Colloquium

Flower color variation: A model for the experimental study of

Michael T. Clegg† and Mary L. Durbin

Department of Botany and Plant Sciences, University of California, Riverside, CA 92521-0124 We review the study of flower color polymorphisms in the morning flower color polymorphisms. Since that time a great deal of glory as a model for the analysis of adaptation. The pathway progress has been made in describing the molecular biology of involved in the determination of flower color phenotype is traced the of flavonoid biosynthesis that determine flower color, from the molecular and genetic levels to the phenotypic level. but we are still some distance from a complete causal analysis Many of the genes that determine the enzymatic components of that connects ecology to phenotype to genes. flavonoid biosynthesis are redundant, but, despite this complexity, We begin by discussing the natural history of the morning it is possible to associate discrete floral phenotypes with individual glory and then turn to a brief account of the genetics of flower genes. An important finding is that almost all of the mutations that color variation in the common morning glory. Next, we describe determine phenotypic differences are the result of transposon the flavonoid biosynthetic pathway that determines flower color, insertions. Thus, the flower color diversity seized on by early and we review pertinent work on the molecular genetics of the human domesticators of this plant is a consequence of the rich genes that encode enzymes within this pathway. Finally, we variety of mobile elements that reside in the morning glory consider progress in the analysis of selection on flower color genome. We then consider a long history of research aimed at variation in natural and experimental populations of the com- uncovering the ecological fate of these various flower phenotypes mon morning glory. in the southeastern U.S. A large body of work has shown that insect pollinators discriminate against white phenotypes when Natural History of I. purpurea. The genus Ipomoea includes white flowers are rare in populations. Because the plant is self- approximately 600 species distributed on a worldwide scale (2) compatible, pollinator bias causes an increase in self-fertilization in that are characterized by a diversity of floral morphologies and white maternal plants, which should lead to an increase in the pigmentation patterns. In addition, a wide variety of growth frequency of white genes, according to modifier theory. habits, ranging from annual species to perennial vines to Studies of geographical distributions indicate other, as yet undis- longer-lived arborescent forms, are represented in the genus. covered, disadvantages associated with the white phenotype. The The common morning glory is an annual bee-pollinated ultimate goal of connecting ecology to molecular genetics through self-compatible vine with showy flowers that is a native of the the medium of phenotype is yet to be attained, but this approach highlands of central Mexico. As the name morning glory may represent a model for analyzing the translation between suggests, the flowers open early in the morning and are available for fertilization for a few hours, after which the these two levels of biological organization. flower wilts and abscises from the vine. The plant is also a common weed in the southeastern U.S., where it is found in flavonoid biosynthesis ͉ transposon ͉ spatial autocorrelation ͉ pollinator association with field corn and soybean plantings, as well as in behavior ͉ morning glory roadside and disturbed habitats. The common morning glory is characterized by a series of flower color polymorphisms that he study of adaptation is a problem that intersects all include white, pink, and blue (or dark blue) phenotypes. A Tdisciplines of biology. The study of adaptation is also among primary pollinator in the southeastern U.S. is the bumblebee the most difficult and challenging areas of experimental research (Bombus pennsylvanicus and Bombus impatiens), but occasion- because a complete causal analysis of adaptation involves a ally plants are visited by honey bees and some lepidopterans translation between different levels of biological organization, (3). The flower color phenotypes are thought to have been the ecological, the phenotypic, and the molecular level (1). In selected by pre-Columbian peoples, perhaps in association this article we review more than 20 years of research on flower with maize culture (4). At some point, the plant was introduced color polymorphisms in the common morning glory [Ipomoea into the southeastern U.S., although the routes and times of purpurea (L.) Roth] that was aimed at exploring this interface. introduction remain uncertain. Early floras of the southeast- When the morning glory research program was initiated in the ern U.S. indicate the presence of I. purpurea populations by the late 1970s, flower color polymorphisms appeared to be a natural late 1600s, providing a minimum estimate of the residence time starting point because (i) they represented simple discrete in this geographical region. phenotypes that were susceptible to genetic analysis; (ii)a substantial body of work existed on the biochemistry of plant Genetics of Flower Color Variants in I. purpurea. At least 21 floral secondary metabolism; (iii) flower color was known to be phenotypes are determined by five genetic loci (5–7). Most of important in insect pollinator behavior; and (iv) selection on these phenotypes have analogous forms in the Japanese morning reproductive performance should be among the most effective forms of selection, and, as a consequence, it should be the This paper was presented at the National Academy of Sciences colloquium ‘‘Variation and component of selection most likely to yield to experimental Evolution in Plants and Microorganisms: Toward a New Synthesis 50 Years After Stebbins,’’ analysis. Virtually nothing was known in the late 1970s about the held January 27–29, 2000, at the Arnold and Mabel Beckman Center in Irvine, CA. molecular biology of the genes that determine the anthocyanin Abbreviations: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3␤-hy- pigments responsible for flower color, so nothing was known droxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase. about the specific mutational changes associated with different †To whom reprint requests should be addressed. E-mail: [email protected].

