DEVELOPMENTAL

BIOLOGY

43, 240-253 (1975)

Mating

Type and the Differentiated

State in

Ph ysarum polycephalum PAUL Department

of Biology,

N. ADLER

Massachusetts

AND CHARLES

Institute

Accepted

E. HOLT

of Technology,

December

Cambridge,

Massachussets

02139

12, 1974

In the acellular slime mold, Physarum polycephalum, the differentiation of amoebae into plasmodia is controlled by a mating type locus, mt. Amoebae carrying heterothallic alleles usually do not differentiate within clones; plasmodia form when two amoebae carrying different alleles fuse and undergo karyogamy. In this paper, we show that amoebae heterozygous for heterothallic alleles can be isolated and maintained as amoebae; the amoebae form plasmodia in clones without a change in ploidy. Plasmodia were also found to be formed, infrequently, by heterothallic amoebae of a single mating type. The plasmodia are healthy and are also formed without a change in ploidy. Thus, the presence of two different heterothallic mating type genes in a single nucleus is compatible with the amoeba1 state and one heterothallic mating type gene is compatible with the plasmodial state, once established. INTRODUCTION

The Myxomycete life cycle includes two strikingly different vegetative forms, small uninucleate amoebae and large multinucleate plasmodia (Gray and Alexopoulos, 1968). Amoebae differentiate into plasmodia by one of two modes: sexual or clonal. In Physarum polycephalum, the mode is controlled by a mating type locus. Amoebae carrying the mth allele, which is derived from the Colonia isolate, form haploid plasmodia in clones. Amoebae carrying heterothallic alleles, such as mt3, do not ordinarily form plasmodia in clones; instead, a diploid plasmodium develops from a zygote formed by the fusion of two haploid amoebae carrying different mating type alleles, such as mt3 and mt4. At least 12 different heterothallic alleles have been identified (Dee, 1973; Collins, 1974). The mth amoebae can also partake in sexual plasmodium formation by mating with heterothallic amoebae (Wheals, 1970; Wheals, 1973; Adler and Holt, 1974a; Cooke and Dee, 197413). Self-sterility systems at least formally similar to those in Myxomycetes have been found in fungi (Raper, 1966), plants (Lewis, 1954) and animals (Morgan, 1942).

It was originally thought that the amoebal and plasmodial states in Myxomycetes were associated with haploidy and diploidy, respectively. It is now clear that this is not necessarily the case. Amoebae of various ploidys have been described (Ross, 1966; S. Kerr, 1968; Mohberg et al., 1973; Therrien and Yemma, 1974; Adler and Holt, 1974b) and haploid, diploid and higher ploidy plasmodia have been shown to exist (S. Kerr, 1968; Mohberg et al., 1973; Cooke and Dee, 1974a). The mechanism of clonal plasmodium formation has been described as homothallit or apogamic in various papers (Gray and Alexopoulos, 1968) but conclusive evidence distinguishing these mechanisms is generally lacking. In the case of a mutant strain of Didymium nigripes, Kerr (N. S. Kerr, 1967) has established by microcinematography that plasmodium formation is not preceded by cell fusion, that is, it is apogamic. Although the Colonia isolate of Physarum polycephalum was originally described as homothallic (Wheals, 1970), the recent demonstration (Cooke and Dee, 1974a) that there is no change in ploidy when plasmodia are formed rules this out. Whether Colonia 240

Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

ADLER AND HOLT

Mating

amoebae develop directly into plasmodia or first fuse with other amoebae (without undergoing karyogamy) is not known, We refer to amoebae that form plasmodia in clones as CPF (clonal plasmodium forming) amoebae without regard to the genetic or cellular basis of their plasmodium-forming behavior. Plasmodia formed clonally by CPF amoebae are designated CPF plasmodia. The present work was stimulated by the finding reported here and by others (Collins, 1961, 1963; Dee, 1966; Haugli, 1971) that platings of spores obtained from plasmodia formed by crossing two heterothallic amoeba1 strains sometimes yield amoeba1 plaques that give rise to plasmodia. One explanation that has been suggested (Dee, 1966; Collins, 1961) is that a single spore, containing two amoebae of different mating types, gives rise to the plasmodiumforming plaques. Indeed, Ross (1967) has found binucleate amoebae emerging from aberrant spores. Such a binucleate amoebae did not develop into a plasmodium directly, but first divided to form two uninucleate amoebae. Collins has suggested mutation at the mt locus in an amoeba within the colony as an explanation in one case (Collins, 1965), but the frequency of plasmodium-forming plaques seems too high for this to be a general explanation. Collins and Ling (1968) have found that heterothallic amoebae of D. iridis can occasionally form plasmodia in clones (we refer to such plasmodia as illegitimate plasmodia), and they suggested this as another explanation for plasmodium-forming clones obtained in spore platings. We have confirmed that such illegitimate plasmodia are formed in P. polycephalum and will describe them in this paper. However, plasmodia are produced in this way only infrequently, after a long time, and by very large populations of amoebae, so we feel it is unlikely that illegitimate plasmodia are responsible for many of the plasmoida yielding clones in

241

Type and Differentiation

spore platings in P. polycephalum. Finally, it is possible that plasmodium-forming plaques are mating type recombinants, but no evidence for this hypothesis has been obtained. Haugli et al. (1971), in analyzing a cross of two heterothalic amoeba1 strains of P. polycephalum, one of which was polyploid, found a large number of progeny clones that would form plasmodia clonally even after recloning. We have isolated and analyzed such CPF clones from similar crosses. When spores from a plasmodium derived clonally from such a progeny clone are analyzed, both heterothallic and CPF amoebae are obtained. In all cases, both of the heterothallic mating types present in the plasmodium that gave rise to the original clone of CPF amoebae are recovered. Thus the parental CPF amoebae are heterozygous for the mating type locus. The DNA content of several of the amoebal strains that are heterozygous for mt has been determined. The strains were found to have a near diploid DNA content. As in clonal plasmodium formation by m th amoebae, clonal plasmodium formation by the strains heterozygous for mating type and illegitimate plasmodium formation do not result in a ploidy change. The frequency of isolation of amoeba1 strains heterozygous for mt, their stability, and the ability of the CPF amoebae to form a plasmodium by mating with other amoebae has been investigated. The frequency of illegitimate plasmodium formation has also been studied. METHODS

