crossing among these lines to create F 1994. Enrichment of microsatellites from the citrus ge- both types of flowers (Baker 1966; Brock- 2 nome using biotinylated oligonucleotide sequences progeny [the progeny design employed by bound to streptavidin-coated magnetic particles. man and Bocquet 1978; Burrows 1960; Dul- Arens et al. (1995)]. For H. acuminata, this BioTechniques 16:657–662. berger and Horovitz 1984; Horovitz and design would take at least 35 years to ac- Kijas JMH, Thomas MR, Fowler JCS, and Roose ML, Gallil 1972; Jolls and Chenier 1989; Koelew- complish, compared to several hundred 1997. Integration of trinucleotide microsatellites into a ijn and Van Damme 1995a; Philipp 1980). linkage map of Citrus. Theor Appl Genet 94:701–706. years for C. alta. The last type is generally called ‘‘gyno- Primmer CR, Raudsepp T, Chowdhary BP, Moller AP, Random physical distributions of mark- and Ellegren H, 1997. Low frequency of microsatellites monoecious’’ or ‘‘partially male sterile.’’ er loci is an important assumption of pop- in the avian genome. Genome Res 7:471–482. The term ‘‘gynodioecy’’ was originally ulation genetic studies as well as a re- Sokal RR and Rohlf FJ, 1981. Biometry, 2nd ed. New coined to describe the plant reproductive quirement for high-resolution genetic York: W.H. Freeman. system with only females and hermaph- mapping. For population genetic studies, Received May 13, 1998 rodites (Darwin 1877). Formally speaking, marker loci should be drawn from as Accepted March 31, 1999 populations containing individuals of the much of the genome as possible so evo- Corresponding Editor: Norman F. Weeden third type should therefore be said to lutionary events (mutation dynamics, phy- have a gynodioecious-gynomonoecious logenetic history, selection, and linkage) breeding system, but normally the occur- Downloaded from https://academic.oup.com/jhered/article/90/5/563/2186975 by guest on 27 September 2021 are averaged across the genome. The use rence of such plants is disregarded and of multiple restriction enzymes or the re- Female and Hermaphrodite the breeding system is still said to be duction of sampling steps when construct- gynodioecious. The standard explanation ing the DNA insert pool may increase the Flowers on a Chimeric of gynomonoecious plants is that they rep- diversity of SSR marker loci available. Gynomonoecious Silene resent the developmental outcome of an These results suggest that SSR loci bear- vulgaris Plant Produce incomplete restoration by nuclear male ing different repeats are not associated fertility genes of male-sterilizing cyto- within the genome, so the use of SSR Offspring with Different plasms (Koelewijn and Van Damme 1995b; marker loci with different repeats may aid Genders: A Case of Louis and Durand 1978; Van Damme 1983; in meeting the assumption of randomness Heteroplasmic Sex Vranceanu and Stoenescu 1978; Wicker- for population genetic studies. Although Determination? sham and Patterson 1980). the genomic distribution of SSR loci is less Silene vulgaris (Moench) Garcke (Cary- of a concern in parentage analysis appli- H. Andersson ophyllaceae), a common weed in ruderate cations (as long as markers are unlinked), habitats throughout Europe, is usually re- using multiple restriction enzymes when In gynodioecious plant species, individu- ferred to as being gynodioecious, though constructing DNA libraries may increase als have either female or hermaphrodite gynomonoecious individuals have fre- the diversity of SSRs available. flowers. However, individuals with both quently been observed (Brockman and From the Smithsonian Institution, National Zoological types of flowers, that is, ‘‘gynomonoe- Bocquet 1978; Charlesworth and Laporte Park, Molecular Genetics Laboratory, Washington, DC. cious’’ or ‘‘partially male steriles,’’ are 1998; Dulberger and Horovitz 1984; Jolls This work was supported by a Smithsonian Institution sometimes found. The standard explana- and Chenier 1989). The full genetic details postdoctoral fellowship to M.B.H. and Friends of the National Zoo. We thank D. Fonseca and L. Shapiro for tion of gynomonoecious individuals is that of the sex determination system in the contributions to the microsatellite cloning methods, I. their male-sterilizing cytoplasm is incom- species is not yet fully understood. There Jones for help picking colonies, and J. Ballou for dis- pletely