7016–7023 ͉ PNAS ͉ June 20, 2000 ͉ vol. 97 ͉ no. 13 Downloaded by guest on September 26, 2021 Fig. 1. Flower color variation in I. purpurea. Loci are described in the text. The locus that determines the phenotype shown is highlighted in bold. Dashes indicate that the phenotype is dominant and only the dominant allele is therefore indicated. In the aa genotype, for example, the A͞a locus is epistatic to the P͞p and I͞i loci; therefore, the albino phenotype determined by the recessive aa is the same regardless of the state of the other loci.

glory (Ipomoea nil), and the genetics of the floral variants in both affects the coloration of the floral display, which attracts polli- COLLOQUIUM species appear to be similar, but not identical (8). A widespread nators. The anthocyanin pigments are therefore important to polymorphism determines pink versus blue flowers (P͞p locus), reproductive success and hence to gene transmission. In addition but the genotype at two other loci modifies the intensity of to pigment production, several side branches of the pathway also expression of the P͞p locus. One modifier is an intensifier locus produce compounds that are important in plant disease defense, (I͞i) that doubles the anthocyanin pigmentation in the recessive pollen viability, microbial interactions, and UV protection (12). ii genotype (9). The second modifier locus is a regulatory locus The flavonoid pathway has a pleiotropic role in plants, and one that determines the patterning and degree of floral pigmentation must consider that a single mutation in the pathway may have (W͞w locus). A fourth locus, the A͞a locus, is epistatic to the multiple phenotypic effects. The pleiotropic role of the flavonoid P͞p, I͞i, and W͞w loci in that the recessive albino phenotype pathway is a complication in the effort to link phenotype to yields a white floral limb independent of genotypic state at the molecular changes, but, in addition, many of the genes of the other loci. The A͞a locus is also characterized by unstable alleles flavonoid pathway are now known to consist of small multigene (denoted a* or af) that exhibit pigmented sectors on an otherwise families (Table 1), so it is essential to associate a mutation in a albino floral limb (10, 11). The pigmented sectors display the particular gene with a phenotype of interest. That is, one must color associated with the P͞p genotype. Finally, the pigmenta- identify the member responsible for the observed tion in the floral tube appears to be controlled separately from phenotype. the outer floral limb, but the genetics of floral tube variation have not been analyzed. Fig. 1 displays the flower color pheno- Molecular Characterization of the Genes of Flavonoid Biosynthesis in types determined by these genetic loci. Ipomoea. The first committed step in the flavonoid biosynthetic pathway is encoded by the enzyme chalcone synthase (CHS), which Flavonoid Biosynthetic Pathway. To put the phenotypic variation catalyzes the formation of naringenin chalcone from three mole- into a biochemical context, it is useful to sketch the main outlines cules of malonyl-CoA and one molecule of p-coumaroyl-CoA (13). of the flavonoid biosynthetic pathway (Fig. 2), which culminates Several lines of evidence suggested that the A͞a locus might encode in the production of anthocyanins, the main pigments respon- a CHS gene. First, the albino phenotype is epistatic to all other sible for flower color. The presence or absence of these pigments flower color variants, suggesting an early point of action in the