AND

MATERIALS

Culture procedures. Amoebae and plasmodia were grown, plasmodia were formed from amoebae either clonally or by crossing, sporulation was induced, and spores were germinated as described previously (Adler and Holt, 1974a; Newlon et al., ( 1973). Strains. Strain numbers, with relevant phenotype and history, are listed in Table 1. Amoeba1 strains expressing the recessive

242

DEVELOPMENTALBIOLOGY TABLE

VOLUME 43, 1975 1

STRAINS Other designation CL CH2 CH4 CH5 CH6 CH9 CHlO CH21

Progeny (or mutant) of

iI73 B174 RSD4 (actA N169) x RSD8 APT 1 (C-50) CH2 x CH9 3rd gen” inbred

Genotype

mth mtl saxmt3 mt4 tml actA N169 mth apt 1 mtl mt3

References

Dee, 1962 Dee, 1966 Dee, 1966 Haugli, Jiminez and Dove, 1972 Wheals, 1973 Adler and Holt, 1974a

mt3 CH22 CH23 CH40-44, CH45a, CH9’7, CH98 CH52 CH53 CH54 CH107 CH153 CH175

CH6 x CH4 CH6 x CH4 CH23 x CH5

mtllmt3 actA N169 mt3 actA N169 mt3/mt4 actA N169/i

CHlO x CH23 x 5th gen” 5th gen”

mtllmt4 mt3/mt4 mt3 mt3 eme E4 mth ebo E4 mt3

CH190

7th gen” inbred

CH5 CH5

mt3 mt3

(CL) 5th gen” inbred

Adler and Holt, 1974a Adler and Holt, 1974a Adler and Holt, 1974a

mt3 mt3

Adler and Holt, 1974a

mt4

Adler and Holt, 1974a

mt3

Adler and Holt, 1974a

mt3 CH195

5th gen” inbred

mt4 CH202

7th gen’ inbred

mt3 0 Gen = generations

inbred to CL (Adler and Holt,

marker actA N169 (Haugli et al., 1972) will form plaques in the presence of 16 pg/ml of actidione. The eboE4 allele was obtained from CL mutagenized with ethylmethane sulfonate and confers partial resistance to ethidium bromide. Amoebae carrying it will produce 2.5 mm plaques after 14 days on plates containing 2 pg/ml of ethidium bromide, while CL will produce no plaques under these conditions. Amoebae carrying the eboE4 allele will also produce plaques at 4 pg/ml of ethidium bromide. of phenotypes. Amoeba1 Determination strains were tested for CPF phenotype by growing them in a “puddle” of E. coli on a dPRM (dilute plasmodial rich medium) plate (Adler and Holt, 1974a). All amoeba1 strains that did not form plasmodia clo-

1974a).

nally on the dPRM plate were tested for their heterothallic mating type specificity by coinoculating them with at least two tester heterothallic strains on dPRM plates (also referred to as mating plates). A strain with a mt3 heterothallic specificity forms plasmodia when cocultured with mt4 or mtl strains, but not with mt3 strains. Plasmodial fusion behavior and drug resistance were determined as described previously (Adler and Holt, 1974a). Actidione (CalBiochem) and ethidium bromide (Boots Pure Drug Co.) were filter sterilized. Cytological procedures. Plates of amoebae were incubated at 0°C overnight to ensure that all amoebae had encysted. Encysted amoebae were washed off the plates in 0°C distilled water with a bent

ADLER AND HOLT

Mating

glass rod (Haugli, 1971) and harvested by centrifugation. The amoebae were washed twice in 0°C distilled water and then fixed in Acetic Alcohol (3:l glacial acetic acid:lOO% ethanol) for 15 min. The fixed cells were put through two changes of absolute ethanol and attached to microscope slides. The amoebae were stained by the Feulgen technique (Humason, 1962) using a 15 min, 25 “C hydrolysis in 6 N HCl. Unhydrolyzed controls were included in all experiments. Basic fuchsin was obtained from Sigma. Coverslips were mounted in Eukitt (Calibrated Instruments) and stain density determined on a Vickers M85 scanning microspectrophotometer at 560 nm with a band width of 35 nm. Plasmodial nuclei were isolated by a modification (Newlon et al., 1973) of the Mohberg procedure (Mohberg and Rusch, 1971), and were treated in the same manner as the washed amoeba1 cells. Staining densities for plasmodial nuclei and amoebae were not comparable using this procedure, perhaps because of the different isolation media. The diameter of isolated plasmodial nuclei was measured under phase optics using a scaled ocular at 1250 x magnification (oil immersion). of position in cell cycle. Determination The time of mitosis was determined cytologically by the procedure of Guttes (Guttes et al., 1961). The timing of DNA synthesis was determined by pulsing pieces of plasmodia for 20 min with tritiated thymidine by the procedure of Muldoon (Muldoon et al., 1971). RESULTS

Analysis of CPF progeny. Analysis of the cross CH23 (mt3 actA N169) x CH5 (mt4 act+) revealed that nine out of 100 progeny amoeba1 strains formed plasmodia in clones. All nine strains maintain the CPF phenotype through repeated recloning, and cells of the strains appear as uninucleate amoebae with no special tendency toward

