Biparental Inheritance of Non-Mendelian Gene Markers in Chlamydomonas Moewusii

BIPARENTAL INHERITANCE OF NON-MENDELIAN GENE MARKERS IN CHLAMYDOMONAS MOEWUSII

ROBERT W. LEE AND CLAUDE LEMIEUX’ Department of Biology, Dalhousie University, Halqax, Nova Scotia B3H 451 Canada Manuscript received March 18, 1985 Revised copy accepted March 4, 1986

ABSTRACT The first two non-Mendelian gene mutations to be identified in Chlamydo- monas moewusii are described. These putative chloroplast gene mutations include one for resistance to streptomycin (sr-nM1) and one for resistance to erythromycin (er-nMI). In one- and two-factor reciprocal crosses, usually over 90% of the germinating zygospores transmitted these mutations and their wild-type alternatives from both parents (biparental zygospores); the remaining zygospores transmitted exclusively the non-Mendelian markers of the mating-type “plus” parent. Among the biparental zygospores, a strong bias in the transmission of non-Mendelian alleles from the mating-type “plus” parent was indicated by an excess of meiotic and postmeiotic mitotic progeny that were homoplasmic for non-Mendelian alleles from this parent compared to those that were homoplasmic for the non-Mendelian alleles from the mating-type “minus” parent. At best, weak linkage was detected between the sr-nMI and er-nM1 loci. Non- Mendelian, chloroplast gene markers in Chlamydomonas eugametos and Chlamy- domonas reinhardtii showed a predominantly uniparental mode of transmission from the mating-type “plus” parent in crosses performed under the same conditions used for the C. moewusii crosses.

uniparental inheritance of chloroplast characters known or likely to THEbe encoded in chloroplast DNA is a pervasive feature of most land plants and all of the few algae that have been studied in this regard (for reviews, see GILLHAM1978; KIRKand TILNEY-BASSETT1978; SEARS1980a; BIRKY1983). The most detailed and extensive analysis of chloroplast gene inheritance has been performed with the alga Chlamydomonas reinhardtii. In this alga, all stable non-Mendelian gene markers, which have been studied by recombination analysis, map to the chloroplast linkage group (SAGER1977; GILLHAM1978; METS and GEIST 1983). The phenotypes of these markers include resistance to and/ or dependence on inhibitors of chloroplast protein synthesis (e.g.,streptomycin, erythromycin, neamine), resistance to herbicides known to act on the photo- synthetic apparatus and deficiency in photosynthesis. Under conditions typically employed in the laboratory when performing crosses with C. reinhardtii, over 90% of the zygospores transmit chloroplast gene markers uniparentally from

’ Present address: Dipartement de Biochimie (Sciences), Pavillon Vachon, Universitk Lava], Quebec. Qu5bec GIK 7P4. Canada.

Genetics 113: 589-600 July, 1986. 590 R. W. LEE AND C. LEMIEUX the mating-type “plus” (mt’) parent, most of the remaining zygospores transmit chloroplast gene markers from both parents (biparental zygospores) and only rare zygospores transmit exclusively chloroplast gene markers from the mating- type “minus” (mt-) parent (SAGER1972; GILLHAM1978). A small fraction of the C. reinhardtii zygotes typically grow mitotically, as vegetative diploids, rather than undergo meiosis. These cells predominantly show biparental inheritance of chloroplast gene markers (GILLHAM1969). Among induced or spontaneously occurring biparental zygospores, SAGERand colleagues have reported an equal contribution of chloroplast alleles from both parents (e.g., SAGERand RAMANIS1976). In contrast, GILLHAMand BOYNTONand colleagues have reported a strong bias favoring the transmission of chloroplast alleles contributed by the mt+ parent (e.g., GILLHAM1969; GILLHAM,BOYNTON and LEE 1974; HARRISet al. 1977; FORSTERet al. 1980; SEARS,BOYNTON and GILLHAM1980) in both zygospores and vegetative diploids. Non-Mendelian, putative chloroplast gene markers have also been reported in other species of Chlamydomonas. MCBRIDEand MCBRIDE(1 975) recovered uniparentally inherited markers for resistance to streptomycin (sr-2) and for dependence on neamine (nd) in Chlamydomonas eugametos and VANWINKLE- SWIFTand AUBERT(1 983) have described a uniparentally inherited mutation for resistance to erythromycin (ery-ul) in the homothallic species Chlamydo- monas monoica. Recently, the sr-2 marker of C. eugametos has been shown to be a chloroplast genetic marker by virtue of its perfect linkage with chloroplast DNA restriction fragment markers encoding 16s ribosomal rRNA (LEMIEUX et al. 1984). In this study we describe the recovery of the first non-Mendelian genetic markers to be identified in Chlamydomonas moewusii, an alga which is interfer- tile with C. eugametos, but not with C. reinhardtii. One of these markers confers resistance to streptomycin (sr-nMI),and the other confers resistance to erythromycin (er-nMI). We show here that these markers are transmitted in a predominantly biparental fashion under conditions in which chloroplast gene markers in C. eugametos and C. reinhardtii crosses are transmitted in a predominantly uniparental fashion. A portion of this work has been published in preliminary form (LEMIEUXand LEE 1980).

