MEIOTIC NONDISJUNCTION AND ANEUPLOIDS IN ...MEIOTIC NONDISJUNCTION IN PARAMECIUM TABLE 1 Stocks and strains of P. caudatum used in this study 78 1 Phenotypes Mating Stocks or strains

Copyright 0 1985 by the Genetics Society of America

MEIOTIC NONDISJUNCTION AND ANEUPLOIDS IN INTERSYNGENIC HYBRIDS OF PARAMECIUM CAUDATUM

YUUJl T S U K I I ' AND KOICHI HIWATASHI

Biological Institute, Tohoku University, Sendai 980, Japan

Manuscript received April 11, 1985 Revised copy accepted August 19, 1985

ABSTRACT

Artificially induced intersyngenic crosses in Paramecium caudatum can produce viable and fertile hybrids. When F1 hybrids of double E mating type (Mti/ Mt' or MtI2 /Mt3) were crossed with mating type 0 (mtlmt), aberrant segregants of double E and single 0 type were produced. This segregation was not explained by ordinary equal or unequal crossing over. Breeding analyses of these segregants by using linkage between Mt and cnrA (a behavioral mutant) revealed that they were produced by meiotic nondisjunction of bivalent chromosomes carrying Mt genes, and thus the double E and single 0 segregants were aneuploids: trisomics (Mt'/Mt'/mt or Mtl2 /MtB/mt ) and monosomics (mt), respectively. An aberrant segregant was also obtained for another locus, tnd 2, independent of both Mt and cnrA, suggesting the occurrence of meiotic nondisjunction throughout hybrid genomes. These aneuploids will be useful for genetic study in this species. The occurrence of meiotic nondisjunction in the intersyngenic hybrids also suggests that syngens of P. caudatum have been reproductively isolated for long enough to develop chromosomal incompatibility in their meiotic process.

HE ciliated protozoan, Paramecium caudatum, consists of a number of subgroups called syngens. Within a syngen, crossbreeding is induced nat-

urally by the mating reaction (sexual cell agglutination via cilia) occurring between cells of complementary mating types. The mating reaction does not usually occur between cells of different syngens, although some exceptional cases of intersyngenic mating reactions have been reported (GILMAN 1949). Thus, syngens are generally considered to be reproductively isolated in nature by the specificity of the mating reaction; however, we recently found that artificially induced intersyngenic crosses in P. caudatum can produce viable and fertile hybrids. Crossbreeding analyses using those fertile hybrids revealed three loci, M t , M A and MB, controlling the syngenic specificity of mating types (TSUKII and HIWATASHI 1983). Codominant multiple alleles at the M t locus control the syngenic specificities of E mating types, and those at two other loci, M A and MB, control the specificities of 0 mating types.

In the course of those experiments, we obtained a few aberrant segregants

' To whom reprint requests should be sent at present address: Laboratory of Biology, Hosei University, Chiyoda-ku, Tokyo 102, Japan

Genetics 111: 779-794 December, 1985

780 Y. TSUKII AND K . HIWATASHI

that, at first, appeared to show that the E mating types of different syngens are controlled not by multiple alleles of a single locus but by different closely linked loci. However, further analyses of those segregants revealed that they were produced by meiotic nondisjunction of bivalent chromosomes, not by recombination of closely linked loci. The meiotic nondisjunction produced trisomic and monosomic progeny. These aneuploids not only breed true veg- etatively but also repeatedly produce aneuploids when crossed to diploid strains.

In this report, we shall present evidence showing the occurrence of meiotic nondisjunction and the subsequent production of aneuploids. We shall also discuss the possible usefulness of the aneuploids for studies on the genetics of this species and on the mechanisms of sexual isolation of syngens.

MATERIALS AND METHODS

Stocks and strains: Three genetic markers, cnrA, cnrB and tnd 2, were employed for crossbreeding analyses in this work. Both cnrA and cnrB are independent behavioral mutants (TAKAHASHI 1979). Gene cnrA was recently found to be closely linked to M t , the mating-type locus controlling type E specificity ( M . TAKAHASHI, personal cornrriunication; Y . TSUKII, unpublished data). Both cnrA and cnrB were also used to discriminate cross pairs from selfing pairs, as described below. The trichocyst nondischarge mutant, tnd 2 (WAI'ANABE and TAKEI 1982) was employed in later parts of this work.

All natural stocks and derived strains used are shown with their markers in Table 1. Sa 7 and Isn 1 in syngen 1, CHB in syngen 3 and Ikz 1 in syngen 12 were natural stocks, and dKI-81 was an offspring from a cross between natural stocks Ksa and I kr 1 in syngen 12. Strain 27aG3 (TSUKII and HIWAI-ASHI 1979) is an exautogamous strain derived from EmIV-3S 27a, a heterozygote of the early mature gene, EmtA (MYOHARA and HIWATASHI 1978). Among strains of syngen 3, K 103, 16A801 and 16A802 are homozygotes of cnrA, and 16B413, 16B318 and 16B911 are homozygotes of cnrB. All CNK strains except K 103 and 16B413 were produced by a series of backcrosses of 16A or 16B to 27aG3. K 103 was derived from selfing conjugation of a mutagenized clone of the cnrA heterozygote produced by a cross between 16A601 and 27aC3. It also carries homozygous cnrC", an allele of the cnrC locus and phenotypically K+-resistant (ENDOH and HI- WATASHI 1981). 16B413 was a selfing derivative of a cnrB heterozygote obtained from a cross between 16B320 and Yt3G1, the latter being an exautogamous clone derived from a natural stock Yt 3 .