Clegg and Durbin PNAS ͉ June 20, 2000 ͉ vol. 97 ͉ no. 13 ͉ 7017 Downloaded by guest on September 26, 2021 nucleotide sequence to the majority of CHS genes character- ized in Petunia. Biochemical analysis of Ipomoea CHS genes A, B, D, and E revealed that only CHS-D and -E are capable of catalyzing the condensation reaction that results in naringenin chalcone (20). The CHS-A and -B genes appear to encode enzymes that produce bisnoryangonin but not naringenin chalcone (H. Noguchi, personal communication). All of the Ipomoea CHS genes (except the ) are expressed, but each displays differential regulatory and developmental control (21, 22). CHS-D is the most abundantly expressed transcript and is now known to be the one CHS gene solely responsible for anthocyanin production in the floral limb (21, 23). As discussed in greater detail below, the identification of CHS-D as the A͞a locus followed the identification of a transposon (23) in CHS-D, which accounted for the sectoring phenotype and loss of pigment. The CHS-E gene is also expressed in the limb but at a much lower level than CHS-D (21, 22). CHS-E is now believed to be responsible for pigment production in the tube of the flower (22). Fig. 2. Flavonoid biosynthetic pathway. Enzymes involved in anthocyanin The next step in the pathway is encoded by chalcone isomerase biosynthesis and side branches leading to related flavonoid pathways are (CHI), which catalyzes the isomerization of the naringenin shown. PAL, phenylalanine ammonialyase; C4H, cinnamate 4-hydroxylase; chalcone product of CHS to the flavanone naringenin. In the 4Cl, coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; absence of functional CHI, spontaneous isomerization may still F3H, flavanone 3␤-hydroxylase; F3ЈH, dihydroflavonol 3Ј hydroxylase; F3Ј5ЈH, occur in vivo because this is known to occur in vitro under dihydroflavonol 3Ј5Ј hydroxylase; DFR, dihydroflavonol reductase; ANS, an- physiological conditions (24). CHI appears to be a single copy thocyanidin synthase; UF3GT, UDP-glucose flavonol 3-0-glucosyl transferase; gene in I. purpurea, as multiple screenings of the genomic library RT, rhamnosyl transferase. have yielded only a single gene (unpublished data). Flavanone 3␤-hydroxylase (F3H) follows CHI in the pathway pathway, and, second, the albino phenotype is consistent with a and hydroxylates flavonones at the 3 position to form dihydrofla- blockage at CHS. In an attempt to show that the A͞a locus encodes vonols, which are required for the synthesis of anthocyanidins a chalcone synthase enzyme, we initiated efforts to clone chalcone and flavonols. F3H consists of at least two copies in the I. synthase genes from a genomic library of I. purpurea. Multiple purpurea genome, both of which are expressed (25). It is not screenings of the library by using a heterologous probe from parsley known at this time whether one of the F3H genes is solely (14) were unsuccessful. Subsequent screening of the library with a responsible for anthocyanin production in the limb (as is the tomato CHS clone (15) resulted in the cloning of four genes case for CHS-D). Also, because they were both identified as (CHS-A, -B, -C, and a pseudogene) initially identified as CHS on F3H on the basis of nucleotide similarity, it is not known the basis of nucleotide sequence similarity to other published CHS whether both are even capable of performing the hydroxyla- gene sequences (16). tion step. One functional copy of F3H and one pseudogene To provide a comparative context for Ipomoea CHS gene have so far been identified in I. nil (ref. 26; S. Iida, personal family evolution, one can look at the Petunia CHS gene family. communication). Petunia is important because both Ipomoea and Petunia are in Another hydroxylase, dihydroflavanol 3Ј hydroxylase (F3ЈH), the same flowering plant order (Solanales), and the Petunia CHS hydroxylates the 3Ј position of the dihydroflavonol produced by gene family had been extensively characterized, with as many as F3H. This results in the eventual production of the red͞magenta 12 genes provisionally identified (17). The four I. purpurea genes cyanidin. F3ЈH has been characterized in both I. purpurea and I. appear to share a common line of descent with an unusual nil (S. Iida, personal communication).‡ Yet another hydroxylase, Petunia gene (CHS-B) that is relatively distant in nucleotide dihydroflavonol 3Ј5Ј-hydroxylase (F3Ј5ЈH), hydroxylates the 3Ј Petunia sequence from other CHS genes. and 5Ј position of the dihydroflavonol produced by F3H. This et al. Subsequently, Fukada-Tanaka (18) cloned and char- product ultimately leads to the production of the blue͞purple acterized two more CHS genes from Ipomoea (CHS-D and -E) delphinidin. F3Ј5ЈH has not yet been characterized in Ipomoea. by using differential display and AFLP-based mRNA finger- The next step in the flavonoid pathway is dihydroflavonol printing (19). These genes proved to be more closely related in reductase (DFR) which reduces dihydroflavonols to leucoan- thocyanidins. In I. purpurea, DFR is a small gene family con- Table 1. Estimated copy number of flavonoid genes in the I. sisting of at least three tandemly arranged copies (DFR-A, -B, purpurea genome and -C) (27). DFR-B has been identified as the gene responsible for anthocyanin production in the floral limb based on work Genes Number of known family members from I. nil in which a transposon disrupts the DFR-B gene, CHS 6 resulting in a sectoring phenotype and loss of pigment (ref. 28; CHI 1 see below). The function of the two other DFR genes in I. F3H 2 purpurea is not known, nor is it known whether they are capable F3ЈH1 of performing the reductase reaction. F3Ј5ЈH? Anthocyanidin synthase (ANS) encodes a dioxygenase and DFR 3 appears to be single copy in I. purpurea. UDP-glucose flavonol ANS 1 3-0-glucosyl transferase glycosylates anthocyanidins and fla- UF3GT 1 vonols on the 3 position. This gene appears to be single copy in RT ?

UF3GT, UDP-glucose flavonol 3-0-glucosyl transferase; RT, rhamnosyl trans- ‡Morita, Y., Hoshino, A., Tanaka, Y. Kusumi, T., Saito, N. & Iida, S. (1999) Plant Cell Physiol. ferase. 40, Suppl., 124 (abstr.).

7018 ͉ www.pnas.org Clegg and Durbin Downloaded by guest on September 26, 2021 Table 2. Transposons and mobile elements in I. purpurea and I. nil associated with genes of the flavonoid pathway Element Location Species Type Reference

Tpn1 DFR-B I. nil En͞Spm 28 Tpn2 CHI I. nil En͞Spm abstr.* Tpn3 CHS-D I. nil En͞Spm abstr.† Tip100 CHS-D I. purpurea Ac͞Ds 23 Tip201 F3ЈH I. purpurea Ac͞Ds S. Iida, personal communication‡ RTip1 ANS flanking I. purpurea Ty3 gypsy 34 MiniSip1 ANS flanking I. purpurea Mini-satellite 34 MiniSip2 MELS DFR flanking I. nil Mites 33 MELS CHS-D flanking I. purpurea Mites 22 Sinelp CHS-D flanking I. purpurea Sine unpublished data

*Hoshino, A. & Iida, S. (1997) Genes Genet. Syst. 72, 422 (abstr.). †Hoshino, A. & Iida, S. (1999) Plant Cell Physiol. 40, Suppl., 26 (abstr.). ‡Morita, Y., Hoshino, A., Tanaka, Y., Kusumi, T., Saito, N. & Iida, S. (1999) Plant Cell Physiol. 40, Suppl., 124 (abstr.).