MATERIALS AND METHODS Strains and their maintenance: The mt+ and mt- wild-type strains of C. moewusii (UTEX 97 and 96) (STARR1978) and the mt+ (or “male”) and mt- (or “female”) wild- type strains of C. eugumetos (UTEX 9 and 10) were provided by C. S. GOWANSof the University of Missouri-Columbia. The sexual equivalency of strains 9 and 97 as mt+ or “male” has been discussed by GOWANS(1 976). A mt+ strain of C. eugumetos carrying the streptomycin resistance marker sr-2 (MCBRIDEand MCBRIDE 1975) was also provided by GOWANS.New sr-2 strains, both mt+ and mt-, with improved mating efficiency were derived from the GOWAN’Sstrain after two generations of crosses in which UTEX 9 was the recurring mt- parent. The strains of C. reinhurdtzi carrying the chloroplast gene markers for resistance to spectinomycin (spr-U-1-27-3)and for resistance to streptomycin (sr-U-2-60)(HARRIS et al. 1977) were provided by N. W. GILLHAMand J. E. BOYNTON of Duke University. The C. eugumetos and C. moewusii strains were maintained on slants at 22” under alternating 12-hr-light (“cool white” fluorescent bulbs, 200 footcandles) NON-MENDELIAN GENE INHERITANCE 59 1 and 12-hr-dark periods on the minimal medium of GOWANS(1960) supplemented with 1.5% Difco Bacto-agar (minimal agar medium). C. reinhardtzz strains were maintained as described previously (LEE and SAPP 1978). Isolation of mutants: For the recovery of streptomycin-resistant mutants, mt+ wild- type cells of C. moewusii were mutagenized by growth in liquid minimal medium containing 10 pg/ml 2-amino-3-phen lbutanoic acid (APBA) as described by MCBRIDEand MCBRIDE(1975). Aliquots of 10Y cells were plated on minimal agar medium supplemented with 12.5 pg/ml streptomycin (from streptomycin sulfate, Pfizer) and were incubated under standard growth conditions (22 " , 400 footcandles continuous light). For the recovery of erythromycin-resistant mutants, mt+ wild-type cells of C. moewusii were mutagenized with methyl methanesulfonate (MMS) by a modification of the method described by HAWKSand LEE (1976). Aliquots of cells were removed from synchronous culture (LEMIEUX,TURMEL and LEE 1980) at the onset of the light period, harvested by centrifugation at 1000 X g for 10 min at 20" and resuspended in 30 mM sodium phosphate buffer, pH 6.8, at a final concentration of lo6 cells per milliliter. The cell suspension was made 30 mM MMS and was then incubated with mild shaking for 30 min at 25" in darkness. Cells were washed once with minimal medium and were resuspended in this medium to a final concentration of about 2 X lo5cells per milliliter. Viable check platings determined survival to be about 50%. The mutagenized cell suspension, in 1.0-ml aliquots, was delivered to each of several culture tubes that were kept at 22" in an incubator flushed with 3% CO2 in air and illuminated continuously under 700 footcandles. During the following 48-hr incubation, cell number increased about 40-fold. A 4-ml aliquot of a 37", 1.25% low-melting agarose (Marine Colloids) solution in minimal medium was added to each tube. Each mixture was then plated on minimal agar medium containing 25 pg/ml erythromycin (from erythromycin lacto- bionate, Abbott) and was incubated under standard growth conditions. Streptomycin- resistant and erythromycin-resistant mutants were recovered after 2 wk of culture on the respective antibiotic media. The resistance level of the mutant strains so recovered was determined on minimal agar medium containing increasing concentrations of streptomycin or erythromycin. Procedure for crosses and genetic analysis: The procedure employed for all crosses with C. moewusii, C. eugametos and C. reinhardtii was adapted from those employed by GOWANS(1 960) and WANC(1 972). The strains to be crossed were first grown separately at 18" on minimal agar medium supplemented with 4 g/liter Difco Bacto-yeast extract under alternating 12-hr-light (500 footcandles) and 12-hr-dark periods for 5 days. An inoculum of each strain was then transferred to minimal agar medium containing %O the normal NH4NOs concentration and was cultured under the above conditions for another 5 days. At the completion of the last dark period, cells from each strain were suspended separately in 0.5-ml sterile distilled water saturated with CaCOs at a density of about 5 X lo6 cells per milliter. Gametogenesis was induced by incubating the cell suspensions at 18" under 700 footcandles for 3-6 hr. Gamete suspensions of opposite mating types were then mixed and allowed to mate for 1-2 hr under the light and temperature conditions used for gametogenesis. After this time, 0.2 ml of each mating- pair suspension was plated on minimal medium and was incubated at 18" under 500 footcandles for 48 hr. The plates were then incubated in darkness at 18" for 6 days. At the end of this period, mature zygospores were scraped from the agar surface, transferred to fresh minimal medium plates and manipulated singly into one end of a series of lanes cut in the agar surface. Germination of zygospores occurred within the following 24-hr period under standard growth conditions. Zygospore manipulation and meiotic product dissection and manipulation were performed with a 0.127-mm tungsten wire (Ventron, Alfa Division) as described by CAINand CAIN (1984). The mating type of meiotic products was determined as described by GOWANS(1960), except that gamete suspensions were prepared as described above. The inheritance of antibiotic resistance markers was scored after zygospores or meiotic products had undergone about 20 cell doublings on nonselective agar medium 592 R. W. LEE AND C. LEMIEUX TABLE 1 Inheritance pattern of streptomycin-resistantand erythromycin-resistant mutant phenotypes in Chlamydomonas moewusii