243

Type and Differentiation

clumping under the phase microscope. Plating efficiency of the strains is approximately loo%, which eliminates the possibility that only mixed colonies can form plaques. All nine strains are sensitive to actidione. The CPF plasmodia formed by these strains have been fusion tested with the parent plasmodium and five of the nine do not fuse compatibly with the parent, showing that segregation of some genes occurred during the formation of at least five of the nine strains (genetically identical plasmodia fuse compatibly (Carlile and Dee, 1967)). A CPF plasmodium of each strain was sporulated, and progeny resulting from the spores were analyzed (Table 2). Both mt3 and mt4 amoebae were recovered from all nine of the CPF plasmodia. Thus all nine strains are heterozygous for the mt locus. Actidione resistant progeny were also recovered from eight of the nine CPF strains (Table 2), showing that eight of the strains were heterozygous for the actA locus and that the actA N169 allele is recessive in amoebae. The varying percentage of CPF progeny recovered from different CPF plasmodia will be dealt with later. Amoeba1 CPF progeny have also been isolated, but at a lower frequency, from crosses involving mtl. One CPF progeny TABLE

2

ANALWS OF PROGENY OF CPF PLASMODIA CPF Plasmodium

Progeny mt3 Acts”

CH40 CH41 CH42 CH43 CH44 CH45a CH53 CH9i CH98

3 4 4 1 4 5 8 2 6

mt4

CPF

ActR

Acts

ActR

4 3 3 0 5 2 0 1 2

3 8 2 4 2 8 9 2 4

2 5 6 2 2 3 0 2 6

Acts 7 0 5 11 5 2 3 13 2

o Growth was tested at 16 pg/ml actidione. sensitive, ActR = resistant.

ActR 1 0 0 2 2 0 0 0 0 Acts =

244

DEVELOPMENTALBIOLOGY TABLE

VOLUME 43, 1975 3

ANALYSIS OF PROGENY OF CPF PLASMODIA Parent of CPF strain

CHlO (mtl) x CH5 (mt4) CH4 (mt3) x CH6 (mtl) CH175 (mt3) x CH195 (mt4) CH175 (mt3) x CH195 (mt4) “This

CPF plasmodium

CH52 CH22 CH175 x CH195:35” CH175 x CH195:2

refers to progeny number 35 from the indicated

Progeny mating behavior mtl

mt3

mt4

CPF

8 3 -

2 4 3

9 1 1

32 12 11

plasmodium.

CH107 has previously been shown to have strain from the cross CHlO(mt1) x CH5 aged and have a DNA content of 2.5 C (mt4) was obtained out of 60 progeny (Adler and Holt, 1974b). Thirteen progeny amoeba1 strains examined, and both mtl clones were isolated and 12 of these are and mt4 progeny were recovered from CPF. Six of these 12 strains form plasspores obtained from CPF plasmodia promodia at a much smaller plaque size than duced by this strain (Table 3). One CPF progeny amoeba1 strain was found in 40 is normally seen in mth strains, and the rate of plasmodium formation is not slowed progeny strains from the cross CHG(mt1) x at 30°C compared to 26°C as is found for CH4(mt3). As expected, mtl and mt3 mth strains (Adler and Holt, 1974a). A progeny are recovered (Table 3) from CPF plasmodium formed from one of the spores of the CPF plasmodium, although strains that was sensitive to ethidium brothe high frequency of CPF progeny from mide has been sporulated and the spores spores of the CPF plasmodium requires analyzed. Both CPF and mt3 strains are that a larger number of progeny be examrecovered in the progeny (Table 4), and ined than in other experiments. most or all of the CPF strains show the mth We have also obtained CPF amoebae phenotype with respect to temperature and from crosses involving mt3 and mt4 strains plaque size at the time of plasmodium that have been extensively inbred to strain formation. Ethidium bromide resistant CL (Adler and Holt, 1974a). Two such CPF strains (CH175 x CH195:2 and CH175 x and sensitive progeny are also recovered showing that the CPF strain is heterozyCH195:35) have been analyzed and in both gous for the ebo locus as well as mating cases mt3 and mt4 progeny were recovered type. The results also show that eboE4 is (Table 3). A varying proportion of progeny recessive. Strains that show the extremely recovered from crosses involving various inbred mt3 and mt4 strains have been rapid plasmodium forming phenotype are CPF. In four cases no CPF progeny were seen routinely at a low frequency in crosses found among the 40-50 progeny examined. In one case two out of 40 were CPF and in TABLE 4 another four out of 40. In one cross (CH175 ANALYSIS OF PROGENY OF PLASMODIUM FORMED FROM AMOEBAE CONTAINING mth AND mt3 x CH195) 32 out of 40 progeny examined were CPF. CPF mt3 Amoebae heterozygous for the mating eboS” eboR eboS eboR type locus, containing mth as one of their two mating type alleles, have also been Number of 7 8 6 7 progeny isolated. Spores from a plasmodium formed by crossing CH153 (mth eboE4) u Growth was tested on 2 pg/ml ethidium bromide. with CH107 (mt3) appeared large and Plates were scored after 2 wk. S = sensitive, R = malformed, which is not surprising as resistant