Culture methods: Culture methods were essentially the same as those of HIWATASHI (1 969). Culture tnediuni used for tube culture was sterilized fresh lettuce juice inoculated with Klebsiella pneumoniae 1 day before use. Ca-poor fresh lettuce juice in which the juice was diluted 1:40 with 2 mM sodiurii phosphate buffer (pH 7.0) instead of DRYL'S solution (DRYL 1959) was used for culturing cells in slide depressions. All cultures were kept at 25" except during isolation procedures, which were done at room temperature.

Crosses: Crossbreedings were performed as previously described (TSUKII and HIWATASHI 1983). In P. caudatum, mating mixtures consisting of cells of complementary mating types E and 0 produce not only cross pairs (heterotypic pairs of type E and 0) but also selfing pairs (homotypic pairs of type E and 0). To isolate only cross pairs, one of the behavior mutants (cnrB or cnrA) was included in one of the complementary mating types in every cross.

About 6 hr after initiation of the mating reaction, conjugating pairs were tested in DRYL'S solution containing 20 n i M KCI. Selfing pairs of wild type react to this solution by a period of straight backward swimming, and CNK selfing pairs by direct forward swimming, whereas cross pairs show spiral backward swimming. Cross pairs were isolated in depression slides containing sterilized physiologically balanced solution (MIYAKE 1958) and were kept in this solution for 2 days to avoid the occurrence of iiiacronuclear regeneration (MIKAMI and HIWATASHI 1975). Sub- sequent procedures are the same as those previously described (TSUKII and HIWATASHI 1983).

MEIOTIC NONDISJUNCTION IN PARAMECIUM

TABLE 1

Stocks and strains of P. caudatum used i n this study

78 1

Phenotypes

Mating Stocks or strains type” Behaviorb Trichocyst‘ Genotypes

Syngen 1

Syngen 3 Sa 7,d ~ s n I d E’ Wild type TD Mt’ +/mt +; +/+; +/+

C H B ~ E’ Wild type TD Mt’ +/MtS +; +/+; +/+ K 103“ E’ CNR TND MtS cnrA/MtS cnrA; +/+; tnd Zltnd 2

16B409‘ E’ CNR TD mt cnrA/mt cnrA; +/+; tnd 2l tnd 2 16A801,” 16A802‘ 0’ CNR TND mt +/mt +; +I+: tnd Pjtnd 2 27aG3f 0’ Wild type TND mt +/mt +; cnrB/cnrB; +/+ 16B413‘ 0 3 CNR TD mt +/mt +; cnrBjcnrB; tnd 2l tnd 2 16B318,’ 168911‘ OS CNR TND

Ikz l,d dKI 81g El2 Wild type T D Mt” +/Mt12 +; +/+; +/+ “For simplicity, odd and even mating types (I and 11) of syngen 1 were designated as 0’ and

Mt’ +/MtS +: cnrB/cnrB; +/+

Syngen 12

El, those of syngen 3 as O’ and E’, etc. CNR, N o backward swimming upon stimulation (TAKAHASHI 1979).

Natural stocks. ‘ TD, Trichocyst discharge; TND, trichocyst nondischarge.

“Strains derived from backcrosses of 16A (cnrA) or of 16B (cnrB) to 27aG3. (For details, see

’A complete homozygote derived from autogamy of an early mature mutant, EM IV-3s 27a

gA laboratory strain derived from a cross between natural stocks, Ikz 1 and Ksa.

MATERIALS AND METHODS.)

(MYOHARA and HIWATASHI 1978).

Three methods f o r obtaining double E-type strains: All double-E-type strains are shown in Table 2 and were obtained according to three different methods described below.

1. Details of this method have already been described (TSUKII and HIWATASHI 1983). Four complementary mating types from two syngens were simultaneously mixed to form mating clumps in a test tube. Although no intersyngenic mating reaction occurs among the syngens used here, appearance of intersyngenic cross pairs was expected because of the nonspecificity of pair formation (HIWATASHI 1951). They were distinguished from intrasyngenic pairs by using cnrB markers for both type 0’ and E’ cells of syngen 3 or by using different cell sizes and cytoplasmic transparencies. As we cannot tell the mating-type composition of a pair by this method, all progeny of the intersyngenic pairs were grown to express their mating types; then, clones of double E type were picked up among them.

2. The details of this method will be described elsewhere (Y . TSUKII, unpublished results). This method is essentially the same as that used in method 1, but nickel paralyzed cells (GELEI 1935; KUZNICKI 1963) of two types of 0 were mixed with living cells of two types of E. When living cells of two types of E from two syngens were mixed with cells of two types of 0 immobilized by nickel ions (100 WM), mating reaction occurred between living E and immobilized 0 cells, but the immobilized 0 cells were unable to enter the second step of cell contact, the pair formation. Thus, pairs were formed only among cells of type E of the two syngens. Intersyngenic cross pairs were discriminated from selfing pairs by using behavioral markers as described in method 1, except for the case of Sa 7 (E’) X CHB (E’), where both parents were phenotypically wild type, and, therefore, double E hybrids (E’E’) were screened after all progeny were grown to the maturity period when they expressed their mating types.