Mutant phenotype Inheritance pattern No. of tetrads sr-50 Mendelian 5 sr-250 Non-Mendelian 15 er-1 OOa Mendelian 5 er-100b Mendelian 5 er-400a Mendelian 4 er-400b Non-Mendelian 7 Phenotypes of antibiotic-resistant mutants are designated by antibiotic (streptomycin = sr, erythromycin = er) and resistance level (micrograms per milliliter). In all crosses the mt+ strain with the indicated antibiotic-resistant phenotype was crossed to the wild-type mt- strain. Mendelian inheritance was revealed by a 2:2 segregation of resistance and sensitivity in tetrads, whereas non- Mendelian inheritance was revealed by all products of each tetrad displaying the resistant phenotype.

to form zygospore- or meiotic-product clones. Cells were then transferred by toothpick to antibiotic medium for the detection of resistant cells. In this same fashion the inheritance of antibiotic resistance markers was scored in progeny subcloned from the meiotic-product clones. The subclones were recovered as colonies on nonselective medium after streaking liquid suspensions of the zygospore- or meiotic-product clones. Minimal agar medium containing 75 rg/ml streptomycin was used for the detection of sr-2 and sr-nMI, and minimal agar medium containing 100 pg/ml erythromycin was used for the detection of er-nM1. The detection of sr-u-2-60 and spr-u-1-27-3 was as described previously (LEEand SAPP 1978).