ADLER AND HOLT

Mating

involving mth and mt3 or mt4. Because of the plaque size at which plasmodia form, it is very difficult to grow up and maintain stocks of such strains and we have not studied them any further. DNA content of CPF amoebae. The cellular DNA content of several strains has been determined by Feulgen staining and microdensitometry. Only encysted amoebae were used to eliminate problems due to cells being in different stages of the cell cycle. Mohberg (Mohberg et al., 1973) has concluded that encysted amoebae are arrested in Gl. Staining density measurements for most strains gave a unimodal peak (see Fig. 1). Histograms of CL, our haploid wild type, CH23, a mt3 strain with a high DNA content, and CH52 and CH42, both of which are heterozygous for the mating type locus, are shown (Fig. 1) for comparison. Mean DNA contents and C values for a large number of strains were determined in one experiment (Table 5). Strains CH2 and CH54 appeared identical to CL and an analysis of variance showed that the interstrain variance is not significantly greater than the intrastrain variance. All of the strains that are heterozygous for mating type (CH52, 42,22,45a, 44, 40, 43) give near diploid DNA contents, with a range of 1.70-2.23 C. Several heterothallic strains (CH4, CH5, CH23) also have a near diploid DNA content. When all of the strains with a near diploid DNA content are compared the interstrain variance is not significantly greater than the intrastrain variance. There is a significant difference when certain combinations, such as CH4 and CH5, or CH44 and CH22, are compared, however, and this may be evidence that at least some of the strains are aneuploid and not diploid. In any case, because of the large number of chromosomes (1 C = 20-25) (Mohberg et al., 1973) in P. polycephalum it is not possible to distinguish between a diploid and a near diploid that is monosomic or trisomic for a few chromosomes by this technique. It is also not possible by this method to show

245

Type and Differentiation

4

8 I2 Densftometer

24

16 20 Reading

28

FIG. 1. Histograms of densitometer readings Feulgen stained slides of four amoeba1 strains. TABLE DNA

for

5

CONTENT OF STRAINS"

Strain

CL (mth) CH4 (mt3) CH5 (mt4) CH6 (mtl) CH23 (mt3) CH22 (mtllmt3) CH40 (mt3/mt4) CH42 (mt3/mt4) CH43 (mt3/mt4) CH44 (mt3/mt4) CH45a (mt3/mt4) CH52 (mtllmt4) CH2 (~1) ‘CH54 (mt3) ‘CH54 (mt3)

Densitometer reading mean

sd

11.96 22.20 26.96 14.23 23.23 27.73 22.08 21.24 24.36 21.29 20.29 24.27 13.14 12.47 12.36

1.37 2.11 1.89 1.79 1.84 2.11 2.79 1.85 2.57 1.16 2.61 1.82 0.97 1.00 1.42

C*

1.00 1.86 2.25 1.19 1.94 2.23 1.85 1.79 2.04 1.78 1.70 2.03 1.10 1.04 1.03

“Cells of all strains in this table gave unimodal distributions of DNA content. * C = staining density of the strain/staining density of CL. ‘Two independent slides of CH54 were run as a check of the reproducibility of the technique.

that a cell with a diploid DNA content is diploid for all chromosomes, and not monosomic for some and trisomic for others. DNA content of CPF plasmodial nuclei. The relative DNA content of plasmodial nuclei has been determined by measuring Feulgen staining density. A complication in measuring the DNA content of plasmodial nuclei arises from the existence in a

246

DEVELOPMENTALBIOLOGY

single plasmodium of nuclei of varying DNA content (Mohberg et al., 1973) and the change in nuclear DNA content seen over time (S. Kerr, 1968; McCullogh et al., 1973). To minimize confusion from the latter phenomena, when possible (for all but illegitimate plasmodia, which are discussed later) measurements were made on nuclei from plasmodia that had developed from amoebae less than 3 wk earlier. To insure that all nuclei were in the same stage of the cell cycle, mitosis was determined cytologically and S period by 3Hthymidine pulses. All Feulgen stained nuclei were isolated from plasmodia in G2. The frequency profile for CL plasmodial nuclei, which have been shown by Cooke and Dee to be haploid, gave a single peak with a shoulder to a 2 C DNA content (Fig. 2). Plasmodia formed clonally from CH52 and CH43, which are heterozygous for mating type, gave a principal peak at twice (2 C) the staining density of the CL peak (Fig. 2). The peak for CH43 shows a

15-

VOLUME 43, 1975

prominent shoulder toward high DNA levels. A plasmodium formed by crossing two amoebae of different mating type (CH202 x CH195) gave peaks at 2 and 3 C (Fig. 21. Plasmodial nuclear ploidy has also been estimated by measurements of nuclear size. It has been reported that plots of nuclear surface area and DNA content are linear and go through the origin (Mohberg et al., 1973). We have confirmed this and used the relationship to get estimates of DNA content. Estimates of DNA content determined by mean nuclear size and mean Feulgen staining density are shown in Table 6. CL has been used as a standard 1 C DNA content for both amoebae and plasmodia (Cooke and Dee, 1974a). The C values for both amoeba1 and plasmodial nuclei for the strains CH52 and CH43 are approximately two. We take this as evidence that the amoebal-plasmodium transition in amoeba1 strains heterozygous for mating type, as in strains carrying mth, does not result in a change in ploidy. Frequency of CPF progeny from CH23 x CH5. The percent of CPF progeny obtained from spores of CPF plasmodia is highly variable (Tables 2 and 3). We have examined this variation in the hope of learning something about the timing of the event

CH52

TABLE

6

PLOIDY ESTIMATES OF AMOEBAE AND PLASMODIA Strain

CL CH52 CH43 CH21-a CHlSO-a CH202 x CH195 I

IIll 4

8 Densltomeler

IllI 12

I6 Readcng

I 20

FIG. 2. Histograms of densitometer readings for slides of Feulgen stained G2 nuclei from five plasmodia.

Plasmodia C from Feulgen”

C from nuclear size”

1 1.86 2.06 1.20 2.36b

1 1.84 1.78 1.11 1.18 2.52

Amoebae C

1 2.03 2.04 1.37 1.08’

“Values are weight averages which are not corrected for the existence of multiple classes. bThis plasmodium contained discrete peaks of both 2 C and 3 C nuclei (see Fig. 2). c Value for CH195; no measurement on CH202 was made.