I. purpurea. Rhamnosyl transferase adds rhamnosyl to glucose to purpurea that is not attributable to the presence of a transposable form rutinoside. This gene is as yet uncharacterized in I. element. purpurea. An En͞Spm-like mobile element termed Tpn1 was charac- terized by Inagaki et al. (28) in the I. nil genome. Tpn1 is a 6.4-kb Most Mutant Phenotypes Appear To Be the Result of Transposon non-autonomous element inserted within the second intron of Insertions. A wide variety of mobile elements (Table 2) have been DFR-B in a mutant termed flecked (a-3flecked). This phenotype identified in the Ipomoea genome, largely because of work from has predominantly white flowers with colored sectors. An un- the laboratory of Shigeru Iida at the National Institute for Basic usual feature of Tpn1 is that it contains a segment of DNA Biology in Okasaki, Japan. Some of these mobile element consisting of four exons that encode part of an HMG-box (High insertions cause phenotypic changes, including those responsible Mobility Group DNA-binding proteins) (29). This finding is for several flower color variants (Table 3). Much of this work has consistent with the observation that transposable elements can concentrated on the Japanese morning glory (I. nil), where the cause rearrangements of the genome (30). Not only did Inagaki rich history of morning glory genetics in Japan has provided an et al. (28) establish that the observed sectoring phenotype was extensive research foundation. Considerable work has also been caused by the movement of this new transposable element, but done both in the U.S. and in Japan on I. purpurea, which is a the finding also established that, of the three DFR genes member of the same subgenus as I. nil. Both species appear to characterized, only one gene family member (DFR-B) is respon- be quite closely related at the DNA sequence level. Molecular sible for pigment production in the floral limb. Another En͞Spm-like element, Tpn2, has also been identified clock calculations indicate that the two species diverged roughly § three million years ago based on synonymous divergence at DFR in I. nil. Tpn2 is a 6.5-kb element with similarity to Tpn1. Tpn2 and CHS genes (unpublished data). These two closely related is inserted in the second intron of CHI in the speckled mutant. species provide a useful comparative framework for the analysis This phenotype is pale yellow with round speckles of pigment on the corolla (31). In addition to the En͞Spm-like transposons, of genetic change over a relatively short period of evolutionary mobile element-like motifs were identified in the flanking

time. COLLOQUIUM regions of the DFR genes. These elements are similar to A 3.9-kb Ac͞Ds-like element (Tip100) has been identified in miniature inverted-repeat transposable elements (22, 32, 33). the intron of CHS-D near the 5Ј junction in flaked (af) mutants Mobile element-like motifs are also found in the DFR region of I. purpurea f of (23). The a flower is white with pigmented sectors I. purpurea (27) and in the CHS-D region of both I. purpurea and on the corolla. Another CHS-D mutant phenotype (a12), which I. nil. Three copies of one of the elements (mobile element-like has white flowers and no pigmented sectors, has been shown to motif 6) from the CHS-D region were also found in the DFR carry two copies of Tip100 in the intron in the opposite orien- region (22). tation (23). In another stable white phenotype (a), a rearrange- Furthermore, three copies of a directly repeated sequence of ment of DNA sequence between exon I and an adjacent Tip100 about 193 bp are found at the 3Ј end of the intron in CHS-D in element has occurred (unpublished data). In yet another mutant some lines of I. purpurea (22). Other lines of I. purpurea contain (a*) of I. purpurea, which has a white corolla with very few four copies of this repeat (unpublished data). In contrast, I. nil pigmented sectors, an additional copy of Tip100 was found in the contains only one copy of this repeat, but it is interrupted by a Ј 5 flanking region of CHS-D (unpublished data). The discovery 529-bp insertion (22). that disruption of the CHS-D gene results in an unpigmented Another En͞Spm-related element termed Tpn3 has been phenotype confirms that CHS-D is the only CHS gene family found in the CHS-D gene of I. nil in the r-1 mutant.¶ This mutant member that is responsible for pigment production in the floral bears white flowers with colored tubes. The insertion of this limb (21, 22). Another Ac͞Ds-like element, Tip201, has also 5.57-kb element into CHS-D results in the accumulation of been discovered in I. purpurea (S. Iida, personal communica- abnormal sizes of CHS-D mRNAs in the floral tissue.¶ tion).‡ Tip201 is found inserted in exon III of F3ЈH, resulting in A long terminal repeat retrotransposon, RTip1, has been a pink phenotype rather than the wild-type blue color corre- reported associated with the ANS gene region in I. purpurea (34). sponding to the P͞p locus (S. Iida, personal communication).‡ The element is 12.4 kb, contains two long terminal repeat A point mutation in F3ЈHinI. nil also results in a red phenotype instead of blue (S. Iida, personal communication). Interestingly, this is the only phenotypic change involving flower color that has §Hoshino, A. & Iida, S. (1997) Genes Genet. Syst. 72, 422 (abstr.). been characterized at the molecular level in either I. nil or I. ¶Hoshino, A. & Iida, S. (1999) Plant Cell Physiol. 40, Suppl., 26 (abstr.).