3. This method employs a temporary change of mating type that is very common in clones of type E in P . caudatum (HIWATASHI 1958; MYOHARA and HIWATASHI 1975). Clones of type E


TABLE 2

Genotypes of double-E-type strains and their origins ~~

Double-E-type strain

E’E’ type A-I, A-2 B-1,“ B-2,’ B-3‘ I- 1 A-1 11

Genotype Source Method”

Mt’lMt’; +/cnrB FI hybrids from Isn 1 X 16B226* (1) Mt‘lMt’; +/cnrB FI hybrids from Isn 1 X 16B409 (1) Mt‘IMt’; +/+ (2) Mt’lMt’; +/cnrB A progeny from A-1 1 derived from (3)

An Fi hybrids from Sa 7 X CHB

A-1 X 16B101 X 27aG3BCSd

Ei2E’ type C- 1 MtI2/Mt’; +/+ An FI hybrid from Ysc 22‘ X Ikz 1 (1) E-1‘ Mti2/MtS; +/cnrB An FI hybrid from 16B409 X Ikz 1 (1) H- 1 Mti2/Mt3; +/cnrB An Fi hybrid from 16B409 X dKI 81 (2) E-2 1 Mti2/Mt’; cnrB/cnrB A progeny from E-2c X 16B409 (3) (2-111, C-112 Mti2/Mt’; +/cnrB A progeny from C-1 1 from C-1 X

16B318 X 16B409

Numbers in parentheses indicate the methods used for inducing double-E-type strains (see MATERIALS AND METHODS).

bDerived from backcrosses of 16B to 27aG3. “These heterozygous double-E-type strains include coconjugant clones (Table 3). dAn ei hth backcross progeny of 27aC6 (an exautogamous E’ strain from Em IV-SS 27a) to

‘Derived from a sibling cross between selfing progeny of Yt 3. 27aC3 (0 8 ).

become unstable for the expression of mating type in the later period of maturity. Cells expressing type E in the early stationary phase change to express type 0 in the late stationary phase. This phenomenon is known to be induced by suppression of the dominant allele Mt (HIWATASHI and MYOHARA 1976). Clones of type El or El2 whose genotypes contain O’-controlling allele(s) were grown to the later period of maturity. When they changed their mating type to 0, they expressed type 0’ and, thus, were capable of crossing to E’ cells. In the progeny, the repressed El or EL* genotype (Mt’ or Mt“) reappeared and, thus, clones of double E type (E’E’ or EI2E’) were obtained. Cross pairs were also isolated by using behavioral markers as described in method 1.

Phenotypic diagnoses: Cultures in the stationary phase were used for scoring three phenotypes. 1. Mating type. About 0.5 ml cultures containing 300-500 cells were pipetted from the top of

tube cultures and were placed in each of four depressions of a disposable tray (Linbro Scientific Inc., Hamden Connecticut). To each depression about equal numbers of mating reactive tester cells of each of the four mating types (e.g., O’, O’, E’ and E’) were added. If the culture being tested was mating-type reactive, mating agglutination between the culture and its complementary testers could be seen immediately. If a culture gave mating reaction with testers of type 0’ and O’, it was scored as double E type, E’E’. If a progeny clone was immature and showed no mating reaction with any of the four testers, tube transfer of the culture was continued until it entered the maturity period and became mating reactive.

2. Behavior. T h e phenotypes of CNK mutants were identified by their response to high con- centrations of K+ ions. A number of cells were transferred to DRYL’S solution (DRYL 1959) containing 20 mM KCI, and their swimming behavior was observed under the binocular microscope. Wild-type cells reacted to this solution by a period of straight backward swimming, but CNR niutants did not respond to this solution and swam forward.

3. Trichocyst. One or two drops of saturated aqueous solution of picric acid were added to an aliquot of cultures containing 100-200 cells in a depression of a disposable tray. Then, they were scored for discharge of trichocysts under the binocular microscope. Cells of wild-type clones (des-

MEIOTIC NONDISJUNCTION IN PARAMECIUM 783

ignated as TD) discharged trichocysts, and cells of mutant clones (designated as TND), which were homozygous for the recessive allele tnd 2, discharged no trichocysts (WATANABE and TAKEI 1982).

RESULTS

As already reported (TSUKII and HIWATASHI 1983), intersyngenic crosses between E mating types of different syngens of P. caudatum produced clones of double E mating type. Appearance of such double-E-type clones in the intersyngenic Fl hybrids suggested that syngenic specificity of type E is controlled by codominant multiple alleles at the locus Mt. Allelism of Mt genes between different syngens was confirmed when the hybrid double E types were testcrossed to type 0 strains that are homozygous for the recessive null allele mt.

When the hybrid E"E3 (double E type of syngen 12 and 3) was testcrossed to O3 (single 0 type of syngen 3), clones of single E types, E" and E3, segregated in the progeny with ratios not significantly different from 1 to 1 (TSUKII and HIWATASHI 1983). This result indicated that Mt" and M t 3 are allelic and it supported the above hypothesis. In the case of E'E3 X 03, however, progeny included a few exceptional segregants of type E'E3 and type 0 (TSUKII and HIWATASHI 1983). At first sight this appeared to indicate that Mt' and M t 3 were not allelic but belonged to different loci of the same linkage group and that the segregants of type E'E3 and type 0 were ordinary recombinants. Alternatively, they could have been produced by unequal crossing over between the alleles Mt' and Mt3 . However, as will be described in the following sections, subsequent analyses showed that the exceptional segregants were produced neither by ordinary nor by unequal crossing over, but were caused by meiotic nondisjunction of bivalent chromosomes carrying Mt genes.

Segregation of double E and type 0 in the progeny of testcrosses, double E X 0: After publishing the previous report (TSUKII and HIWATASHI 1983), more testcrosses of double E to 0 were performed. All results of the testcrosses, including those previously reported, are shown in Table 3. As seen in the table, the majority of the testcrosses (14 of 18) produced aberrant segregants of double E and type 0. In each testcross, the proportions of the aberrant segregants varied and seemed to be associated with cytoplasmic parentage. In Table 3, double E strains, B-lc, B - ~ c , B-3c (E'E3) and E-lc (E"E3), were derived from cytoplasmic parents of syngen 3; and their coconjugant clones, B-lw, B-2w and B - ~ w , were from parents of syngen 1 and E-lw from a parent of syngen 12, respectively. The proportion of aberrant segregants in testcrosses of the former clones (cross 5 , 7, 9, 13) was apparently smaller than that in crosses of the latter clones (cross 6, 8, 10, 14). These results suggest that some nucleocytoplasmic interactions are involved in the appearance of aberrant segregants.