restored by nuclear male fertility is, however, strong evidence that both nu- cussion of G tests. W. J. Kress provided tissue samples of H. acuminata and helpful discussion. Two anony- genes. Silene vulgaris, the bladder cam- clear and cytoplasmic factors are involved mous reviewers provided comments that improved the pion, is usually referred to as being gyno- in the determination of sex (Charlesworth manuscript. Address correspondence to M. B. Hamil- ton, Georgetown University, Department of Biology, dioecious, though gynomonoecious indi- and Laporte 1998; Marsden-Jones and Tur- Reiss Building STE 406, Box 571229, Washington DC viduals have frequently been observed. In ill 1957). 20057-1229, or e-mail: [email protected]. an attempt to determine if the segregation In a survey of S. vulgaris plants in natu- ᭧ 1999 The American Genetic Association of flower types on gynomonoecious plants ral populations from southern Sweden, gy- of S. vulgaris is associated with a differ- nomonoecious individuals were found to- ence in offspring gender, a series of cross- gether with females and hermaphrodites. References es was performed. The results show that Seeds were collected from the populations Arens P, Odinot P, van Heusden AW, Lindhout P, and Vosman B, 1995. GATA- and GACA-repeats are not even- female and hermaphroditic flowers on the and plants were raised under standard ly distributed throughout the tomato genome. Genome same plant produce offspring of all three conditions in a greenhouse. At flowering 38:84–90. types, but the frequencies with which they the frequencies of gynomonoecious, fe- Armour JAL, Neumann R, Gobert S, and Jeffreys AJ, do so differ. If incomplete restoration was male, and hermaphroditic plants were de- 1994. Isolation of human simple repeat loci by hybrid- ization selection. Hum Mol Genet 3:599–605. the relevant explanation in this case, these termined. In the different populations gy- Bell CJ and Ecker, JR, 1994. Assignment of 30 microsa- results would not have occurred. Instead nomonoecious plants ranged in frequency tellite loci to the linkage map of Arabidopsis. Genomics an intraindividual segregation of one or from 0 to 25%, while female plant frequen- 19:137–144. many genetic factors that affect the sexual cy ranged from 0 to 90%. Three types of Fleischer RC and Loew S, 1995. Construction and phenotype of flowers and their subse- flower stalks could be observed on the gy- screening of microsatellite-enriched genomic libraries. In: Molecular zoology: advances, strategies and proto- quent offspring is proposed. nomonoecious individuals: stalks with cols (Ferraris J and Palumbi S, eds). New York: Wiley- only female flowers, stalks with only her- Liss; 461–468. Three different genders are frequently maphrodite flowers, and stalks with a mix- Goldstein DB and Pollock DD, 1997. Launching micro- satellites: a review of the mutation processes and found in gynodioecious species: plants ture of both types of flowers. The ratio of methods of phylogenetic inference. J Hered 88:335–342. with only female flowers, plants with only female to hermaphrodite flowers on the Kijas JMH, Fowler JCS, Thomas MR, and Garbett CA, hermaphrodite flowers, and plants with gynomonoecious plants varied, as did the

Brief Communications 563 Table 1. The gender of offspring to four sets of crosses between a chimeric gynomonoecious plant (A) and two hermaphrodite test plants (M and N)ofS. vulgaris

Offspring gender Test of homogeneity Gynomo- Herma- Crosses Female noecious phrodite Total ␹2 p

Figure 1. Typical flower arrangements in chimeric 1. A (self-fertilized) branches of gynomonoecious S. vulgaris plants. Filled a. Female flowers 50 6 8 64 13.0 Ͻ.01 circles represent hermaphroditic flowers; unfilled cir- b. Hermaphroditic flowers 44 28 8 80 cles represent female flowers. 