ADLER AND HOLT

Mating

that causes a CPF amoeba to be produced. Unless otherwise stated, all of the spores used in these frequency studies came from CH23 x CH5 plasmodia that had been grown up and sporulated directly after being transferred from mating plates, so as to eliminate possible complications due to plasmodial aging. Spores resulting from a single plasmodium have been assayed several times (Table 7) over a period of several months. Only random fluctuations were seen with a mean of 60.5% CPF progeny. In a second experiment, spores from eight plasmodia, that had been formed at the same time from the same stock cultures of CH5 and CH23, and grown and sporulated under the same conditions, were assayed for CPF progeny (Table 8). The frequency of CPF progeny ranged from lo-82%, and the results were distributed in a decidedly nonrandom fashion. The data in Tables 7 and 8 suggest that a premeiotic event, the effects of which can replicate during the growth of the plasmodia, is responsible for the production of the CPF progeny. A possible explanation is that mitotic failure early in the development of the plasmodium or multiple amoebal fusions at the time of plasmodial formation (Ross and Cummings, 1970) would create a plasmodium with polyploid nuclei. The polyploid nuclei could undergo meiosis to give progeny heterozygous for the mating type locus. Both parental amoeba1 strains have a high DNA content (Table 5) which would seem to preclude any need for either of the events mentioned above to create polyploid plasmodial nuclei. DNA measurements, however, were made 6-9 mo after the frequency studies were done, and both strains are known to have undergone an increase in nuclear DNA content (Adler and Holt, 1974b) during the interval. The frequency of CPF progeny seems to increase as spores from successively older plasmodia are tested. On one occasion spores obtained from the first, second, and

247

Type and Differentiation TABLE

7

FREQUENCV OF CPF PROGENY FROM MULTIPLE SAMPLINGS OF A SPORE PLATER Sample number

Number of CPF

% CPF

1

29

2 3 4

30 35 27

58 60 70 54

30.25

60.5

mean

0 Fifty progeny were examined in each experiment. TABLE

8

FREQUENCY OF CPF PROGENY FROM EIGHT INDEPENDENT SPORE PLATES~ Plate number

‘3%CPF

1

29

58

2 3 4

41 18

5 6

6 34 5 28

82 36 22 12 68

7 8

mean “Fifty spores.

Number CPF

11

21.5

progeny were examined

10 56 43%

from each batch of

third passage plates (not enough viable spores could be obtained from later passage plates, as plasmodia of this strain senesce very rapidly) were assayed for the percentage of CPF progeny. No difference was seen between the first (71% CPF) and second (70% CPF) passage spores; however, an increase was seen in the third passage (89% CPF) spores. This correlates well with the increase in nuclear DNA content upon aging (McCullogh et al., 1973); however, since only one plasmodium has been followed, it is premature to make any general conclusions. Stability of the heterozygous state. During the initial stages of the work with the amoeba1 strains heterozygous for mating type it became clear that the heterozygous state was relatively stable, as no hetero-

248

DEVELOPMENTALBIOLOGY

thallic segregants were seen among over 1000 clones examined. We have examined the stability of heterozygosity at the actA locus, by looking for the production of actidione resistant amoebae from three sensitive heterozygotes (CH42, CH43, and CH441. In these experiments, amoeba1 clones of about lo6 cells each were plated at l-2 x 10’ cells per plate on LIA containing 16 pg/ml actidione. The frequency of actidione resistant segregants in the 11 clones tested ranged from about 0.5 to 20 x 10m5 with all three of the strains tested yielding similar frequencies. Ability of a mating type heterotygote to mate. Amoebae of strain CH22 (mtllmt3 actA N169) were mixed with an excess of CH5 (mt4 act+) amoebae on mating plates. Strain CH22 is actidione resistant and is therefore either homozygous or hemizygous for act N169. Two of the four plasmodia derived from the mating plates were found not to fuse with a clonally formed CH22 plasmodium, suggesting that they were the result of a cross. One of the two presumptive crossed plasmodia was sporulated, and progeny clones from the spores analyzed (Table 9). Fifty-two of 60 progeny were CPF. Among the remaining eight progeny were amoebae of mtl, mt3 and mt4. Both actidione resistant and sensitive recombinants were found. One explanation for these data is that amoebae heterozygous for mating type can mate with amoebae of a third mating type. A second interpretation of these data is that mtl and mt3 hemizygotes or homozygotes segregated from the mtllmt3 heterozygotes on the mating plate, that these crossed with the mt4 amoebae, and that the resulting plasmodium was a heterokaryon resulting from somatic fusion of plasmodia, with individual nuclei containing only two mating type genes. If heterozygosity at the mating type locus shows a stability similar to that of the actA locus, then the second interpretation would be unlikely. Plasmodia containing one heterothallic

VOLUME 43, 1975 TABLE

9

ANALYSIS OF PROGENY OF CH22 x CH5 CPF CPF CPF mtl mtl mt3 mt3 mt4 mt4

Acts” ActR Act not determined ActR Acts ActR Acts ActR Acts

total

10 13 29 2 1 1 0 1 -3 60

a Actidione sensitivity tested by growth on plates containing 16 pg/ml actidione. S = sensitive, no growth; R = resistant, growth.