Clegg and Durbin PNAS ͉ June 20, 2000 ͉ vol. 97 ͉ no. 13 ͉ 7019 Downloaded by guest on September 26, 2021 Table 3. Mutations linked to phenotype in I. purpurea and I. nil Phenotype Mutant allele Location Reference

Mutations identified in I. purpurea Numerous pink sectors on white corolla af1 Tip100 in intron of CHS-D 23 Few purple sectors on white corolla a* Tip100 in 5Ј flanking and intron of CHS-D unpublished data Stable white a12 Two copies of Tip100 in intron of CHS-D 22 Stable white a Genomic rearrangement of exon 1 of CHS-D unpublished data Pink corolla p Tip201 in exon III of F3ЈH S. Iida, personal communication† Mutations identified in I. nil Magenta corolla p Point mutation at F3ЈH S. Iida, personal communication† Round spots on unpigmented corolla speckled Tpn2 insertion in CHI abstr.‡ Colored flecks and sectors a-3f Tpn1 insertion in DFR-B 28 Double corolla dp Tpn-botan into C class MADS-box gene abstr.§ White corolla r-1 Tpn3 in CHS-D abstr.¶

†Morita, Y., Hoshino, A., Tanaka, Y., Kusumi, T., Saito, N. & Iida, S. (1999) Plant Cell Physiol. 40, Suppl., 124 (abstr.). ‡Hoshino, A. & Iida, S. (1997) Genes Genet. Syst. 72, 422 (abstr.). §Nitasaka, E. (1997) Genes Genet. Syst. 72, 421 (abstr.). ¶Hoshino, A. & Iida, S. (1999) Plant Cell Physiol. 40, Suppl., 26 (abstr.).

sequences of about 590 kb, and appears to be a defective Ty3 37). We speculate that this pattern is a consequence of the gypsy-like element. The RTip1 element resides within yet an- introduction of horticultural forms selected for flower color other element (MiniSip1), which is described as a minisatellite. diversity into the U.S. However, we do not know the source of Another minisatellite, MiniSip2, has also been described and is these introductions. located within the Rtip1 element. Thus, there are three elements There are at least two plausible introduction scenarios. One piggybacked on one another in the ANS 3Ј flanking region. scenario posits a northward migration of the common morning Although no phenotypic changes have been linked to these glory along with maize culture over 1,000 years ago. A second elements, DNA rearrangements in the vicinity of ANS are scenario posits an introduction of the common morning glory attributed to their presence (34). into the southeastern U.S. by European settlers of this region as A sine-like element (SineIp) has also been identified in the 5Ј a horticultural plant. The genetic evidence points to a strong flanking region of CHS-D (unpublished data) in some lines of I. founder effect associated with the introduction of the common purpurea. This element is 236 bp and contains the Pol III morning glory into the southeastern U.S. Maize does not appear promoters at the 5Ј end of the element. It has 15 bp direct to have experienced so extreme a founder effect, and it is not terminal repeats. No phenotypic changes have yet been associ- obvious why the two species would experience different popu- ated with this element. lation restrictions during a common migration process, so we Another floral mutation in I. nil, not related to the flavonoid regard the first scenario as less likely. Under either scenario, we pathway, but which is caused by a transposon insertion, is the may regard the southeastern U.S. populations as a crude series duplicated mutant. In this mutant phenotype, sexual organs are of experiments in the microevolution of flower color determin- replaced by perianth organs (petals and sepals) resulting in a ing genes. double floral whorl. An En͞Spm-like insertion, termed Tpn- Epperson and Clegg (35) conducted geographic surveys of botan, was found in a C class MADS-box-like geneʈ that is flower color variation within the southeastern U.S. at three evidently responsible for this phenotype. A similar phenotype different spatial scales. The smallest spatial scale (the intrap- has also been described in I. purpurea (11), although the mo- opulation scale) was analyzed via spatial autocorrelation statis- lecular basis for the mutation in I. purpurea is unknown. tics (38); second, the sub regional scale (defined as local The vast majority of the phenotypic variation in Ipomoea populations that range from 0.8 to 32 km apart) was analyzed via characterized to date at the molecular level appears to be caused gene frequency distances between populations (39); and third, by the insertion or deletion of transposable elements (Table 3). the regional scale ranging from 80 to 560 km between popula- It is apparent that a wide variety of mobile elements exist in the tions, and including much of the southeastern U.S., was also Ipomoea genome, and these are evidently quite active based on analyzed by using genetic distance statistics. A strong result of the relatively modest period of evolutionary time that separates these analyses is that there is no correlation between genetic I. nil and I. purpurea. We now turn to studies of the population distance and geographic distance at subregional or regional genetics of some flower color phenotypes in I. purpurea. scales for either the W͞wortheP͞p loci. Populations separated by a few kilometers are as differentiated from one another with Geographic Distribution of Flower Color Polymorphisms in I. purpurea. respect to the P͞p and W͞w flower color determining loci as The geographic distribution of genetic diversity in I. purpurea those separated by hundreds of kilometers. Such a pattern is appears paradoxical. Levels of flower color polymorphism are consistent with the hypothesis that the flower color variants were high in the southeastern U.S. whereas Mexican populations are randomly introduced into multiple locations in the southeastern frequently monomorphic for the blue color form (4, 35). The U.S. Analyses of spatial distributions within local populations led situation is reversed for biochemical and molecular variation. to two major conclusions: first, spatial autocorrelation statistics Mexican populations have levels of isozyme polymorphism that were heterogeneous between the W͞w and P͞p loci within the are similar to other annual plants (4, 36), but U.S. populations same local populations; and, second, analyses of spatial corre- ͞ are depauperate in isozyme variation. Surveys of ribosomal lograms for the P p locus revealed genetic neighborhood sizes DNA restriction fragment variation and samples of gene se- consistent with an isolation-by-distance model of population quence data for the (CHS-A) locus also reveal reduced levels of structure (35). We begin by elaborating on the second conclu- variation in U.S. populations relative to Mexican populations (4, sion; we then turn to the importance of the first conclusion in establishing that the W͞w locus is subject to selection within local populations. ʈNitasaka, E. (1997) Genes Genet. Syst. 72, 421 (abstr.). For clarity, it is useful to review a few elementary definitions