RESULTS On minimal agar medium, the threshold resistance level of C. moewusii both to streptomycin and to erythromycin is about 5 Pg/ml. Mutants resistant to at least 12.5 pg/ml streptomycin were recovered with an incidence of about among mt' wild-type cells after APBA mutagenesis. The majority of these mutants (about 90%) showed a streptomycin resistance level of 50 &ml, and rare mutants (about 10%) were resistant to 250 Pg/ml. No erythromycin- resistant mutants were recovered after APBA mutagenesis; however, after MMS mutagenesis, four independent mutants were recovered among 7 x lo6 treated cells. Two were resistant to 100 pg/ml, and two were resistant to 400 Pg/ml. One mutant from each of the streptomycin resistance-level classes and all four erythromycin-resistant mutants were crossed to the mt- wild-type strain, and the progeny were scored by tetrad analysis. The results are shown in Table 1. The sr-50, er-lOOa, er-lOOb and er-400a mutations behaved as single Mendelian genes showing a 2:2 segregation of resistance and sensitivity in tetrads. The sr-250 and er-400b mutants behaved as non-Mendelian genes. Their respective resistant phenotypes were transmitted to all four products of each tetrad scored; however, some products were obviously heteroplasmic for NON-MENDELIAN GENE INHERITANCE 593 TABLE 2 Segregation of non-Mendelian allele pairs in tetrads from two different Chlamydomonas moewusii crosses

Frequency of tetrads

+ mt+ + mt+ X X Segregation pattern er-nM1 mt- sr-nMI mt- OS:OR:4H 0.58 (8) 0.69 (1 1) 1S:OR:SH 0.07 (1) 0.19 (3) 2S:OR:2H 0.14 (2) 0.06 (1) 3S:OR:1 H 0.14 (2) 0.00 4S:OR:OH 0.00 0.06 (1) OS:lR:3H 0.07 (1) 0.00 Total 1.00 (14) 1.00 (16) The number of tetrads showing the indicated type of segregation pattern is given in parentheses. Abbreviations: S = homoplasmic for sensitivity allele-no resistant cells detected in the meiotic-product clone; R = homoplasmic for resistance allele-no sensitive colonies detected among 50 subcloned colonies of the meiotic-product clone; H = heteroplasmic for sensitivity and resistance alleles-resistant cells detected in the meiotic-product clones and one or more sensitive colonies detected among 50 subcloned colonies of the meiotic- product clone. the resistance and sensitivity alleles. Such heteroplasmic products were revealed by the appearance of resistant cell clusters within a background of dead cells after transfer of the meiotic-product clones from nonselective medium to antibiotic medium. The segregation pattern of the non-Mendelian alleles for resistance and sensitivity was further examined in tetrads obtained from two monofactor crosses in which the sr-250 and the er-400b mutations, designated sr-nM1 and er-nMI, were carried by the mt- parent. Table 2 shows that all 30 zygospores scored from the two crosses, with one exception, were biparental, i.e., heteroplasmic for the non-Mendelian resistance and sensitivity alleles of the parents; moreover, most meiotic products were themselves heteroplasmic (99 of 120). Indeed, the most frequent tetrad class was one in which the four meiotic products were heteroplasmic. Among the homoplasmic products that were detected (21 of 120), all but one expressed the sensitivity allele of the mt+ parent. This biased output in favor of the alleles for sensitivity from the mt+ parent and against the alleles for resistance from the mt- parent is also seen in an analysis of the frequency of sensitive cells in clones derived from the heteroplasmic meiotic products of these crosses (Table 3). Most of the 99 heteroplasmic meiotic-product clones from the two crosses yielded an excess of subclones that expressed only the antibiotic-sensitive allele of the mt+ parent. The most common heteroplasmic meiotic product clone contained fewer than 2% resistant subclones. In order to document further the biparental inheritance of the sr-nMI and 594 R. W. LEE AND C. LEMIEUX TABLE 3 Frequency of antibiotic-resistantcells in meiotic-product clones derived from heteroplasmic meiotic products recovered from the C. moewusii crosses of Table 2