2. A ϫ M a. Female flowers 64 2 30 96 30.6 Ͻ.001 degree of maleness of the individual her- b. Hermaphroditic flowers 32 22 46 100 maphroditic flowers (as judged by the 3. A ϫ N number and size of anthers). The segre- a. Female flowers 60 2 37 99 10.9 Ͻ.01 b. Hermaphroditic flowers 52 15 32 99 gation of female and hermaphroditic flow- 4. A (self-fertilized) ers on chimeric stalks often showed a Downloaded from https://academic.oup.com/jhered/article/90/5/563/2186975 by guest on 27 September 2021 a. Female flowers 49 20 6 75 — — strikingly symmetrical pattern (Figure 1). b. Hermaphroditic flowers — — — — Even though the ratio of female to her- maphroditic flowers on gynomonoecious In crosses 1, 2, and 3 the female flowers came from female stalks and the hermaphroditic flowers from hermaph- rodite stalks. Castrations were made in crosses 2 and 3 to avoid self-fertilization. The homogeneity between off- plants varied from one period of flowering spring classes was investigated with a chi-square test with two degrees of freedom. In the fourth cross, the flowers to the next, their gender expression was used to set seeds came from chimeric stalks; no seeds were produced by the hermaphroditic flowers. The pollen used for self-fertilizing plant A (crosses 1 and 4) came from hermaphroditic flowers on hermaphroditic flower stable over years. stalks. To determine if the segregation of flow- ers on gynomonoecious plants of S. vul- garis is associated with a difference in the analogous to the second cross but in hermaphroditic flowers produce more gy- offspring types produced, a series of which plant N was used as the pollen do- nomonoecious offspring than female flow- crosses was performed. A typical gyno- nor, the same differences in offspring gen- ers do. When the flowers from chimeric monoecious individual (A) and two her- der were seen. In general, the proportion stalks are studied, it is seen that female maphroditic test plants (M and N) were of hermaphrodites among the offspring in- flowers from such stalks produce more gy- used as parents in the crosses. The pater- creased when plants M and N were used nomonoecious offspring than female flow- nal plants were chosen from the green- for fertilization, in comparison to when ers on stalks with only females. house-grown material described above. plant A was self-fertilized. If the occurrence of gynomonoecious The fourth set of crosses were identical plants in S. vulgaris were strictly due to to the first crosses, except that the female incomplete restoration of a male-sterile The Crossing Experiments and hermaphrodite flowers that were fer- cytoplasm by one or many nuclear loci, as Four sets of crosses were performed, iden- tilized with A pollen this time came from has been suggested for other species tified as 1, 2, 3, and 4 in Table 1. Seeds chimeric flower stalks. In this case only (Koelewijn and Van Damme 1995b; Louis were obtained from all crosses except the female flowers set any seeds. The self- and Durand 1978; Van Damme 1983; Vran- from hermaphroditic flowers on chimeric fertilized female flowers on chimeric stalks ceanu and Stoenescu 1978; Wickersham stalks (4b). The derived seeds were ger- and the self-fertilized female flowers on and Patterson 1980), the observed differ- minated and cultivated in the greenhouse nonchimeric stalks (results from experi- ences between the offspring to the two and the offspring plants could, when flow- ment 1a) produced progeny that differed flower types would not have existed. Nu- ering, be classified into the same three significantly in gender proportions (␹2 ϭ clear genes are inherited on the female gender classes as the plants from the nat- 7.0, df ϭ 2, p Ͻ .05). The difference was side in the same way, irrespective of ural populations. primarily due to the flowers on chimeric whether they are transmitted via a female When female and hermaphroditic flow- branches producing a higher frequency of or a hermaphroditic flower. The frequency ers on plant A were fertilized with pollen gynomonoecious offspring. of offspring genotypes with an unstable from the same plant (set 1, Table 1), both sex expression should therefore have female and hermaphroditic flowers pro- been the same for the two kinds of flow- Interpretation duced offspring of all three gender class- ers. es, but they did so in different propor- These results show that female and her- On the other hand, cytoplasmic factors tions. The most obvious difference was maphroditic flowers on nonchimeric are capable of segregating within single in- that hermaphroditic flowers produced stalks produce offspring that differ signif- dividuals. Since a nucleocytoplasmic de- many more gynomonoecious offspring icantly from each other. Both female and termination of sex is proposed for S. vul- than female flowers, while female flowers hermaphroditic flowers can produce off- garis (Charlesworth and Laporte 1998; produced more female offspring than her- spring