7020 ͉ www.pnas.org Clegg and Durbin Downloaded by guest on September 26, 2021 in spatial statistics. A spatial autocorrelation measures the pollinators against visiting white flowers when white is less than correlation in state of a system at two points that are separated 25% of the population (3, 43, 44). In contrast, there is no by x distance units. For example, in the common morning glory evidence of pollinator discrimination among P͞p (blue͞pink) or case, the state may be the flower color determined by the P͞p I͞i (dark͞intense) locus phenotypes (43, 45). The under visita- locus x distance units apart. The autocorrelation is often graphed tion of white phenotypes is correlated with an increased fre- as a function of distance (x), and the resulting graph is called the quency of self-fertilization by white maternal parents based on correlogram [y ϭ f(x)]. For discrete characters, the convention estimates using isozyme marker loci (3, 43). However, Schoen is to transform the autocorrelation into a standard normal and Clegg (45) discovered that pollinator visitation rates and deviate with mean 0 and variance 1, and this transformed outcrossing estimates did not differ between white and pig- function is graphed as the correlogram. Let us again take the mented phenotypes when the two forms were in equal frequency. concrete example of the common morning glory P͞p locus. Later, Epperson and Clegg (3) and Rausher et al. (44) showed Three graphs result from the autocorrelation of blue with blue that the pollinator discrimination against whites, and the reduced (ybb), pink with pink (ypp), and blue with pink (ybp), each as a outcrossing rate of white maternal plants, was frequency- function of distance. When graphed as a function of distance, the dependent and disappeared when the frequency of the white data from many local I. purpurea populations in the southeastern phenotype approached 50%. These observations and experi- ͞ Ͼ U.S. revealed a common pattern for the P p locus ypp or ybb ments indicate that white loci act as mating system modifier loci ϽϪ 2 and ybp 2 initially (for the smallest values of x). This result and consequently bias their own transmission to subsequent indicates a positive autocorrelation over short spatial distances generations. among like phenotypes (blue with blue or pink with pink) and a There is substantial theoretical literature on the population negative autocorrelation at short spatial scales among unlike genetics of modifier loci (46–48). According to this literature, phenotypes (blue with pink). The distance (x), where the auto- white genes that increase the frequency of self-fertilization are correlation first crosses the x axis (y ϭ 0), provides an operational expected to increase in frequency to fixation within popula- definition of patch size. These analyses revealed substantial tions because the white gene should be transmitted differen- patchiness for the P͞p locus and yielded a minimum estimate of tially to progeny of white maternal plants via self-fertilization. about 120 pink (recessive homozygous) plants per patch. The This assumes that the selfing (white) gene is also transmitted estimates of patch size are highly consistent with simulations of to the outcross pool in proportion to its frequency in the spatial distributions based on a pattern of pollinator flight population, where it can fertilize ovules of non-white maternal distances that is strongly biased toward nearest neighbor moves phenotypes (absence of pollen discounting). [‘‘Pollen dis- (40, 41). counting’’ refers to the situation in which pollen transmission In contrast to the P͞p locus pattern, the W͞w locus genotypes to other (nonmaternal) plants is reduced by a gene promoting show little or no spatial patchiness (that is, y ϭ 0 at the smallest self-fertilization, as might be the case when pollinators avoid values of x, in populations in which y Ͼ 2orϽϪ2 initially for particular floral displays.] The degree of advantage of self- the P͞p locus). Because both loci are transmitted to successive fertilization genes, if any, clearly hinges on several additional generations within populations through the same mating pro- factors, including (i) the extent of pollen discounting; (ii) cess, we expect homogeneous spatial patterns. Heterogeneous differential maternal fertility associated with autopollination; spatial patterns argue that isolation-by-distance is not the sole and (c) reduced viability of progeny derived through self- factor governing spatial patterns within local populations. Other fertilization because of inbreeding depression. A number of forces must be invoked to explain the W͞w locus pattern, and the experiments have been conducted to investigate each of these most likely appears to be selection against white homozygous additional factors. plants. Epperson (42) has carried out extensive simulation Rausher et al. (44) found no evidence for pollen discounting studies that tend to validate this conclusion, and, further, the in experimental populations of morning glory and instead found simulations suggest an intensity of selection against white ho- a nonsignificant excess of white gene fertilizations of ovules of

