The genetic stelium mucoroides," Develop. Biol. 32, 321-330. damage that caused these mutations may be so great as to 4. Erdos, G. W., Raper, K. B. & Vogen, L. K. (1973) "Mating adversely influence the viability of the macrocysts whose types and macrocyst formation in Dictyostelium discoi- progeny carry both markers. This is particularly true of the deum," Proc. Nat. Acad. Sci. USA 70, 1828-1830. Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 72 (1976) Sexuality in Dictyostelium 973

5. Clark, M. A., Francis, D. & Eisenberg, R. (1973) "Mating slime mold Dictyostelium discoideum in liquid nutrient types in cellular slime molds," Biochem. Biophys. Res. medium," J. Gen. Microbiol. 25, 375-378. Commun. 52, 672-678. 10. Wallace, M. A. (1974) "Cultural and genetic studies of the 6. Singh, B. N. (1947) "Studies on soil Acrasiae. 1. Distribution macrocyst in Dictyostelium discoideum," M.S. thesis, Uni- of species of Dictyostelium in the soils of Great Britain versity of Wisconsin, Madison. and the effect of bacteria on their development," J. Gen. 11. Gauger, W. (1961) "The germination of zygospores in Rhizo- Microbiol. 1, 11-21. pus stolonifer," Amer. J. Bot. 48, 427-429. 7. Nickerson, A. W. & Raper, K. B. (1973) "Macrocysts in 12. Wenrich, D. H. (1954) in Sex in Micro-organisms (American the life cycle of the Dictyosteliaceae. I. Formation of the Association for the Advancement of Science, Washington, macrocysts," Amer. J. Bot. 60, 190-197. D.C.), pp. 134-265. 8. Weber, A. T. & Raper, K. B. (1971) "Induction of fruiting 13. Aldrich, H. C. (1967) "The ultrastructure of meiosis in three in two aggregateless mutants of Dictyostelium discoideum," species of Physarum," Mycologia 59, 127-148. Develop. Biol., 26, 606-615. 14. MacInnes, M. & Francis, D. (1974) "Meiosis in Dictyo- 9. Sussman, M. (1961) "Cultivation and serial transfer of the stelium mucoroides," Nature 251, 321-324. Downloaded by guest on October 1, 2021