To determine if the same kind of aberrant segregants also occur in intrasyngenic crosses, E3 homozygotes (Mt3 /Mt3) were crossed to O3 (mtlmt). In these crosses, the only aberrant segregants that could be detected were of the single 0 type, because the phenotype of the double E equivalent (Mt3/Mt3) should be indistinguishable from that of ordinary E3 heterozygotes (Mt 3 /mt) .

784 Y. TSUKII AND K. HIWATASHI

TABLE 3

Allelism tests fo r Mt genes of dzfferent syngens: segregation of phenotypes among progeny from E'E' X 0' and from E12E3 X 0'

Mating type % of B/

C:ross" A B (A + B) cnrB marker

E ' E ~ x O3 1. A-1 X 16B101 2. A-1 X 168318 3. A-2 X 16B101 4. A-2 X 16B318 5. B-lcd X 16B413 6. B-lwd X 16B413 7. B-2cd X 16B413 8. B-2wd X 16B413 9. B-3cd X 16B413

10. B-3wd X 16B413 11. A-I l l X 16B318

E' E' 7 10 9 11 4 6

12 9 144 142

19 23 90 107 34 32 89 112 47 51 53 43

E'E3 Ob W T " (+/cnrE) CNR (cnrB/cnrE) 1 0 6 9 9 1 1 9 8 14 1 0 9 2 9 2 3 19 9 17 1 1 13(1) 8 153 157 3 3 13 25 23 7 12 ( 1 ) 9 103 113 5 5 13 43 33 6 7 6 108 106 5 11 ( 1 ) 14 59 55 0 0 0 47 49

EI2E8 X 0' E12 E3 ~12E3 Ob

12. C-1 X 16B338 47 59 0 0 0 106 0 13. E-lcd X 16B413 87 62 0 0 0 63 86 14. E-lwd X 16B413 50 41 1 4 5 34 62 15. H-1 X16B413 30 33 2 5 10 35 35 16. E-21 X27aC3 67 59 0 0 0 126 0 17. C - I l l X16B413 19 17 1 0 3 18 19 18. C-112 X 16B413 15 26 1 0 2 23 19

"Data of crosses 1-4, 12 and 13 were already reported (TSUKII and HIWATASHI 1983), and data of crosses 5 , 7 and 9 are composed of those already reported as well as new ones. All others are new data.

bType 0 progeny were mostly single 0 (Os), but numbers in parentheses are double 0 (0'0') (see TSUKII and HIWATASHI 1983). Numbers outside the parentheses are totals of single 0 and double 0.

' W T , Wild type. dB-lc, B - ~ c , B-3c and E-lc were derived from cytoplasmic parents of syngen 3, and their

toconjugant clones, B-lw, B-2w and B - ~ w , were from parents of syngen 1 and E-lw from a parent of syngen 12, respectively.

Among 2 15 progeny clones derived from the above crosses, no type O3 segregants appeared, and all were type E3 (data not shown). This result suggests that in intrasyngenic crosses the aberrant segregants do not appear or that, if they were to appear, their frequencies would be very low.

Segregation of mating type among progeny from backcrosses of the aberrant double E segregants to type 0: I f the cause of the occurrence of these double E aberrant segregants was recombination, the double E segregants should have two M t in cis position on the same chromosome and the type 0 segregants should have no Mt. If so, backcrosses of these double E to ordinary type 0 should produce a majority of double E and type 0 with only a few single E recombinants. However, when 21 of 42 E'E3 segregants and three of five E"E3 segregants were backcrossed to type Os, in no case did segregation patterns conform to the recombination hypothesis (Table 4).


TABLE 4

Distribution of mating type among progeny from backcrosses of aberrant double E segregants to type 0'

Cross % of clones carrying

Mating type M t gene Behavior

Aberrant EIES X 0' Type I

AIR1 X 16B214 A2R1 X 27aG3 B l R l X 27aG3 B1R2 X 27aG3

B1R3 X 27aG3 B1R4 X 27aG3 B1R5 X 27aG3 B1R6 X 27aG3 B1R7 X 16B413 B2R1 X 27aG3 B2R2 X 27aG3 B2R3 X 27aG3 B2R4 X 27aG3 B3R1 X 27aG3 B3R2 X 27aG3 B3R3 X 16B413 B3R4 X 16B413 B3R5 X 16B413 B3R6 X 16B413 B3R7 X 16B413 B3R8 X 16B413

Type I 1

E'

3 0 0 0

7 6

14 6

10 21 I O 14 6 5

14 13 8

15 17 3

13

Aberrant E'%' X 0' El2

E l R l X 27aG3 0

C - I l l R I X 27aG3 6 3

Type I

Type I 1

C-112R1 X 16B413 48

E'

0 31 23 10

8 3

17 4 9

16 14 7 4 3 4 8 3 9

15 1

16

E'

1 1

48

E'E'

0 0 0 0

7 1 7 2 3

10 1 1 2 1 2 2 6 3 3

10 1

1 1

ELZE'

0

36

0

4 28 26 29

12 (3)b 7

12 7

15 10 7 5 6 2 8 (2)* 4 ( a b

15 14 22

5 18

0

3

42 48 34 40

Mt I

43 0 0 0

41 41 42 42 35 54 50 57 41 58 57 61

44 42 40 41

Mt"

0

52

38

Mt' Wild type CNR

0 3 53 59 47 49 26" 39

44 34 24" 17 48 50 32 19 32" 1 1 46 57 60 42 32 28 29 17 42 12 21" 28 45 14 21" 18 29 14 39 25 20 3 47 24

MtS

78" 14

44 189

4 0 0 0

0 0 0 0

26 0 0 0 0 0 0

17 11 27 39

7 34

0

0 48 48 66 104

Deviation from 50% is statistically significant. 'Numbers in parentheses are double 0 (0'0') (see TSUKII and HIWATASHI 1983). Numbers

outside the parentheses are totals of single 0 and double 0.