mating type. To see whether illegitimate plasmodium formation occurs in P. polycephalum, heterothallic amoebae were inoculated onto dPRM plates and incubated at 26°C. Plates were examined at weekly intervals for 4 wk. Under these conditions net growth is complete in 4-6 days. Illegitimate plasmodia generally appear during the second and third weeks of incubation on a varying percentage of plates. A few illegitimate plasmodia have appeared during the fourth week, and none has ever been seen at 1 wk of incubation. We have not attempted to determine if more would appear if the plates were incubated for more than 4 wk. Plates on which illegitimate plasmodia appear contain only one or two areas of plasmodium formation. When CPF plasmodia are formed by mth amoebae at 26°C or by amoebae heterozygous for mating type, plasmodia appear all over the plate. A similar result is found for crossed plasmodium formation. Progeny spores obtained from seven illegitimate plasmodia formed by mt3 amoebae have been analyzed (Table 10). Only mt3 progeny have been obtained. Some of these “illegitimate plasmodia” arose after mutagenesis of amoebae; however, the data suggest they were not mutants. Ploidy of illegitimate plasmodia. The ploidy of illegitimate plasmodia has been

ADLER AND HOLT

Mating

Type and Differentiation

249

mate plasmodium formation by different strains has been examined (Table 11). Several things are apparent. There is quite Plasmodian Progeny Mutagenized a bit of variation in the frequency of mtx mt3 Total illegitimate plasmodium formation between strains such as CH21, CH54, and 0 33 CH54-a 33 + 0 20 20 CH54-b + CH190, even though these strains are ge12 0 12 CH54c + netically similar. A possible explanation 20 0 20 CH54-d + for this is that CH21 and CH190 were aging 20 0 20 CH21-a while CH54 was not, and perhaps there are 0 20 20 CHZl-b aneuploid states which have a high fre20 0 20 CHlSO-a quency of illegitimate plasmodium formaa -a, -b, etc. distinguish plasmodia isolated indetion. Aneuploidy per se probably does not pendently from the same amoeba1 strain. promote illegitimate plasmodium formation since CH4 and CH5, both of which estimated by Feulgen staining density and have a high DNA content (Table 5), form nuclear size. Plasmodial nuclei of an illeillegitimate plasmodia at a low frequency. gitimate plasmodium formed by strain Collins and Ling (1968) reported variaCH21 (Fig. 2) gave a peak at a staining tions in the frequency of illegitimate plasdensity slightly higher than that of the CL modium formation between amoeba1 subpeak. Estimates from nuclear size agreed lines in D. iridis. We have examined six and both estimates are in the range sublines of CH190 and seen (Table 12) only 1.11-1.20 C (Table 6). An estimate of random variations among sublines. Perploidy by nuclear size for an illegitimate haps our stock culture had been recloned plasmodium of CH190 (Table 6) gave a more recently than theirs had, so that value of 1.18 C. The amoebae of both CH21 sufficient heterogeneity did not build up. and CH190 were early in the process of Cold shock, heat shock, UV irradiation, aging (Adler and Holt, 1974b) during the and fresh medium after 1 wk did not formation of the illegitimate plasmodia, SO stimulate illegitimate plasmodium formathat the values of about 1.2 C may reflect tion. aneuploidy. TABLE 11 Yemma and Therrien (1972) have preFREQUENCY OF FORMATION OF ILLEGITIMATE sented Feulgen measurements on plasPLASMODIA~ modial nuclei of illegitimate plasmodia in PlasStrain Percentage of cultures D. iridis. They pointed out that their data with plasmodia modial are compatible with either haploid nuclei viabilityb Expt. Expt. Expt. in G2 or diploid nuclei in Gl. They favored 1 2 3 the latter interpretation and assumed that the illegitimate plasmodia had an ex- CH4 (mt3) 2 0 18 2 2 + tended Gl. Gl has not been reported in P. CH21 (mt3) 0 CH54 (mt3) 0 polycephalum (Rusch, 1970) and they did 18 12 14 1 CH190 (mt3) not determine whether Gl indeed occurred 0 CH5 (mt4) 0 in their plasmodia. Our Feulgen staining 0 CH195 (mt4) 0 data were obtained from plasmodia which 8 CH2 (mtl) * 14 had finished S phase, so we know that the CH54-c5 (mt3) nuclei were in G2 and can therefore connIn each case, 50 replicate amoeba1 cultures on clude that they are haploid or near haploid. dPRM agar were incubated for 4 wk at 26°C. Factors affecting illegitimate plasmoD + = vigorous growth, and easy sporulation. ~ = very limited growth. * = growth, but no sporulation. dium formation. The frequency of illegitiTABLE

10

ANALYSIS OF PROGENY OF ILLEGITIMATE PLASMODIA

250

DEVELOPMENTALBIOLOGY TABLE

12

FREQUENCY” OF FORMATION OF ILLEGITIMATE PLASMODIA BY SUBCLONESOF CH190 Subclone

A B C D E F Average

Percentage of cultures with plasmodia” 22 12 10 12 18 14 14.67

0 See Table 11.

Illegitimate plasmodia formed by amoebal strains such as CH21 and CH190, which have been inbred to CL (Adler and Holt, 1974a), are vigorous. The illegitimate plasmodia formed by CH2 and CH4 grow only to several cm2 and cannot be maintained in culture. This could be due to the fact that CL plasmodia. being naturally haploid, do not contain recessive genes reducing viability. In CH2 and CH4, which are derived from naturally diploid plasmodia, there is evidence for deleterious genes (Poulter, 1969; Adler and Holt, 1974a). Interestingly, illegitimate plasmodia produced by CH54-c:5, itself the progeny of an illegitimate plasmodium will grow but not sporulate. Perhaps this is due to aneuploidy, as CH54-c:5 produces fuzzy plaques, which we have correlated with amoeba1 aging (Adler and Holt. 1974b). In recent studies. spores of a large number of illegitimate plasmodia formed by mtl, mt2, mt3 and mt4 amoeba1 strains, all inbred to CL, have been analyzed. In nearly all cases, germination of the spores yielded amoebae only of the mating type used to form the plasmodium. However, a few of the plasmodia seem to have resulted from mutations; an analysis of these putative mutants is in progress. Attempt to detect recombination at the mating type locus. In many of the earlier papers (Dee, 1962; Dee, 1966; Haugli et al., 1972) dealing with the genetics of P. poly-

