Journal of Heredity Advance Access originally published online on May 21, 2008
Journal of Heredity 2008 99(5):558-563; doi:10.1093/jhered/esn031
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Brief Communications |
Genetic Control of Floral Morph in Tristylous Pickerelweed (Pontederia cordata L.)
From the Department of Agronomy Plant Genetics and Breeding, University of Florida Institute of Food and Agricultural Sciences IFAS, 304 Newell Hall, Box 110500, Gainesville, FL
Address correspondence to Lyn A. Gettys at University of Florida Center for Aquatic and Invasive Plants, 7922 NW 71 Street, Gainesville, FL 32653, or e-mail: lgettys{at}ufl.edu.
Pickerelweed (Pontederia cordata L.) is a diploid (2n = 2x = 16) tristylous aquatic perennial. Populations usually contain 3 floral morphs that differ reciprocally in style length and anther height (referred to as the long-, mid-, and short-styled morphs, hereafter L-, M-, and S-morphs). The floral polymorphism promotes disassortative mating among the 3 floral morphs and is maintained in populations by negative frequency-dependent selection. The objective of this study was to determine the number of loci, number of alleles, and gene action controlling floral morph in pickerelweed. Three parental lines (one each of the L-, M-, and S-morph) were used to create S1 and F1 populations. F2 populations were produced through self-pollination of F1 plants. Progeny ratios of S1, F1, and F2 generations revealed that tristyly is controlled by 2 diallelic loci (S and M) with dominant gene action. The S locus is epistatic to the M locus, with the S-morph produced by plants with the dominant S allele (genotype S _ _ _). Plants with recessive alleles at the S locus were either L-morph (ssmm) or M-morph (ssM_). The results of this experiment demonstrate that the inheritance of tristyly in pickerelweed is the same as previously reported for several tristylous species in the Lythraceae and Oxalidaceae.
Tristyly is a floral polymorphism that promotes disassortative mating among floral morphs and encourages insect-mediated cross-pollination among different morphs (Darwin 1877; Crowe 1964; Vuilleumier 1967; Ganders 1979; Barrett 1993). Populations of tristylous species have members that display 1 of 3 distinct floral morphs, each with a unique set of characters. Floral morph differences include length or density of stigmatic papillae, style coloration, and pollen exine sculpturing (Barrett 1988), but the most obvious visible difference among the morphs is style length. There are 3 positions within each flower, and each position is occupied by either a single style or 1 of 2 sets of stamens. Floral morph designation is determined by style length. Flowers with long styles are L-morphs, whereas those with styles in the mid or short positions are classified as M-morphs or S-morphs, respectively (Figure 1). Reciprocal positioning of anthers and stigmas occurs so that each plant produces flowers with anthers borne at the same level as the stigma of the other morphs. This arrangement promotes insect-mediated cross-pollinations between anthers and stigmas of equivalent height, resulting in disassortative mating and seed set (Charlesworth and Charlesworth 1979; Barrett and Glover 1985; Lloyd and Webb 1992; Jesson and Barrett 2002; Thompson et al. 2003). Darwin (1877) referred to this as "legitimate pollination," whereas "illegitimate pollinations" between anthers and stigmas at different levels result in reduced seed production in some tristylous species.
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Tristyly is thought to be one of the most complex breeding systems in plants; the system has an elaborate developmental basis and is rare, occurring in only 6 angiosperm families (Barrett 1993). This suggests that the evolution of the trait may be difficult (Charlesworth 1979; Richards and Barrett 1987; Kohn et al. 1996). Inheritance of style length in tristylous systems is controlled by 2 diallelic loci (S and M) in the species studied thus far (e.g., Barlow 1923; Fisher and Mather 1943; Ornduff 1964; Anderson and Ascher 1995; also see review of Lewis and Jones 1992). The S locus is epistatic to the M locus and prevents expression of alleles at the M locus. The dominant S allele is present only in plants of the S-morph (genotype S_ _ _). The M-morph phenotype is due to a recessive condition at the S locus and the presence of at least one dominant allele at the M locus (genotype ssM_), whereas the L-morph phenotype is produced by the completely recessive genotype ssmm. Progeny ratios resulting from self-pollination of S-morph and M-morph plants are dependent upon the genotype of the parent plant, but L-morph plants are true-breeding, and self-pollinations produce only L-morph progeny.