As seen in the table, patterns of mating-type segregation can be grouped into two categories, designated type I and type 11. In type I segregation, only one of the two single E types appeared in the progeny, and E and 0 segregated in a ratio of 1:l. When some E' progeny from a cross producing type I segregation (A2R1 X 27aG3 in Table 4) were again backcrossed to type 03, E3 and 0' clones segregated in a ratio of 1: 1. This shows that the E' in the type I segregation has the genotype Mt3/mt . On the other hand, when their parental double E, A2R1, was crossed to another strain, type E' progeny was segregated with very low frequency (1 of 105, Table 10). These results suggest that the parental double E clone has three M t genes (Mt ' /Mt3/mt) in its micronucleus, and one of the two M t genes, M t ' , tends to be eliminated during


the first backcross. By contrast, in the type I1 segregation, two single E’s, double E and type 0 appeared with various frequencies. If no Mt were eliminated during the crosses, the ratio of the two dominant Mt alleles in the progeny would be 50% for each Mt. As seen in Table 4, however, various frequencies from 20-61 % were observed, and many of them were significantly different from 50%. This suggests that elimination of Mt alleles occurred also in the crosses producing the type I1 segregation. When three double E progeny clones from the cross, B1R3 X 27aG3 (see Table 4) were backcrossed again to type 03, the four mating types segregated and their segregation ratios were similar to that of their parental cross (data not shown), indicating that the characteristic segregation patterns were transmitted in the double E descend- ants in successive generations.

Hypothesis f o r the appearance of the aberrant segregation: For the appearance of the aberrant double E and type 0 segregants and the abnormal segregation patterns (type I and type 11) of mating types in the backcross progeny of the double E segregants, two alternative hypotheses can be proposed: nondisjunction of bivalent chromosome carrying Mt or transposition of Mt at meiosis.

I n the first hypothesis, the nondisjunction model, F1 double E hybrids should have a pair of homologous chromosomes carrying Mt of different syngens. If some of these homologous chromosomes failed to separate at the next meiotic division, some gametic nuclei would receive two Mt-carrying chromosomes and some others would receive no Mt chromosome. When such gametic nuclei are fertilized with normal ones containing mt-carrying chromosome of type 0 mates, synkaryons will become aneuploids, either trisomics or monosomics for the Mt locus. Trisomics will express double E type and monosomics type 0. In this model, the difference between the type I and I1 segregation patterns can be explained by the stability of the three chromosomes; for example, if the Mtl-carrying chromosome were eliminated and the Mt3 and mt chromosomes were transmitted to the progeny, type I segregation of E3 and 0 would appear. On the other hand, if the trivalents were stable and the three Mt chromosomes were assorted at random, the gametic nuclei would receive various combinations of Mt chromosomes. Some would receive only one, while others would receive two. Thus, when stable double E trisomics are crossed with type 0 (mtlmt), progeny will segregate four different mating types with a ratio of 2:2:1:1 as type I1 segregation (see Table 5).

I n the second hypothesis, the transposition (or translocation) model, two M t genes coming from different syngens should be located at the same locus in FI double E hybrids, but one of them would be translocated to some other chromosome at the next meiosis. Thus, in the aberrant double E segregants, two Mt genes would be located at independent loci. If the aberrant double E segregants were backcrossed again, progeny would segregate four mating types, two single E’s, double E and type 0, with a ratio of 1:1:1:1 as type 11 segregation. If elimination or repression of one of the two Mt genes occurred in the micronucleus after the transposition, a backcross of the aberrant double E would result in type I segregation.


TABLE 5

Frequency of Mt genes in testcross progeny when Mt chromosomes forming trivalent were segregated randomly in meiosis

Mating types Frequency in progeny Genotypes of of testcross

Parental genotype gametes progeny M t ’ Mt’

TABLE 6

Appearance of aberrant double E and type 0 segregants and their genotypes expected from nondisjunction hypothesis

Aneuploids produced by meiotic nondisjunction

Cross (genotypes) Trisomy Monosomy

(I) E’E’, wild type X Os, CNR Aberrant E’E’, wild type Os, CNR (mt cnrA)

Os, wild type (mt +; +/cnrE)

(Mt’ + / M t s +) (mt cnrA/mt cnrA) (Mtl + / M t S +/mt cnrA)

Aberrant EIEs, wild type (Mtl + / M t s + /mt +; +/cnrB)

(11) E’E’, wild type X O’, CNR (Mt’ +/Mt’ +; +/+) (mt +/mt +; cnrB/cnrB)

In the following experiments, the nondisjunction hypothesis was supported and the transposition hypothesis was rejected.

Evidence for the nondisjunction hypothesis: The simplest method for testing the nondisjunction hypothesis would be cytological observations of chromosomes at meiotic metaphase. In P. caudatum, however, the 100 or more meiotic chromosomes are small and similar in size (see WICHTERMAN 1953), and it is practically impossible to identify aneuploids morphologically.

