Precise spatio-temporal regulation of the anthocyanin biosynthetic pathway leads to petal spot formation in Clarkia gracilis (Onagraceae).

Petal spots are widespread in Angiosperms and are often implicated in pollinator attraction. Clarkia gracilis petals each have a single red-purple spot that contrasts against a pink background. The position and presence of spots in C. gracilis are determined by the epistatic interaction of alleles at two, as-yet-unidentified loci. We used HPLC to identify the different pigments produced in the petals, and qualitative and quantitative RT-PCR to assay for spatio-temporal patterns of expression of different anthocyanin pathway genes. We found that spots contain different pigments from the remainder of the petal, being composed of cyanidin/peonidin-based, instead of malvidin-based anthocyanins. Expression assays of anthocyanin pathway genes show that Dfr2 has a spot-specific expression pattern and acts as a switch for spot production. Co-segregation analyses implicate the gene products of the P and I loci as trans-regulators of this switch. Spot pigments appear earlier in development due to early expression of Dfr2 and F3′h1. Pigments in the background appear later, due to later expression of Dfr1 and F3′5′h1. The evolution of this spot production mechanism appears to have been facilitated by duplication of the Dfr gene and to have required substantial reworking of the anthocyanin pathway regulatory network. Keywords: Clarkia, Dfr, flower color, petal spot, pigment pattern Introduction A major objective of evolutionary developmental biology (‘EvoDevo’) is to elucidate the genetic changes that result in the evolution of novel characters. One unresolved issue is whether novel characters can arise through changes in the activity of genes that are already active in developmental programs or if they require recruitment of additional genes into those programs (Keys et al., 1999). In addition, while the evolution of novel morphologies is sometimes associated with gene duplication (Xiao et al., 2008, Parker et al., 2009), it is unclear how frequently duplication contributes to the emergence of novel traits. Because of the apparent simplicity and ecological relevance of wing spotting patterns, the developmental control of evolutionarily novel spots in animals, particularly butterflies, has been used to address these issues (Nijhout 1980, Carroll et al, 1994, Keys et al. 1999, Beldade et al. 2002, Reed & Serfas, 2004). These investigations have tentatively indicated that new wing spots have arisen by the modification of existing developmental programs. In plants, petals with large spots— discrete depositions of visible pigments that contrast with the background coloration of the flower—occur in the families Liliaceae, Orchidaceae, Asteraceae, Papaveraceae, Fabaceae, and many others, indicating that they have evolved independently numerous times. Moreover, field studies have frequently demonstrated that spots are important in mediating interactions with pollinators (Jones, 1996; Johnson & Midgley, 1997; Van Kleunen et al., 2007; Goulson et al., 2009). Nevertheless, the genetic and developmental control of petal spots, and more generally, of petal color patterning, have seldom been investigated. Studies in model species such as petunia and snapdragon have clarified the genetic control of some patterns, namely vein-associated pigmentation (Schwinn et al., 2006; Shang et al., 2011) or variation in pigment intensity in different regions of the corolla (Jackson et al., 1992; Schwinn et al., 2006; Albert et al., 2011). However, our general understanding of how floral pigment patterns develop and are regulated at the molecular level, specifically in relation to petals spots, remains poor. In this report, we describe experiments that begin to elucidate the biochemical, genetic and developmental basis of petal spots in the California wildflower, Clarkia gracilis. Clarkia gracilis (Onagraceae) is native to northern California, Oregon and Washington. It is the only polyploid in section Rhodanthos. Based on cytological analyses, (Abdel-Hameed & Snow, 1972) suggested that this allotetraploid derived from hybridization between two diploid species, one closely related to C. amoena ssp. huntiana and one related to C. lassenensis and C. arcuata. However, the parentage of C. gracilis has not been tested adequately by modern molecular systematic methods. Clarkia gracilis is composed of four different subspecies that have somewhat overlapping ranges: C. g. ssp. albicaulis, C. g. ssp. gracilis, C. g. ssp. sonomensis, and C. g. ssp. tracyi. The subspecies differ in their floral morphologies, particularly with respect to the presence and position of petal spots. Subspecies albicaulis and tracyi have a single spot at the base of each