Pickerelweed (Pontederia cordata L.) is a diploid (2n = 2x = 16) tristylous herbaceous perennial found in marshes, swamps, streams, ditches, and the shallow water along the margins of lakes and ponds (Lowden 1973; Godfrey and Wooten 1979; Bell and Taylor 1982; Tobe et al. 1998). The species is hardy in USDA zones 4–11 and has a North American geographic range that extends from Prince Edward Island to the Florida Keys (Godfrey and Wooten 1979) and is also found in Central America, Brazil, the West Indies, and Argentina (Lowden 1973; Godfrey and Wooten 1979). Pickerelweed reproduces by both sexual and vegetative means, with sexual reproduction influenced by tristyly. There is no published information describing the inheritance and genetic control of floral morph in pickerelweed. Barrett and Anderson (1985) assessed a small set of S1 progenies (20 seedlings) produced from controlled self-pollinations and found that 2 of the 4 populations under investigation segregated for style length; however, the small population size precluded speculation about the inheritance of style length in pickerelweed. The objective of this study was to determine the type of gene action and number of loci controlling tristyly in pickerelweed.
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Materials and Methods |
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 Top Materials and Methods Results and DiscussionReferences |
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The plants used in this experiment were part of a population maintained for genetic and breeding studies at the University of Florida in Gainesville. All plants were grown in 1-l nursery containers filled with a commercially available potting mix; nutrition was supplied by the incorporation of 10 g of controlled-release fertilizer per container. Plants were subirrigated and kept in a pollinator-free glasshouse with air temperature maintained at 27 °C (day) and 16 °C (night). During preliminary experiments, we observed that some genotypes were more floriferous when grown under long days; therefore, supplemental lighting was employed to artificially extend day length to 16 h in this study.
One S-morph parent (coded S), one M-morph plant (coded M), and one L-morph plant (coded L) were used in this experiment. Parent M was purchased from a commercial source, whereas S and L were collected from natural populations throughout southern Florida. Each collected parent was selected from a different geographically isolated location, so it is unlikely they share a common ancestor. None of the parents used in this study had been subjected to breeding or improvement programs and should thus be considered representative samples of the species. Herkogamy (the physical separation of reproductive organs) often restricts self-pollination in heteromorphic species. However, self-incompatibility in pickerelweed is not tightly controlled, especially in the M-morph (Barrett and Anderson 1985). Some difficulty was encountered when initial self-pollinations of L- and S-morphs were undertaken for this project, but these problems were overcome by using specialized pollination techniques developed for this and other research (Gettys 2005). Each parent was self-pollinated to create the S1 families S 
, M , and L . Cross- and reciprocal pollinations were performed between parents to create the F1 families S x M, S x L, and M x L. Anthers borne superior to stigmas (i.e., anthers borne on the long filaments of the parent M and all anthers of the parent S) were removed to facilitate access to the stigmatic surface and to prevent self-pollination when appropriate. Representative samples were selected from each F1 family and self-pollinated to generate F2 families. Cross- and reciprocal pollinations to generate F1 populations were performed between December 2001 and April 2002, whereas self-pollinations of parents and F1 plants (to create S1 and F2 populations, respectively) were conducted between January and June 2003.
Each inflorescence was pollinated from the commencement to completion of flowering (ca. 7–12 days), and all flowers borne by a given inflorescence were pollinated using the same method. Each completed inflorescence was enclosed in a small mesh bag and secured with a plastic-covered twist tie until fruits were ripe (usually 23–30 days after completion of pollinations). Fruits were collected in their mesh bag and air dried for approximately 7 days and then dehusked using a rubber-covered rub board. Cleaned seeds were immediately germinated under approximately 5 cm of water in glass half-pint (250 ml) bottles in the greenhouse under the temperature and light conditions described above. Germinated seeds were transplanted into 612 cell packs, where they remained until seedlings were approximately 30 cm tall. Seedlings were then transplanted into 1-l nursery containers, subirrigated, and kept in a pollinator-free glasshouse under the conditions described above and in Gettys (2005). No evidence of inbreeding depression was noted in seed germination, seedling vigor, days to flowering, or other life cycle indicators. All plants were grown to reproductive maturity (between 56 and 127 days from germination to flowering) and evaluated for floral morph.