The nondisjunction hypothesis was tested by two different series of breeding analyses using the recently found linkage between M t and cnrA (M. TAKA- HASHI, personal communication; Y. TSUKII, unpublished data). In the first crossbreeding analysis, FI double E hybrids, E’E’, which were homozygous for the wild-type allele of the cnrA locus (Mt’ +cnrA/Mt3 +cnrA), were testcrossed with the cnrA mutant of 0’ (mt cnrA/mt cnrA). According to the nondisjunction hypothesis, the double E segregants of this cross should have three Mt alleles and three cnrA alleles as seen in the genotype Mt’ +/Mt’ +/mt cnrA. Thus, the double E should be wild type for the cnrA locus, and the result conformed to this expectation (Table 7). However, the type 0 segregants should have only one mt and one cnrA; thus, type 0 segregants should always show the cnrA mutant phenotype (Table 6, cross I). On the contrary, according to the

788 Y . TSUKII AND K . HIWATASHI

TABLE 7

Evidence for the nondisjunction hyfothesis: results of testcrosses, E'E', wild type X 03, cnrA and E'E' , wild type X Os, cnrB

Segregation of phenotypes among progeny (behavior/niating type)

Cross Wild type CNR

( I ) Mt' +/Mt' + X mt cnrA/mt cnrA E' E' E'E' 0 E' E' E'E' 0 1-1 X 16A801 56 41 4 0 0 0 0 Z ( 1 ) " B-2c X 16A802 6 0 4 9 7 0 0 0 0 1

Total 116 90 1 1 0 0 0 0 3

(11) Mt' +/Mts +; +/+ X mt +/mt +; cnrB/cnrB

1-1 X 16B911 63 65 9 10 (2)" 0 0 0 0

"Numbers in parentheses are double 0 (0'0') (see TSUKII and HIWATASHI 1983). Numbers outside the parentheses are totals of single 0 and double 0.

transposition hypothesis, the type 0 segregants should be heterozygous for the cnrA locus (+/cnrA) and, therefore, should be phenotypically wild type. As shown in Table 7, cross I, among 220 progeny obtained from two different crosses, three were type 0 and all of them showed the CNR phenotype as expected. These results indicate that the type 0 segregants were really monosomics for the chromosome carrying mt and cnrA, supporting the nondisjunction hypothesis.

In a second breeding analysis, an F1 double E hybrid and a type 0 strain both containing only wild-type alleles for cnrA were used (Table 6, cross 11). An F1 double E hybrid, 1-1 (see Table 2), was obtained from an intersyngenic cross between wild-type stocks of type E' and type E', which was induced by nickel-paralyzed cells of type 0 (see MATERIALS AND METHODS). The double E (1-1) was crossed to type 0 homozygous for +cnrA and also for cnrB, another behavioral marker. All progeny were phenotypically wild type. As shown in Table 7, cross 11, ten type 0 segregants were obtained from this cross. When seven of the ten type 0 segregants were crossed to cnrA homozygote of type E' (MtJ cnrA/Mt3 cnrA), five among the seven crosses segregated progeny of CNR phenotype (Table 8). These CNR progeny should be cnrA monosomics, because the type 0 parents in these crosses should be +CnrB/cnrB and would never produce cnrB/cnrB. Appearance of these CNR progeny cannot be expected according to the transposition hypothesis. These results strongly suggest that the type 0 segregants were really monosomics for the chromosome carrying Mt and cnrA loci and support the nondisjunction hypothesis. The progeny segregated wild type and CNR with a ratio close to 1:l in crosses 1 and 2 as expected (Table 8); however, the segregation ratio was biased in crosses 3, 4 and 5, and no mutant progeny appeared in crosses 6 and 7. These deviations from the expected 1:l ratio cannot be attributed to the monosomy condition of the M t chromosome, because all Mt chromosomes came from the same cnrA


T A B L E 8

Segregation of CNR mutants among progeny from crosses between type 0, wild-type segregants and type E', cnrA (Mt' cnrA/Mt' cnrA)

Segregation of phenotypes

Cross Mating type and behavior Trichocyst

O'," wild type, X E', CNR, T N D

(mt +; +/tnd 2)" T D (Mt' cnrA/Mt' cnrA;

tnd 2 l tnd 2 ) 1. 0 - w - 1 X K 1 0 3 2. 0 - w - 2 " X K 1 0 3 3. 0-W-3 X K 103 4. 0 -W-4 X K 103 5. 0-W-5 X K 1 0 3 6. 0-W-6 X K 1 0 3 7. 0-W-7 X K 1 0 3

E', C N R E', wild type T D T N D (Mt' cnrA)' (Mt' cnrA/mt +) (+ / tnd 2 ) (tnd 2 l tnd 2 )

2 5 2 5 1 7 33 2 0 30 2 8 22

6 1 3 7 1 2 4 2 4 16 1 2 1 9 5 5 0 19 10 9 0 8 4 4

'Type 0, wild-type segregants derived from a cross 1-1 X 16B911 in Table 7. Genotypes of monosomics expected from nondisjunction hypothesis.

'Type 0'0' derived from 1-1 X 16B911 was used only in this cross.

TABLE 9

Evidence for trisomy: results of backcrosses of the aberrant type E'E' segregants' to type 0', cnrA

Segregation of phenotypes

Cross Mating type and behavior Trichocyst

E'E', wild type X O', C N R E' E' E'E' 0 3 TD TND W T C N R W T C N R W T CNR W T CNR

(A) (Mt' +/Mt'/mt +; (mt cnrA/mt cnrA; +/ tnd 2)' tnd 2 l tnd 2 ) 1. I l R 1 " X 16A801 7 0 1 2 0 1 9 0 3 0 20 21 2. l l R 2 " X 16B801 1 5 0 17 0 9 0 1 5 0 25 31

(B) (Mt ' +/Mt' +/mt (mt cnrA/mt cnrA; cnrA; +l tnd 2 ) b tnd 2 l tnd 2 )

3. B2R5" X 16A801 0 0 1 3 1 0 0 2 12 1 5 1 3 4. I l R 3 " X 16A801 33 1 2 3 1 2 6 0 3 22 60 50 5. B2R6" X 16A801 7 0 6 2 1 0 0 1 5 2 4 7 6. B2R7" X 16A801 1 1 0 12 1 1 2 0 2 5 2 3 2 0

" I l R 1 and I1R2 were derived from a cross 1-1 X 16B911; I1R3 from 1-1 X 16A801; and B2R5, B2R6 and B2R7 from B-2c X 16A802 in Table 7.