No. meiotic-product clones

+ mt+ + mt+ Frequency of X X resistant cells er-nM1 mt- sr-nMI mt- <0.02 12 19 0.02-0.08 7 9 0.10-0.18 6 7 0.20-0.28 1 8 0.30-0.38 4 3 0.40-0.48 2 4 0.50-0.58 4 0 0.60-0.68 3 1 0.70-0.78 1 2 0.80-0.88 2 0 0.90-0.98 2 2 Total 44 55

~ ~ ~~

TABLE 4 Recombination between non-Mendelian gene markers in zygospore subclones of C. moeuncsii

No. subclones

Cross mt+ x mt- % BPZ" E + ES S M Total % R er-nM1 + X + sr-nM1 97 (91) 521 106 166 117 85 995 27 + sr-nM1 X er-nM1 + 87 (109) 60 78 101 700 61 1000 18 + + X er-nM1 sr-nM1 91 (94) 139 442 237 72 0 890 24 Ten biparental zygospores (BPZ) were recovered from each cross, and about I00 subclones from each BPZ were scored for their phenotype: E = resistant to erythromycin and sensitive to streptomycin; + = sensitive to erythromycin and to streptomycin; ES = resistant to erythromycin, to streptomycin and to a mixture of erythromycin and streptomycin; S = resistant to streptomycin and sensitive to erythromycin; M = mixed, resistant to erythromycin and to streptomycin but sensitive to a mixture of erythromycin and streptomycin. % R = percentage of subclones with recombinant phenotype (+ or ES). "Total number of zygospores scored from each cross is given in parentheses. Biparental zygospores from the repulsion crosses were identified as those transmitting a resistance marker from both parents, those from the coupling cross were identified as those transmitting both resistance markers from the mt- parent. er-nM1 markers and to test for possible linkage between them, crosses were performed between singly resistant cells, in reciprocal, and between wild-type mt+ cells and mt- doubly resistant cells. Inheritance of the non-Mendelian markers was scored by zygospore clone analysis (ADAMSet aE. 1976). The results of all three crosses, as shown in Table 4, agree with those of previous crosses in showing both a high percentage of biparental zygospores and a strong bias favoring the transmission of alleles from the mt+ parent. This bias occurred regardless of whether the mt +-derived alleles were for resistance or NON-MENDELIAN GENE INHERITANCE 595 TABLE 5 Linkage retention of non-Mendelian gene markers contributed by the mt- parent

% subclones recombinant for Cross mt+ X mt- Selected marker unselected marker

er-nM1 + X + sr-nMI er-nMl+ 39 sr-nM1 45 + sr-nM1 X er-nM1 + er-nM1 45 sr-nMl+ 34 + + X er-nM1 sr-nM1 er-nM1 37 sr-nM1 23 sensitivity to erythromycin or for resistance or sensitivity to streptomycin and, hence, cannot be explained by allele-specific effects. Moreover, the favored transmission of mt+-derived alleles persisted, despite the fact that subclones were scored from zygospore-colonies that revealed the strongest transmission of mt--derived resistance alleles; such selection was performed in an attempt to enhance opportunities for recombination. The percentage of subclones that were recombinant (nonparental) for the non-Mendelian allele pairs was 27% and 18% in the repulsion crosses and 24% in the coupling cross (average 23%). Using these same data, however, recombination frequencies averaged from the three crosses increased to 42% (Table 5) if recombination was scored by the paternal (mt-) marker selection method of METS and GEIST(1983). With this method, recombination is scored as the percentage of subclones that inherited at least one marker from the mt- parent (selected marker) but were recombinant for the other marker (unselected marker). The predominantly biparental transmission of non-Mendelian gene markers in the C. moewusii crosses described here contrasts with the predominantly uniparental and strictly uniparental transmission patterns reported for similar markers in C. reinhardtii and C. eugametos crosses, respectively. To test if there might be an environmental basis for this difference, the transmission of non- Mendelian gene markers in C. reinhardtii and C. eugametos crosses was followed using the conditions of gametogenesis, mating, zygospore development and zygospore germination used here for the C. moewusii crosses. In the C. reinhardtii cross, a mt+ strain with the spr-u-1-27-3 chloroplast marker for spectinomycin resistance was crossed to a mt- strain with the sr-u-2-60 chloroplast marker for streptomycin resistance. For C. eugametos, reciprocal monofactor crosses were performed between strains that differed for the sr-2 chloroplast marker. The results in Table 6 show that the C. reinhardtii chloroplast markers were transmitted uniparentally from the mt+ parent by over 90% of the zygospores, as is typical under crossing procedures commonly employed with this species. For the C. eugametos crosses, a predominance of uniparental zygospores was also evident. The sr-2 marker when contributed by the mt+ parent was transmitted by all zygospores, but when contributed by the mt- parent, it was transmitted by 10% of the zygospores in one cross and by 20% of the zygo- 596 R. W. LEE AND C. LEMIEUX TABLE 6 Transmission of chloroplast gene markers in C. reinhardtii and C. eugametos crosses performed under the conditions used earlier for the C. moewusii crosses