No maternal effects were noted, so data for each family were pooled within each cross/reciprocal set. Data from S1 and F1 families were used to develop a working model to explain the type of gene action and number of loci controlling floral morph in pickerelweed. Development of this model allowed the assignment of genotypes to parents; the model was then verified by analyses of F2 populations. All data were analyzed using goodness-of-fit (chi-square or 
2) tests (Steel et al. 1997). A test for heterogeneity of the data for F2 populations from different crosses was performed to determine whether it was appropriate to pool data for all F2 progenies.
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Results and Discussion |
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TopMaterials and MethodsResults and DiscussionReferences |
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Self-pollination of parent L yielded 56 L-morph progenies, whereas self-pollination of parents S and M resulted in segregating S1 populations. The family S
was composed of 7 S-morph and 2 M-morph S1 progenies, which was not different from a 3S:1M ratio (P = 0.8494). The family M had 32 M-morph and 12 L-morph S1 progenies, which was not different from a 3M:1L ratio (P = 0.7277). Cross-pollinations resulted in segregating progenies in all F1 families. Offspring from the F1 families S x M and S x L segregated in patterns that were not statistically different from a 1S:1M ratio (Table 1), whereas segregation of progeny from the F1 family M x L was not statistically different from a 1M:1L ratio (Table 1).
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These S1 and F1 data were compared with the 2-loci diallelic model responsible for the control of floral morph in other tristylous species and then genotypes were assigned to all 3 parents using the proposed model and segregation of S1 and F1 progenies. We hypothesized that the S-morph parent S was heterozygous at the epistatic locus and homozygous dominant at the hypostatic locus (SsMM). The M-morph parent M was homozygous recessive at the epistatic locus and heterozygous at the hypostatic locus (ssMm), whereas the L-morph parent L was homozygous recessive at both loci (ssmm). This model for the genetic control of tristyly in pickerelweed is similar to that described by Barlow (1923), Fisher and Mather (1943), Ornduff (1964), and Eckert and Barrett (1993) for other tristylous species. The model has 2 diallelic loci with dominant gene action at each locus and expression influenced by epistasis. The L-morph phenotype is produced by the completely recessive genotype (ssmm), whereas the M-morph phenotype is due to a recessive condition at the S locus and at least one dominant allele at the M locus (ssM_). The presence of a dominant allele at the epistatic S locus conditions the S-morph phenotype (S _ _ _) and prevents expression of alleles at the M locus.
Fifteen F1 plants (7 S-morphs and 8 M-morphs) from the family S x M were self-pollinated to produce 15 F2 families. S-morph F1 plants had genotype SsMm or SsMM, whereas M-morph plants had genotype ssMM or ssMm. Four F2 families from S-morph F1 plants had offspring that represented all 3 floral morphs and were derived from F1 plants with genotype SsMm. Tests for heterogeneity were not significant (P = 0.2442), so these F2 progenies were pooled and segregated in a 12S:3M:1L ratio as expected (Table 1). The remaining 3 F2 families from S-morph F1 plants had only S-morph and M-morph progenies and were derived from F1 plants with genotype SsMM. Heterogeneity chi-square analysis was not significant (P = 0.2617), so these F2 progenies were pooled and segregated in a 3S:1M ratio as expected (Table 1). Four F2 families from M-morph F1 plants produced only M-morph progenies and were derived from F1 plants with the genotype ssMM. The remaining 4 F2 families from M-morph F1 plants had both M-morph and L-morph progenies and were derived from F1 plants with the genotype ssMm. Tests for heterogeneity were not significant (P = 0.8095), so these F2 progenies were pooled and segregated in a 3M:1L ratio as expected (Table 1).
Twelve F1 plants (6 S-morphs and 6 M-morphs) from the family S x L were self-pollinated to produce 12 F2 families. All 6 F2 families from S-morph F1 plants (genotype SsMm) had offspring that represented all 3 floral morphs. Heterogeneity chi-square analysis was not significant (P = 0.1754), so these F2 progenies were pooled and segregated in a 12S:3M:1L ratio as expected (Table 1). All 6 F2 families from M-morph F1 plants (genotype ssMm) were composed of M-morph and L-morph plants. Tests for heterogeneity were not significant (P = 0.1436), so these F2 progenies were pooled and segregated in a 3M:1L ratio as expected (Table 1).