mozygotes (ww) of approximately 10%. Despite this conclusion, non-white parents. In a different set of experiments, Epperson COLLOQUIUM the frequency of white alleles varies from a low of 0% to a high and Clegg (unpublished data) found a nonsignificant deficit of of 43% across local populations with a mean value of about 10% white gene fertilizations of ovules of non-white parents. Much (35). Because the various local populations can be thought of as larger experiments with greater statistical power are needed to quasi-independent experiments in which selection has operated provide a definitive answer about the role of pollen discounting, for a number of generations, the persistence of white alleles but at present there is no clear evidence that the advantage of argues strongly for countervailing selective forces favoring the white genes is offset by pollen discounting. Epperson and Clegg retention of white phenotypes. (unpublished data) have also measured the fertility of white The spatial pattern of albino alleles, including unstable alleles maternal parents subjected to autopollination and find a reduc- (a*), is of interest because this locus also determines a white tion in maternal fertility that could act to moderate or eliminate recessive phenotype that may experience selective pressures the transmission advantage of white genes. Experiments to common to those experienced by the ww phenotype. Limited measure inbreeding depression provide evidence for a depres- sampling across the southeastern U.S. indicates very low fre- sion in fitness associated with self-fertilization, but the magni- quencies of albino alleles (Ͻ1%) in most local populations (11). tude is not sufficient to offset the selfing advantage of white In addition, unstable alleles also appear to be rare in most local genes (49). Other studies have suggested heterogeneity among populations. An exceptional local population near Athens, GA families in segregation bias favoring the transmission of either is the only population sampled with moderate frequencies of white or dark alleles in different heterozygous parents (50). both albino (a) and unstable (a*) alleles (11). Finally, overdominance in seed size among the progeny of white maternal parents has also been documented, but this evidently Selection on Flower Color Phenotypes. Because flowering plants does not translate into a fitness advantage (51). often depend on insect pollinators for reproductive success, a In summary, there is clear and convincing evidence that white natural question to investigate is whether pollinators discrimi- phenotypes suffer some disadvantage in natural populations nate among the various flower color phenotypes in morning based on spatial autocorrelation analyses, and there is clear and glory populations. A number of experiments conducted in convincing evidence that white genes have a transmission ad- different years and by different investigators in both natural and vantage when white maternal plants are infrequent in popula- experimental populations agree in revealing a bias by bumblebee tions. The transmission advantage is associated with pollinator