Genotypes of trisomics expected from the nondisjunction hypothesis.

parent. The deviation might be attributed to low viability of the cnrA progeny, probably due to their genetic background (data not shown).

Evidence for trisomy: Table 9 shows the results of various testcrosses of aberrant double E segregants derived from the crosses in Table 7. I l R l and I1R2 from the cross 1-1 X 16B911 (see Table 7) were presumed to have three wild- type alleles of cnrA (Mt' + / M t 3 +/mt +), and I1R3 from 1-1 X 16A801 as well


TABLE 10

Segregation oftriple-E-type progeny from crosses E'E' X E12E3 and E 1 E 3 X E"

Cross" Segregation of phenotypes among progeny

E ' E ~ X E1'E3 E'E~E'* E'E' E'"' E ' E ' ~ E' E' El2 0 Wild type CNR 1 . A2R1 X C-112R1 1 0 15 1 0 33 38 17 50 55 2. B l R l X C-112R1 1 3 0 1 1 3 2 1 5 8 3. D-l X C - l I l R 1 7 5 4 1 3 4 1 2 10 17

4. B l R 3 3 X E-11 2 3 1 4 3 1 3 1 E'E' X E'*

"A2R1, B l R I , C-112R1 and C - I l l R I were double E trisomics derived from crosses shown in Table 3. D-1 was from the testcross of a triple E progeny from A2R1 X C-112R1 to type Os (27aC3), B1R33 from B1R3 X 27aG3 (Table 4) and E-11 from E-lwr X 16B413 (Table 3).

as B2R5, B2R6 and B2R7 from B-2c X 16A802 were presumed to have two wild-type alleles and one mutant allele of cnrA locus (Mt' + / M t 3 +/mt cnrA). The segregation of phenotypes among progeny obtained was consistent with those expected from the presumed parental genotypes (Table 9). In testcrosses of double E trisomics with three wild-type alleles, no CNR mutant progeny appeared, but those of trisomics with two wild-type alleles and one mutant allele segregated progeny expressing type E and CNR (Mt cnrA/mt cnrA). These are supposed to be produced by a recombination between the chromosome carrying M t (Mt' or Mt3) and and that carrying mt and cnrA. These results show the trisomy of the double E clones and support the nondisjunction hypothesis.

Construction of triple E-type clones: Triple E-type clones (E1E3E'') were obtained from crosses between two different double E's (E1E3 X E'*E3) or a cross between double E (E'E') and single E (E") (Table IO). In those triple-E-type clones, single cells expressed three different E types. This was confirmed using three different 0 type testers (O', O3 and 0") marked with carmine particles, india ink and polystyrene latex particles. A single triple-E-type cell clumped simultaneously with the three different 0 type cells. These triple-E-type clones (Mt'/Mt'/Mt' ' or M t ' /Mt3/Mt1' /mt) were also able to produce triple E progeny when crossed to ordinary type O3 (mtlmt) (data not shown), suggesting the occurrence of nondisjunction of trivalent chromosomes (Mt'/Mt3/Mt "). When these triple E were testcrossed, the three M t genes were mostly assorted at random. In some triple E, however, a certain pair of M t genes tended to be assorted into different gametic nuclei, which resulted in deviation from the segregation ratio of mating types expected from random assortment of the Mt- carrying chromosomes.

DISCUSSION

Meiotic nondisjunction in intersyngenic hybrids of Paramecium caudatum: In P. caudatum, artificially induced conjugation between different syngens (sibling species) produced viable and fertile hybrids (TSUKII and HIWATASHI 1983). When intersyngenic hybrids were crossed to ordinary strains, they occasionally showed meiotic nondisjunction. Progeny of cells undergoing nondisjunction

MEIOTIC NONDISJUNCTION IN PARAMECIUM 79 1

became aneuploids, either trisomics or monosomics. In this article, the aneuploids found in the Mt-carrying chromosome were reported. However, prelim- inary study also shows the occurrence of aneuploids of the tnd 2 locus, which controls trichocyst discharge (Y. TSUKII, unpublished data). Thus, meiotic nondisjunction seems widespread throughout the hybrid genomes.

The frequencies of meiotic nondisjunction varied depending on the strains used. In the double E mating-type hybrids between syngen 1 and syngen 3, nondisjunction occurred at high frequency up to 19%, but in those between syngen 12 and syngen 3 it occurred much less frequently or did not occur at all. However, recombination frequencies between Mt and cnrA in the hybrids between syngen 3 and syngen 12 were nearly the same as those seen in intrasyngenic crosses in syngen 3, but they were greatly reduced in the hybrids between syngen 1 and syngen 3 (Y. TSUKII, unpublished data). Thus, a de- crease in recombination ratios between M t and cnrA seems to be correlated with an increase in meiotic nondisjunction. A similar phenomenon has been demonstrated in recombination-defective meiotic mutants in other organisms (BAKER et al. 1976).

Testcrosses of double E hybrid clones cytoplasmically derived from syngen 1 or syngen 12 showed nondisjunction of higher frequency than those of their coconjugant clones derived from syngen 3 cytoplasmic parents. As the two coconjugant clones should have an identical genotype, this result suggests that some nucleocytoplasmic interactions are involved in the occurrence of meiotic nondisjunction.