- - ~ ~ ~~ Cross mt+ X mt- Frequency of zygospores UP+ BP UP- C. reinhardtzi spr-u-1-27-3 + X + sr-u-2-60 0.94 0.05 0.01 (78) Transmitting sr-2 allele

C. eugametos sr-2 X + 1.00 (69) + X sr-2 0.10 (140), 0.20 (287) - ~ ~ ~ ~ The number of zygospores scored in each cross is given in parentheses. UP+ = spectinomycin-resistant, streptomycin-sensitive; UP- = spectinomycin- sensitive, streptomycin-resistant; BP = spectinomycin-resistant, streptomycin- resistant. spores in a replica cross performed at a different time. The usual two-sample test for equality of binomial probabilities shows that such a large difference in the replica crosses is unlikely to occur by chance (P = 0.01).

DISCUSSION The sr-nMI and er-nM1 non-Mendelian gene markers of C. moewusii and their wild-type allelic alternatives were transmitted biparentally by the great majority of zygospores; the remaining zygospores transmitted only the non- Mendelian alleles of the mt+ parent. Among the progeny of biparental zygospores, there was a strong bias favoring the transmission of non-Mendelian alleles derived from the mt+ parent. As discussed earlier, a similar bias in the transmission of chloroplast alleles derived from the mt+ parent has been described for biparental zygospores of C. reinhardtii (e.g., GILLHAM,BOYNTON and LEE 1974). We note, however, that the direction of this bias in the two species has no significance because the mating-type designations of C. moewusii are defined arbitrarily relative to those of C. reinhardtii (GOWANS1976). Three, two-factor crosses with the er-nM1 and sr-nMI resistance markers in coupling and repulsion were performed in order to assess possible linkage between these sites. When linkage was scored by zygospore clone analysis (ADAMSet al. 1976), a method that has been successfully employed to obtain a reliable and consistent ordering of chloroplast gene markers in C. reinhardtii (HARRISet al. 1977), about 23% of the subclones from biparental zygospores were recombinant for these markers. However, as is typical in crosses with C. reinhardtii, about half or more than half of the subclones scored revealed the non-Mendelian (nonrecombinant) phenotype of the mt+ parent. This excess, which results from the bias in the transmission of non-Mendelian alleles from the mt+ parent, enhances artificially the linkage of the markers followed. We decided, therefore, to use the approach of METS and GEIST (1983) to score NON-MENDELIAN GENE INHERITANCE 597 subclones that inherited at least one marker from the mt- parent and to de- termine the percentage of these that were recombinant for the other marker. When scored in this manner, the percentage of recombinants increased to about 42%, a value that is close to the 50% normally observed for unlinked nuclear genes. Unequal mixing of the parental chloroplast genomes and limited rounds of pairing for recombination could produce a maximum percentage of recombinants that is less than 50%. In this connection, an upper recombina- tional limit of 20-25% has been described for mitochondrial gene markers in Saccharomyces cerevisiae (DUJON,SLONIMSKI and WEILL 1974). Moreover, if cytoplasmic mixing is incomplete in Chlamydomonas zygospores, as discussed by METS and GEIST (1983), markers in the same sector of unmixed cytoplasm could show linked inheritance even if they are encoded by separate non-Men- delian genomes (e.g., chloroplast and mitochondrial). Because of these uncer- tainties, therefore, the mechanistic basis for the rather weak, linked inheritance of the sr-nM1 and er-nm1 loci is unclear. The great excess of C. moewusii zygospores that transmit putative chloroplast gene markers biparentally, as reported here, contrasts with the predominantly uniparental and strictly uniparental transmission of known chloroplast gene markers in C. reinhardtii and C. eugametos, respectively, as reported in the literature. This difference could result from environmental conditions associ- ated with the crosses or from inherent differences between the species. The