Clegg and Durbin PNAS ͉ June 20, 2000 ͉ vol. 97 ͉ no. 13 ͉ 7021 Downloaded by guest on September 26, 2021 preferences and a consequent increased rate of self-fertilization Another fascinating result of this work is the overwhelming among white maternal parents, but this advantage is one-sided importance of mobile elements as generators of phenotypic and should diminish to zero as the frequency of white maternal diversity. All except one of the flower color phenotypes analyzed types approaches 50%. Many lines of evidence argue for a so far in both I. purpurea and I. nil are the result of transposon compensatory disadvantage of white types to account for the insertions. This strongly implies that transposable elements are global frequency in the southeastern U.S. of approximately 10%, the major cause of mutations that yield an obvious phenotype in but the precise nature of the disadvantage is unresolved. It may these plant species. Because the distribution of transposons be that a number of separate components such as reduced appears to be heterogeneous across plant genomes, it seems maternal fertility under autopollination, inbreeding depression, reasonable to speculate that some species may experience higher and segregation bias all sum to provide the needed balance. mutation rates and therefore may be more flexible in adapting Because small effects demand very large experiments to provide to environmental changes than other plant species. In the case adequate statistical power, it may not prove feasible to measure of I. purpurea, a significant environmental change was the each component of fitness with sufficient accuracy to identify all appearance of human plant domesticators who selected and of the factors that comprise a balance. It is also important to note propagated unusual phenotypes for esthetic and perhaps other that, despite much experimental effort over two decades, no purposes. The plant has clearly been successful, at least in the evidence has emerged for differential selection among other short term, by virtue of its ability to adapt to this circumstance. flower color phenotypes such as those determined by the P͞p A number of questions remain to be resolved before this work and I͞i loci. can be seen as complete. First, the gene that is clearly subject to selection in nature is not a structural gene determining an Conclusions enzyme in the anthocyanin pathway but, instead, a regulatory We began this work with the conviction that a full understanding gene that determines the floral distribution of pigmentation of the mechanisms of adaptation would entail an integration of (W͞w locus). To date, this gene has not been characterized at the ecological and the genetic dimensions of biology. This the molecular level, and we do not know the nature of the wisdom was by no means original but, rather, was embodied in mutational changes that cause the white phenotype. We do know Ledyard Stebbins’ monumental contributions to plant evolution- a lot about the A͞a locus, which also determines a white (albino) ary biology. As Stebbins emphasized, the point of contact phenotype, and we can probably assume that this phenotype is between these two levels of biological organization is the phe- also discriminated against by insect pollinators, but the albino notype. Whether a phenotype is adapted to a particular envi- phenotype is rare in populations. So major gaps exist in our ronment depends on a host of biotic and abiotic factors that present knowledge, but we have every reason to expect these collectively translate into survival and reproductive success. It is gaps to fill in over time. Interestingly, we do know the molecular often difficult to identify the environmental elements that bases for the P͞p locus phenotypes, but so far there is no occasion phenotypic success. We began with the notion that evidence of selection in nature at this locus, although in a flower color provided a simple model for the study of adaptation broader sense it is clear that man has selected this polymorphism because one aspect of the environment seemed, a priori,tobe for propagation. predominant—the behavior of insect pollinators. To a large A second limitation is that most population work has focused extent a substantial body of experimental work has validated this on the southeastern U.S., where the plant is introduced, rather assumption, but along the way we have been forced to begin to than on native populations in the central highlands of Mexico. confront the full complexity of plant reproductive biology. In a The southeastern U.S. is not the geographical region in which the similar vein, the phenotypic bases of flower color variation species evolved, and it is not possible to make inferences about appeared to be susceptible to analysis because much was already the longer term ecological circumstances that shaped the evo- known about the biochemistry and genetics of flower color lution of this species based on investigations in other geograph- determination and because the tools for molecular analysis were ical and ecological settings. Despite this reservation, the south- rapidly appearing on the horizon. What we did not anticipate was eastern U.S. populations represent a crude series of experiments the remarkable ecology of plant genomes in which most genes that trace to introductions within the last several hundred to one are redundant and in which mobile elements are a major player thousand years, and the ability to place temporal limits on the in the generation of phenotypic diversity. history of these populations is a strength of the morning glory One complexity that may be more apparent than real is the program. Common patterns among populations are indicative of problem of genetic redundancy. As noted in this article, most of common initial conditions or common selective forces. Thus, the the genes of flavonoid biosynthesis occur in multiple copies, and lack of spatial autocorrelation for the W͞w locus phenotypes sorting through this redundancy to find the particular genes over populations is strongly indicative of a disadvantage that is responsible for individual flower color phenotypes appeared independent of location. Similarly, the case for an isolation-by- daunting at first sight. The actual findings are encouraging, in distance model of population structure for the P͞p locus is that particular gene copies of CHS and DFR are shown to be strongly supported by the consistency of this result over local causally responsible for particular flower color phenotypes. In populations. the case of CHS, we know that the other redundant copies are A third consideration is the problem of statistical power. predominantly expressed at other stages in development (e.g., Because the structure of statistical hypothesis testing is deliber- CHS-E in the floral tube) or they have diverged in catalytic ately biased against accepting the alternative hypothesis (power properties (e.g., CHS-A, -B, and -C). is usually much less than the size of the critical region, ␣, under Another complexity is the pleiotropic nature of biosynthetic the null hypothesis), very large experiments must be carried out pathways. At first sight it appears that each mutation in a gene to detect moderately large effects. Put another way, magnitudes encoding a pathway component is likely to have many pheno- of selection that can be quite effective from an evolutionary typic effects. Is it possible to associate a main effect with one standpoint cannot be detected in experiments of reasonable size. aspect of phenotype? Perhaps, if gene redundancy and special- This may explain the failure to detect pollen discounting and the ization in expression patterns approximates a one-to-one corre- failure to detect significant inbreeding depression in experimen- spondence between gene and phenotype. Whether the unravel- tal populations of I. purpurea. Limited statistical power can in ing of phenotypic determination will be so simple remains to be part be overcome by experiments that run over many generations seen, but there is some reason to be optimistic based on flower in which the effects are cumulative. This is the strength of color analyses. the geographical and population studies in the southeastern

7022 ͉ www.pnas.org Clegg and Durbin Downloaded by guest on September 26, 2021 U.S., which represents the cumulative outcome of many gener- phenotype and in penetrating the complexities associated with ations rather than the result of single generation experiments. the coordinated action of many genes. The determination of Despite the limitations noted above, the study of model floral color development is an area of special promise because evolutionary systems is just as important as the study of other the translation between genes and phenotype is tractable. Sim- model systems. Model systems should reveal the important ilarly, the translation between environment and phenotype is questions and provide a basis for inventing novel approaches that more transparent for flower color than in most other cases, and can then be extended to more refractory systems. As one there is still much to be learned from this research strategy. example, Stebbins pioneered the analysis of plant development as an essential prerequisite to understanding the determination We thank Dr. Shigeru Iida of the National Institute for Basic Biology in of phenotype and hence to the understanding of adaptation. This Okazaki, Japan for his critical review and suggestions and for sharing his unpublished data. We also thank Dr. Hiroshi Noguchi from the School approach is just as important today as it was more than 40 years of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan for ago, when Stebbins began his classic work with barley awn sharing his unpublished data on the substrate specificity testing of the I. development as his model system. Today we have a rich suite of purpurea CHS genes. This work was supported in part by a grant from molecular tools that assist in unraveling the determination of the Alfred P. Sloan Foundation.

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