of all three types, but the frequen- Marsden-Jones and Turill 1957), intraindi- maphroditic flowers. cies with which they do so differ. The vidual segregation of one or many genetic The same result was obtained in the presence of hermaphroditism appears to factors affecting sex determination is a second set of crosses, where female and be associated with the production of gy- possible explanation of the results report- hermaphroditic flowers on A were fertil- nomonoecious offspring. For example, the ed here. It has the advantage of explaining ized with pollen from test plant M, but greatest difference between hermaphrodit- both the mosaic of flowers on the chimer- here the recorded differences were even ic flowers on hermaphroditic stalks and fe- ic individuals and the differences among greater. Also, in the third cross, which was male flowers on female stalks is that the offspring from the different flower types. If

564 The Journal of Heredity 1999:90(5) a fertilized egg is heteroplasmic for male- Burrows C J, 1960. Studies in Pimeliea I—the breeding July and January (P Ͻ .01), whereas no system. Trans R Soc N Z 88:29–45. developing and/or male-sterilizing mito- cline was found for the mitochondrial Charlesworth D and Laporte V, 1998. The male-sterility chondrial factors, then successive cell di- of Silene vulgaris: analysis of genetic data from two marker. Parallel clines in MDH allele fre- visions could lead to a sorting-out process populations and comparison with Thymus vulgaris. Ge- quencies have now been found on four where mitochondria of different types end netics 150:1267–1282. continents, and the Mdh 80 allele has been up in different parts of the plant. The sym- Darwin C, 1877. Different forms of flowers on plants of shown to be less heat stable in vitro than metrical pattern often seen in chimeric the same species. London: John Murray. the other alleles. We conclude that this is stalks could be the result of such segre- Dulberger R and Horovitz A, 1984. Gender polymor- very strong evidence that the MDH-1 phism in flowers of Silene vulgaris (Moench) Garcke gational cell division events. (Caryophyllaceae). Bot J Linn Soc 89:101–117. clines observed in honeybees are due to Thus it seems as if some plants consist Godelle B and Reboud X, 1997. The evolutionary dy- temperature-dependent selection. of a mixture of mitochondrial genomes, a namics of selfish replicators: a two-level selection mod- part of which signals the development of el. J Theor Biol 185:401–413. Based on morphological and ethological male function in the given nuclear back- Horovitz A and Galil J, 1972. Gynodioecism in east Med- characters, honeybees (Apis mellifera) iterranean Hirschfeldia incana, Cruciferae. Bot Gaz 133: ground, while other parts inhibit it. Pro- 127–131. have been broadly divided into three ra- gressively during growth the proportion of Jolls CL and Chenier TC, 1989. Gynodioecy in Silene cial groups, the African, the northern Eu- Downloaded from https://academic.oup.com/jhered/article/90/5/563/2186975 by guest on 27 September 2021 the two mitochondrial types change, lead- vulgaris (Caryophyllaceae): progeny success, experi- ropean, and the eastern European (Rutt- ing to different sexual phenotypes in dif- mental design, and maternal effects. Am J Bot 76:1360– ner 1968). These groupings are now well 1367. ferent parts of the plant, as determined by supported by mitochondrial (Smith 1991; Koelewijn HP and Van Damme JMM, 1995a. Genetics of the most common mitochondrial geno- male sterility in gynodioecious Plantago coronopus. I. Garnery et al. 1992) and microsatellite (Es- type. Presumably female flowers on chi- Cytoplasmic variation. Genetics 139:1749–1758. toup et al. 1995) evidence. meric stalks contain a higher proportion Koelewijn HP and Van Damme JMM, 1995b. Genetics of Two simple molecular markers are use- of male-fertile mtDNA molecules than, for male sterility in gynodioecious Plantago coronopus. II. ful for distinguishing these groups. First, example, female flowers on purely female Nuclear genetic variation. Genetics 139:1759–1775. mitochondria originating from the A. m. stalks. This effect is seen in the present Louis JP and Durand B, 1978. Studies with the dioe- mellifera (northern European) and A. m. li- cious angiosperm Mercuroalis annua L. (2n ϭ 16): cor- experiment as the difference between relation between genic