VOLUME 43, 1975

cephalum, amoeba1 progeny that could not be classified with respect to mating type were obtained from crosses at a high frequency. Such progeny would not cross with either of the two parental mating type tester strains. These unclassifiable progeny could have been due to the inefficient crossing techniques in use at the time or to recombination. With improved methods for crossing amoeba1 strains (Collins and Tang, 1973), it has now been possible to reexamine this problem. Over 600 progeny of mt3lmth and mt4l mth plasmodia have been examined (Table 13). Mating types segregated in a 1:l ratio and all of the heterothallic progeny were unambiguously classified as having a mt3 or mt4 heterothallic specificity. More than 600 progeny of mt3/mt4 plasmodia, obtained either by crossing or clonally from amoebae heterozygous for mating type, have been examined (Table 13). Three types of amoeba1 progeny were obtained: mt3, mt4, and CPF. Eleven of the CPF progeny were shown to be mt3lmt4. We can therefore say that the production of new heterothallic mating types by recombination occurs at a frequency of less than 0.4%. It seems likely that the earlier reports of unclassifiable progeny with respect to mating type were due to the use of inefficient crossing techniques. DISCUSSION

The data in this paper show that amoebae that are heterozygous for the mating type locus exist, are viable, and can be passaged indefinitely as amoebae. The TABLE

13

SEGREGATIONOF MATING TYPE How formed

Number progeny

mt3/mth mt4/mth mt3/mt4

cross cross cross

356 310 375

mt3/mt4

CPF

290

Plasmodial genotype

Classification of progeny 180 mth, 176 mt3 156 mth, 154 mt4 165 mt3, 176 mt4, 35 CPF 106 mt3, 94 mt4, 90 CPF

ADLER AND HOLT

Mating

amoebae were isolated from spores of plasmodia formed by crossing two heterothallic amoebae of different mating types and have the property of plasmodium formation in clones. All such CPF strains that we have examined have been heterozygous for mating type. We therefore tentatively conclude that it is heterozygosity at the mating type locus which is responsible for the strains being CPF. Such amoebae may be the main explanation of earlier reports of plasmodia-forming plaques produced by spores derived from heterothallically formed plasmodia (Collins, 1961, 1963; Dee, 1966; Wheals, 1973). The data on illegitimate plasmodia in this paper, and earlier work of others (Collins and Ling, 1968; Yemma and Therrien, 1972), show that a plasmodium containing one heterothallic mating type is viable, and can sporulate. The frequency at which such plasmodia are formed is low, but once formed they can be vigorous. The work of Cooke and Dee (1974a) demonstrated that there was no difference in ploidy in amoebae and plasmodia of CL. We have shown that plasmodia formed clonally from amoebae heterozygous for the mating type locus have the same C value as the amoebae they were derived from. Similarly, there appears to be no change in ploidy in forming an illegitimate plasmodium. We interpret the-data in this paper as supporting the concept that the mating type locus is a regulatory locus. The probability of plasmodium formation in a clone of amoebae appears to be a function of the mating type alleles present in the amoebae. In order of increasing probability of differentiating we find mthlmtn > mtnl m tm > mth >> mtn (where n, m refer to different heterothallic alleles). It has long been assumed (Dee, 1973; Ross et al., 1973) that heterothallic mating type functions by preventing fusion of amoeba1 cells of the same mating type or by inducing fusion of amoeba1 cells of different mating

Type and Differentiation

251

types. It is interesting to note, however., that if our interpretation of the role of the mating type locus in determining the rate of plasmodium formation is correct, then fusions between two mt3 cells would yield a diploid mt3 cell which would still have only a very low probability of differentiating into a plasmodium. It is premature, of course, to limit the function of the mating type locus to controlling only the probability of an amoeba differentiating into a plasmodium. Further studies will be needed to further define the function(s) of the mating locus. The assumption that a cell containing mth, or heterozygous for the mt locus, can differentiate into a plasmodium without fusing with another cell (Kerr, 1967) is implicit in much of the discussion in this paragraph. However, the possibility that plasmogamy without karyogamy occurs cannot be ruled out at the present time. The demonstration that near diploid amoebae heterozygous for the mating type locus exist raises the possibility that they are a normal intermediate in crossed plasmodium formation by heterothallic amoebae. Preliminary attempts to isolate such diploid amoebae heterozygous for the mating locus from mixed cultures of mt3 and mt4 amoebae have not been successful; however, this may be due to technical problems. The relationship of mth to the heterothallic alleles is not clear. The fact that mthlmt3 amoebae form plasmodia in clones much more rapidly than mth or mt3 amoebae suggests that the mth allele has a functioning product. The mth allele could be the result of a nonreciprocal recombination between two heterothallic mating types, yielding a chromosome with two tightly linked heterothallic mating type loci. A second possibility is that mth is a “very leaky” heterothallic mating type, which allows illegitimate plasmodia to form at a high frequency. In theory fine structure genetic mapping of the mating