petal, ssp. sonomensis has a single spot in the center of each petal, and ssp. gracilis lacks petal spots (Fig. 1). In all cases, mature petal spots are dark reddish-purple concentrations of pigment on a pale pink background. However, there is some variation within some subspecies: C. g. sonomensis and C.g. albicaulis can be found in either spotted or unspotted morphs. Fig. 1 Flowers of subspecies in the C. gracilis species complex. From left: C. g. ssp. tracyi, C. g. ssp. sonomensis, C. g. ssp. gracilis, and C. g. ssp. albicaulis. The ecological significance of petal spots in C. gracilis has been studied in some detail. In mixed populations of central spotted and unspotted C. gracilis ssp. sonomensis, spotted plants have higher fitness, producing as much as 32% more seed than their unspotted counterparts in some growing seasons and sites (Jones, 1996). This difference might reflect pollinator preference, since petal spots were also shown to influence pollinator foraging behavior (Jones, 1996). In another species of Clarkia, C. xantiana, the maintenance of both spotted and unspotted morphs has been attributed to pollinator-based negative frequency-dependent selection (Eckhart et al., 2006). Previous investigations have established that C. gracilis petal spots are under simple genetic control. Gottlieb & Ford (1988) identified two independent loci that interact epistatically to give rise to all of the morphs present in this species complex. One locus, P, has co-dominant alleles that determine the position of the spot within the petal: the PB allele causes the production of basal spots, and the PC allele causes the production of central spots. The other locus, I, affects the presence of basal spots: the IA allele (or I, in (Gottlieb & Ford, 1988)) suppresses basal spots, while the IP allele (i, in Gottlieb & Ford, 1988) allows basal spots to form. Neither allele at the I locus affects the formation of central spots. Gottlieb & Ford (1988) determined that C. gracilis ssp. gracilis generally has a PBPBIAIA genotype, whereas spotted plants of ssp. sonomensis often have the genotype, PCPCIPIP. However, a variable dominance relationship was also found between IA and IP suggesting that additional modifiers or alternative alleles are present in different backgrounds. The identity of the pigments underlying this phenotype can provide important clues as to the genes that may be involved in spot formation. Species in the genus Clarkia produce anthocyanin pigments, mostly derivatives of malvidin and cyanidin, although not all species produce these pigments (Dorn & Bloom, 1984; Soltis, 1986). Production of pelargonidin and derivatives is rare in the genus, being known only in C. unguiculata (Robinson & Robinson, 1931), suggesting that the biochemical pathway branch leading to pelargonidin production is inactive in most species (Fig. 2). In C. gracilis flowers, the anthocyanidins malvidin, cyanidin and delphinidin (a precursor of malvidin) have been reported (Soltis, 1986). Fig. 2 Simplified schematic representation of the anthocyanin biosynthetic pathway. Our objectives in this investigation were to identify genes associated with spot formation in C. gracilis and characterize their pattern of regulation. Specifically, we sought to answer the following questions: What pigments are produced in C. gracilis flowers? Do pigments in spots differ from the rest of the petal? Do pigments in central spots differ from those in basal spots? Is transcriptional regulation of specific anthocyanin biosynthetic genes involved in petal spot formation? If so, does any gene correspond to the spot position P locus? Materials and Methods Plant growth and crosses Seeds from C. gracilis (Piper) A. Nelson & J. F. Macbride were germinated in vermiculite at 15°C in the dark, and transferred to a 1:1 metromix:peatmoss mixture once seedlings emerged. Seedlings were then transferred to 5-inch pots and were grown in a glasshouse (20–24°C) under natural light. Plants were periodically randomized to avoid possible light-induced changes in flower color. Several individuals from various populations were grown, and only those from monomorphic (with respect to spots) populations were used as the parents for crosses. Two independent crosses were carried out to separate the P and I loci. For the ‘P’ cross, a central-spotted C. g. sonomensis was crossed to a basal-spotted C. g. albicaulis. For the ‘I’ cross, an unspotted C. g. gracilis was crossed to a basal-spotted C. g. albicaulis. Voucher information is available in Supporting Information Table S1. Pollen recipient flowers were emasculated several days before full stigma receptivity to avoid self-pollination. Stigmas were pollinated by touching them with a dehiscing anther from the designated sire. F1 individuals were selfed to obtain F2 progeny.