and cytoplasmic male sterility, gustica (eastern European) subspecies can crosses 1 and 4. sex segregation and feminizing hormones (cytokinins). be distinguished using a DNA restriction Of course, various complicated systems Mol Gen Genet 165:309–322. fragment length polymorphism (Garnery invoking epigenetic inheritance, imprint- Marsden-Jones EM and Turill WB, 1957. The bladder et al. 1992; Hall and Smith 1991; Nielson et campions. London: Ray Society. ing, and/or maternal effects could also be al. 1994). Second, the frequency of the Philipp M, 1980. Reproductive biology of Stellaria lon- proposed to explain the results. However, gipes goldie as revealed by a cultivation experiment. three common alleles at the MDH-1 locus all such explanations are difficult to rec- New Phytol 85:557–569. (Mdh65, Mdh80, and Mdh100) is also broadly oncile with the fact that female flowers on Van Damme JMM, 1983. Gynodioecy in Plantago lanceo- discriminatory (Badino et al. 1983; Cor- chimeric stalks produce offspring that are lata L. II. Inheritance of three male sterility types. He- nuet 1979). The mellifera subspecies is redity 50:253–273. more similar to that of hermaphroditic monomorphic for the Mdh80 allele, where- flowers on hermaphroditic stalks than to Vranceanu AV and Stoenescu FM, 1978. Genes for pol- as this allele is absent from ligustica (Cor- len fertility restoration in sunflowers. Euphytica 27: that of female flowers on female stalks. 617–627. nuet 1979; Badino et al. 1984). Thus this Segregating heteroplasmy opens up the Wickersham DS and Patterson FL, 1980. Male-fertility in locus has been frequently used as a mo- possibility of intraindividual competition crosses of R5 with soft red winter wheats. Crop Sci 20: lecular marker for hybridization events between different types of cytoplasm with 100–102. (e.g., Badino et al. 1984; Del Lama et al. far-ranging effects on sex determination Received January 25, 1999 1990; Lobo et al. 1989; Oldroyd et al. 1992, Accepted May 3, 1999 and sex ratio evolution (see, e.g., Godelle 1995). and Reboud 1997). It would be of interest Corresponding Editor: Norman F. Weeden Latitudinal clines have been demon- to investigate if the surprising effect re- strated at the MDH-1 locus in natural pop- ported herein also occurs in other species ulations in Italy (Badino et al. 1984) and with gynomonoecious plants. in introduced populations in North and South America (Del Lama et al. 1990; Lobo From the Department of Genetics, Lund University, So¨l- Evidence for Temperature- vegatan 29, S-223 62 Lund, Sweden. Address correspon- et al. 1989; Nielsen et al. 1994). The pop- dence to Helene Andersson at the address above or e- Dependent Selection for ulations in these areas have different ge- mail: [email protected]. Thanks are due to Malate Dehydrogenase Allele Bengt O. Bengtsson for helpful comments on the manu- netic antecedents and therefore these par- script. This research was supported by grants from the Frequencies in Honeybee allel clines provide strong evidence that Jo¨rgen Lindstro¨m Fund to H. Andersson and the Swed- MDH-1 experiences natural selection (End- ish Natural Science Research Council to B. O. Bengts- Populations son. ler 1986; Oakshott et al. 1982). Further ev- S. Hatty and B. P. Oldroyd idence for this notion comes from the dif- ᭧ 1999 The American Genetic Association ferences in the thermostability of MDH-1 The MDH-1 genotype and a mitochondrial allozymes (Cornuet et al. 1995). However, References DNA haplotype was determined for feral the clines have also been regarded as ev- Baker H G, 1966. The evolution of floral heteromor- honeybees (Apis mellifera L.) collected idence for as yet incomplete hybridization phism and gynodioecism in Silene maritima. Heredity from 10 sites in southern New South events, not selection (Badino et al. 1984; 21:689–692. Wales, Australia. The frequency of the Del Lama et al. 1990; Lobo et al. 1989; Brockman I and Bocquet G, 1978. O¨ kologische einflu¨sse Mdh 65 allele was positively correlated, and Smith and Glenn 1995). auf die geschlechtsverteilung bei Silene vulgaris 80 (Moench) Garcke (Caryophyllaceae). Ber Deutsch Bot the Mdh allele negatively correlated with A. m. mellifera was established in Aus- Ges Bd 91:217–230. increasing average daily temperature for tralia in the early 19th century and A. m.

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