latter seems to be the more important factor because, with the protocol used here for the C. moewusii crosses, chloroplast gene markers in C. reinhardtii and C. eugametos were still transmitted in a predominantly uniparental fashion. The spr-U-1-27-3 and sr-U-2-60chloroplast gene markers of C. reinhardtii were transmitted biparentally by fewer than 10% of the zygospores, a value consistent with those observed under the range of conditions normally employed when performing such crosses (SEARS,BOYNTON and GILLHAM1980). However, the sr-2 chloroplast gene marker of C. eugametos was transmitted biparentally by about 10% of the zygospores in one cross and by about 20% of the zygospores in a replica cross. These results contrast with those of MCBRIDEand MCBRIDE (1975), who found no biparental transmission of the same marker in mass plating studies with about 3000 zygotes. The different level of biparental chloroplast gene transmission in our replica crosses suggests an effect of culture conditions on this phenomenon, despite our efforts to standardize these conditions. The inability of MCBRIDEand MCBRIDE(1975) to detect such biparental inheritance could be due to differences in culture conditions and/or to genetic differences in the C. eugametos strains employed. In C. moewusii, evidence for an influence of culture conditions on chloroplast DNA (cpDNA) transmission has been described recently by COLEMANand MAGUIRE(1 983). They interpreted fluorometric staining patterns of chloroplast nucleoids in nutrient-starved zygospores of C. moewusii as showing a complete loss of the nucleoids derived from one parent (one-sided distribution) in over 95% of the zygospores. However, zygospores that received a fresh supply of nutrients had not become as completely one-sided (62% one-sided, 3% two- sided, 35% with normal-sized nucleoids on one side and tiny nucleoids on the 598 R. W. LEE AND C. LEMIEUX other). These results suggest that good zygospore nutrition promotes biparental, but unequal, transmission of cpDNA. Such an effect may have contributed to our results, which also employed zygospores grown on a complete nutrient medium. The higher fraction of zygospores that were scored as biparental in our study, using the genetic approach, compared to those detected cytologically by COLEMANand MAGUIRE(1 983) with the nutrient-fed population, might be explained by the greater sensitivity of the genetic approach and/or by the numerous differences in the culture conditions employed in the two studies. Clearly, it would be of interest to score the transmission of the sr- nM1 and er-nM1 markers from the same C. moewusii zygospore population in which cpDNA is assayed cytologically. Finally, in C. reinhardtii, the inheritance pattern of chloroplast genes can be influenced by the environmental conditions to which zygospores and mitotic zygotes are subjected (VANWINKLE-SWIFT1978; SEARS1980b). Moreover, UV- irradiation of mt+ gametes increases the transmission of chloroplast genes from the mt- parent to zygospore progeny (SAGERand RAMANIS1967). Other environmental or chemical treatments of gametes before mating can also affect the inheritance pattern of chloroplast genes in C. reinhardtii crosses (SAGER and RAMANIS1973; WURTZ,BOYNTON and GILLHAM1977; SEARS,BOYNTON and GILLHAM1980). At this stage, it is difficult to evaluate the evolutionary significance of differences in the rates of biparental chloroplast gene transmission among related species of Chlamydomonas under one set of laboratory conditions. However, one may begin to understand the genetic basis for this phenomenon from an analysis of progeny from interspecific crosses between C. moewusii, a species with high biparental chloroplast gene transmission, and C. eugumetos, a species with low biparental chloroplast gene inheritance.

We thank L. METS, M. ROSE,J. VANDER MEER and E. ZOUROS for helpful discussion. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.

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Communicating editor: J. E. BOYNTON