Twenty-four F1 plants (12 M-morphs and 12 L-morphs) from the family M x L were self-pollinated to produce 24 F2 families. All 12 F2 families from M-morph F1 plants (genotype ssMm) were composed of M-morph and L-morph progenies. Heterogeneity chi-square analysis was not significant (P = 0.4047), so these F2 progenies were pooled and segregated in a 3M:1L ratio as expected (Table 1). All F2 progenies from L-morph F1 plants (genotype ssmm) from the family M x L bore only L-morph flowers (Table 1).
F2 progenies from M-morph F1 plants (genotype ssMm) from the families S x M, S x L, and M x L were subjected to heterogeneity chi-square analysis (Table 2). The test for heterogeneity was not significant, so these F2 progenies were pooled and segregated in a 3M:1L ratio as expected (Table 2). Tests for heterogeneity among F2 progenies from S-morph F1 plants (genotype SsMm) from the families S x M and S x L revealed no differences (Table 3), so these F2 progenies were pooled. Pooled progeny segregated in a 12S:3M:1L ratio as expected (Table 3).
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Progeny in our experiment segregated as expected under a model with 2 diallelic loci influenced by epistasis. These results differ from those of other workers who have reported that ratios may be skewed by linkage or by poor representation of the S-morph in populations of some tristylous species. Barrett et al. (1989) reported that self-pollination of heterozygous S-morph plants of Eichhornia paniculata, a close relative of pickerelweed, produced progeny ratios with fewer S-morph plants than expected under the diallelic 2-loci model and suggested that genetic factors could play a role in the fitness of the S-morph. These workers and others (e.g., Mather and de Winton 1941; Strobeck 1980; Richards 1998) postulated that deleterious alleles may be tightly linked to the S locus in some heterostylous species and that homozygosity at the S locus could increase inbreeding depression as a result of the accumulation of these deleterious alleles, but experiments by Manicacci and Barrett (1996) provided no evidence that homozygosity at the S locus was detrimental to fertility of the S-morph of E. paniculata. Our results provide additional support to the theory that homozygosity at the S locus is not associated with a direct decrease in fitness, at least in pickerelweed, because we found no evidence of inbreeding depression in this study.
Differing degrees of genetic linkage between the S and M loci have been noted in tristylous species; for example, tight linkage has been noted in Oxalis spp. (Fyfe 1950; Weller 1976) and in E. paniculata (Barrett et al. 1989), but Fisher and Mather (1943) found that the S and M loci were independent in Lythrum salicaria. Our experiment did not reveal linkage between the S and M loci in pickerelweed and provides further evidence that tristyly in this species is controlled by 2 independent diallelic loci. One might expect the linkage relationship in pickerelweed to be similar to that of the closely related species E. paniculata; however, the development of tristyly is widely considered to be polyphyletic in origin among angiosperm families (Ganders 1979). Although it is unlikely, it is possible that the syndrome arose independently in these species.
Our study is the first to provide strong experimental evidence regarding the inheritance of the tristylous syndrome in pickerelweed. Our results suggest that tristyly in pickerelweed is controlled by 2 independent diallelic loci, with expression influenced by epistasis. Other workers have identified linkage—either between the S and M loci or with unrelated deleterious alleles—in other tristylous species; however, we found no evidence of any type of linkage in the system controlling tristyly in pickerelweed. These results contribute to the body of knowledge available regarding the inheritance and genetic control of this unusual breeding system that encourages outcrossing as a means to maintain genetic diversity.
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Acknowledgments |
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This research is presented by the senior author as partial fulfillment for the Doctor of Philosophy degree and was supported by the Florida Agricultural Experiment Station. We would like to thank David Sutton, Paul Pfahler, William Haller, and Eric Ostmark for their contributions to this experiment.
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Footnotes |
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Corresponding Editor: John Burke
Received November 19, 2007
Accepted April 10, 2008
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References |
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