Patterns of assortment of Mt-carrying chromosomes in double E trisomics: In this report, the aberrant double E segregants derived from the crosses, double E X 0 were shown to be trisomics containing three Mt chromosomes (e.g., Mt', Mt', mt). If the three chromosomes formed a stable trivalent at meiosis and were assorted randomly, the gametic nucleus would have one of the following six different genotypes: Mt'lMt', Mt'lmt, Mt3/mt, Mtl, Mt' and mt. When the trisomics were backcrossed to type 0 (mtlmt), progeny would segregate four mating types, E', E3, E'E3 and 0, with a ratio of 2:2: 1 : 1 (Table 5) . The results obtained, however, were different from the expected ratio (Table 4). Segre- gation patterns of mating types in the backcrosses seem to differ from cross to cross, indicating that the three Mt chromosomes were assorted in different ways in each trisomic, even if derived from the same parents. Patterns of the mating-type segregations after the crosses of double E trisomics to 0 were tentatively divided into two groups designated type I and type 11. In type I segregation, only one of the two type E's appeared in the progeny, and E and 0 were segregated with nearly equal frequencies. In subsequent testcrosses, other type E's never appeared in the progeny, suggesting that one of the M t chromosomes was lost during meiosis in the trisomics. However, in type I1 segregation, four mating types-two single E's, double E and type O-were segregated with various frequencies. As mentioned above, if three chromosomes carrying the M t locus were assorted with random combinations of two to one, the segregation ratio of the four mating types would be 2:2: 1 : 1 (Table 5). Among 19 type I1 segregations in Table 4, however, 11 showed significantly


different segregation ratios from the expected ratio (data not shown). Fur- thermore, if none of the three M t chromosomes were lost and they were transmitted with equal frequencies, the ratio of the two dominant Mt alleles in the progeny would be 50% for each Mt (Table 5); however, this was not the case. In some second backcrosses, frequencies of Mt alleles from 20-61 % were observed (Table 4); this indicates that Mt chromosomes were occasionally eliminated during meiosis in some double E trisomics of type I1 segregations. However, the reasons why patterns of assortment of Mt chromosomes were so different in each trisomic are unknown.

Inheritance of the state of aneuploids: As shown in Tables 8 and 9, both monosomics and trisomics can produce the same kinds of aneuploid progeny when crossed. Monosomics can be reproduced in successive generations by alternating two different crosses. A cross between monosomics of mt +cnrA and diploid strains of M t 3 cnrA/Mt3 cnrA will produce monosomic progeny of Mt3 cnrA. Then, when the newly produced monosomics are crossed to mt +cnrA/mt +rnrA , progeny will segregate monosomics of mt +cnrA and ordinary diploids of Mt' cnrA/mt +cnrA. According to this procedure, we have obtained monosomic progeny in five successive generations.

On the other hand, trisomics for the M t chromosomes (e.g., Mt' /Mt' /mt) can produce trisomic progeny when crossed with type 0 (mtlmt). Moreover, when trisomics of Mt'/Mt'/mt were crossed with another trisomic, such as Mt'/Mt'?/ mt, progeny segregated the triple E type of Mt'/Mt'/Mt' ' or Mt'/Mt3/MtI2/mt that expressed E'E'E'? in single cells. These triple E types were also able to produce triple E progeny when crossed to type 0 (mtlmt) (data not shown). Thus, these triple E progeny were assumed to be tetrasomics of Mt'Mt3/MtI2/ mt. These results suggest the possibility that, by combining various type E's of different syngens, we can significantly increase the number of Mt chromosomes.

Applications of the aneuploids for Paramecium genetics: The aneuploids will be useful for various experimental genetic systems. Monosomy can be used for the isolation of recessive mutations on the chromosome carrying Mt and cnrA or tnd 2. On the other hand, trisomics or tetrasomics will be useful to examine gene dosage effects, as with the use of segmental aneuploidy in Drosophila (LINDSLEY et al. 1972; O'BRIEN and GETHMANN 1973; STEWART and MERRIAM 1974).

The species problems: Syngens in ciliates are considered reproductively isolated groups in a taxonomic species (SONNEBORN 1957). Recent advances in com- parative studies, especially of isozyme patterns, gave a firm basis for defining syngens as real biological species, and thus, species names were given to syngens of P. aurelia and Tetrahymena pyrformis (SONNEBORN 1975; NANNEY and McCoy 1976).

In our previous report (TSUKII and HIWATASHI 1983), however, we demonstrated that intersyngenic crosses artificially induced in P. caudatum produced highly viable and fertile hybrid progeny. The demonstration of such hybrid fertility immediately threw doubt on the concept of syngen in this species. In P. caudatum, the concept of syngen has been accepted solely based


on an assumption that genes do not flow between different syngens where a mating reaction does not occur. Since the distributions of the syngens overlap (Y. TSUKII and K. HIWATASHI, unpublished data) and intersyngen matings are possible in the laboratory, it is possible that genes could be exchanged between different mating groups even in natural conditions. In that case they should not be designated as different syngens, but should be included within a single biological species.

As reported here, however, there is some incompatibility when syngens are crossed. The incompatibility results in chromosomal nondisjunction in intersyngenic hybrid clones. This suggests that syngens of P. caudatum have been reproductively isolated for enough time to cause meiotic incompatibility among them. The incompatibility is, however, not so extensive as to produce sterile hybrids.

We thank DONALD L. CRONKITE and anonymous reviewers for their critical reading of the manuscript and for their invaluable comments. This research was supported by grant 5648000 1 from the Ministry of Education, Science and Culture.

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Communicating editor: S. L. ALLEN

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MEIOTIC NONDISJUNCTION AND ANEUPLOIDS IN ...MEIOTIC NONDISJUNCTION IN PARAMECIUM TABLE 1 Stocks and strains of P. caudatum used in this study 78 1 Phenotypes Mating Stocks or strains