252

DEVELOPMENTALBIOLOGY

type locus would distinguish between these possibilities. The lack of genetic markers linked to the mating type locus precludes the possibility of detailed studies at the present. The existing data on the lack of recombination at the mating type locus gives no evidence for complexity, such as has been found in some higher fungi (Raper, 1966). It is possible that some small percentage of the CPF progeny of plasmodia formed by two heterothallic amoebae are not heterozygous for mt but are the result of a crossover at the mt locus. We have not encountered such amoebae, however. The near diploid amoebae in this paper should be of value as a genetic tool. Dominance and cis/trans tests in amoebae can be accomplished with such strains, and indeed, two drug resistance markers have been shown in this paper to be recessive in amoebae. A mitotic mapping system should also be feasible. The segregation of actidione resistant progeny from sensitive heterozygous amoebae supports this idea. The authors are grateful to Lance Davidow and Susan Selvig for many helpful discussions, to Alan Wheals for comments on the manuscript, to John Kirk of Vickers Ltd. for the use of a microdensitometer, and to Gary Grove for advice on Feulgen staining. This work was supported by Grant No. GB23022/1 from the National Science Foundation, P.A. is a predoctoral trainee supported by N.I.H. Training Grant No. 5-TOl-GM00710 to the Department of Biology. REFERENCES ADLER, P. N., and HOLT, C. E. (1974a). Genetic analysis in the Colonia strain of Physarum pol.ycephalum: Heterothallic strains that mate with and are partially isogenic to the Colonia strain. Gqzetics. 78, in press. ADLER, P. N., and HOLT, C. E. (1974b). Change in properties of physarum polycephalum amoebae during extended culture. J. Bacterial. 120,532%533. CARLILE, M. J., and DEE, J. (1967). Plasmodial fusion and lethal interaction between strains in a myxomycete. Nature (London) 215, 832-834. COLLINS, 0. R. (1961). Heterothallism in two myxomycetes. Amer. J. Bot. 48, 674-683. COLLINS, 0. R. (1963). Multiple alleles at the incompatibility locus in the myxomycete didymium iridis. Amer. J. Bot. 50, 477-480.

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COLLINS, 0. R. (1965). Evidence for a mutation at the incompatibility locus in the slime mold Didymium iridis. Mycologia 57, 314-315. COLLINS, 0. R. (1974). Mating type in five isolates of physarum polycephalum. Mycologia, in press. COLLINS, 0. R., and LING H. (1968). Clonally-produced plasmodia in heterothallic isolates of didymium iridis. Mycologia 60, 858-868. COLLINS, 0. R., and TANG, H. C. (1973). Physarum polycephalum: pH and plasmodium formation. Mycologia 65, 232-236. COOKE, D. J., and DEE, J. (1974a). Plasmodium formation without change in nuclear DNA content in physarum polycephalum. Genet. Res. Camb. 23, 307-318. COOKE, D. J., and DEE, J. (1974b). Methods for the isolation and analysis of plasmodial mutants in physarum polycephalum. Genet. Res. Camb. 24, in press. DEE, J. (1962). Recombination in a Myxomycete, physarum polycephalum Schw. Genet. Res. Camb. 3, 11-23. DEE, J. (1966). Multiple alleles and other factors affecting plasmodial formation in the true slime mold physarum polycephalum Schw. J. Protozool. 13,610-616. DEE, J. (1973). Aims and techniques of genetic analysis in physarum polycephalum. Ber. Deut. Bot. Ges. 86, 93-121. GRAY, W. D. and ALEXOPOULOS,C. J. (1968). “Biology of the Myxomycetes.” Ronald Press, New York. GUTTES, E., GUTTES, S., and RUSCH, H. P. (1961). Morphological observations on growth and differentiation of physarum polycephalum grown in pure culture. Deuelop. Biol. 3, 588-614. HAUGLI, F. B. (1971). Mutagenesis, selection and genetic analysis in physarum polycephalum. Ph.D. Thesis, Univ. of Wisconsin. HAUGLI, F. B.. DOVE, W. F., and JIMINEZ, A. (1972). Genetics and biochemistry of cycloheximide resistance in physarum polycephalum. Molec. Gen. Genet. 118, 97-107. HUMASON, G. L. (1962). “Animal Tissue Techniques,” pp. 293-298. W. H. Freeman and Co., San Francisco. KERR, N. S. (1967). Plasmodium formation by a minute mutant of the true slime mold, didymium nigripes. Exp. Cell Res. 45, 646-655. KERR, S. (1968). Ploidy level in the true slime mould didymium nigripes. J. Gen. Microbial. 53, 9-15. LEWIS, D. (1954). Comparative incompatibility in angiosperms and fungi. in “Advances in Genetics” (M. Demerec, ed.), Vol. 6, pp. 235-285. Academic Press, New York. MCCULLOUGH, C. H. R., COOKE, D. J., FOXON, J. L., SUDBERY, P:E., and GRANT, W. D. (1973). Nuclear DNA content and senescence in physarum polycephalum. Nature New Biol. 245, 263-265.

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ROSS,I. K., and CUMMINGS,R. J. (1970). An unusual pattern of multiple cell and nuclear fusions in the heterothallic slime mold didymium iridis. Protoplasma 70, 281-294. Ross, I. K., SHIPLEY,G. L., and CUMMINGS,R. J. (1973). Sexual and somatic cell fusions in the heterothallic slime mould didymium iridis. 1. Fusion assay, fusion kinetics and cultural parameters. Microbios 1, 149-164. RLJSCH,H. P. (1970). Some biochemical events in the life cycle of Physarum polycephalum. in “Advances in Cell Biology” (D. M. Prescott, ed.), Vol. I. pp. 297-328. New York: Appleton-Century-Crofts. THERRIEN,C. D., and YEMMA,J. J. (1974). Comparative measurements of nuclear DNA in a heterothallit and a self-fertile isolate of the myxomycete, didymium

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WHEALS,A. E. (1973). Developmental mutants in a homothallic strain of physarum polycephalum. Genet. Res. Camb. 21, 79-86. YEMMA,J. J., and THERRIEN,C. D. (1972). Quantitative microspectrophotometry of nuclear DNA in selfing strains of the myxomycete didymium iridis. Amer. J. Bot. 59. 828-835.