Ancient Gene Duplications, Rather Than Polyploidization, Facilitate Diversification of Petal Pigmentation Patterns in Clarkia gracilis (Onagraceae).

It has been suggested that gene duplication and polyploidization create opportunities for the evolution of novel characters. However, the connections between the effects of polyploidization and morphological novelties have rarely been examined. In this study, we investigated whether petal pigmentation patterning in an allotetraploid Clarkia gracilis has evolved as a result of polyploidization. Clarkia gracilis is thought to be derived through a recent polyploidization event with two diploid species, C. amoena huntiana and an extinct species that is closely related to C. lassenensis. We reconstructed phylogenetic relationships of the R2R3-MYBs (the regulators of petal pigmentation) from two subspecies of C. gracilis and the two purported progenitors, C. a. huntiana and C. lassenensis. The gene tree reveals that these R2R3-MYB genes have arisen through duplications that occurred before the divergence of the two progenitor species, that is, before polyploidization. After polyploidization and subsequent gene loss, only one of the two orthologous copies inherited from the progenitors was retained in the polyploid, turning it to diploid inheritance. We examined evolutionary changes in these R2R3-MYBs and in their expression, which reveals that the changes affecting patterning (including expression domain contraction, loss-of-function mutation, cis-regulatory mutation) occurred after polyploidization within the C. gracilis lineages. Our results thus suggest that polyploidization itself is not necessary in producing novel petal color patterns. By contrast, duplications of R2R3-MYB genes in the common ancestor of the two progenitors have apparently facilitated diversification of petal pigmentation patterns.

Floral traits influence the opportunity for selection among male gametophytes: independent and combined effects of style length and petal area.

PREMISE: Strong correlations between traits can obscure their independent effects on components of reproduction. Style length (SL) and petal area (PA) vary within species, for example, but their independent effects on the opportunity for selection among pollen genotypes are poorly understood. Previous work in Clarkia detected a positive effect of SL on pollen receipt, potentially intensifying selection. However, this apparent effect of SL may be influenced by a correlated trait, such as PA. Here, we examine the independent effects of these two traits on pollen receipt and performance. METHODS: We collected petals and styles from wild populations of two insect-pollinated Clarkia taxa and estimated the independent and combined effects of SL and PA on pollen receipt and performance. RESULTS: In both taxa, SL and PA are positively correlated. In C. unguiculata, both traits positively and independently affect pollen receipt, but in C. xantiana ssp. xantiana, the two traits act only in combination to affect pollen receipt. In both taxa, pollen receipt positively affects the numbers of pollen tubes entering and penetrating the style, as well as pollen tube attrition. CONCLUSIONS: The effects of SL and PA on pollen receipt and performance are taxon specific. In C. unguiculata, both traits may be independent targets of selection due to their effects on pollen receipt. In C. xantiana ssp. xantiana, by contrast, the combined (but not independent) effects of SL and PA influence pollen receipt. Ecological differences between these taxa require exploration to understand the mechanisms by which these traits affect pollinator behavior.

Ecological sorting and character displacement contribute to the structure of communities of Clarkia species.

Despite long-standing interest in the evolutionary ecology of plants that share pollinators, few studies have explored how these interactions may affect communities during both community assembly (ecological sorting) and through ongoing, in situ evolution (character displacement), and how the effects of these interactions may change with community context. To determine if communities display patterns consistent with ecological sorting, we assessed the frequency of co-occurrence of four species of Clarkia in the southern Sierra foothills (Kern County, CA, USA). To investigate potential character displacement, we measured pollination-related traits on plants grown in a greenhouse common garden from seed collected in communities with one, two or four Clarkia species. Among the four species of Clarkia in this region, the two species that are often found in multi-species communities also co-occur with one another more frequently than expected under a null model. This pattern is consistent with ecological sorting, although further investigation is needed to determine the role of pollinators in shaping community assembly. Patterns of trait variation in a common garden suggest that these two species have diverged in floral traits and converged in flowering time where they co-occur, which is consistent with character displacement. Trait variation across community types also suggests that the process and outcome of character displacement may vary with community context. Because community context appears to affect both the direction and magnitude of character displacement, change in more species-rich communities may not be predictable from patterns of change in simpler communities.

Two genetic changes in cis-regulatory elements caused evolution of petal spot position in Clarkia.

A major premise in evolutionary developmental biology is that regulatory changes, often involving cis-regulatory elements, are responsible for much morphological evolution. This premise is supported by recent investigations of animal development, but information is just beginning to accumulate regarding whether it also applies to the evolution of plant morphology1-4. Here, we identify the genetic differences between species in the genus Clarkia that are responsible for evolutionary change in an ecologically important element of floral colour patterns: spot position. The evolutionary shift in spot position was due to two simple genetic changes that resulted in the appearance of a transcription factor binding site mutation in the R2R3 Myb gene that changes spot formation. These genetic changes caused R2R3 Myb to be activated by a different transcription factor that is expressed in a different position in the petal. These results suggest that the regulatory rewiring paradigm is as applicable to plants as it is to animals, and support the hypothesis that cis-regulatory changes may often play a role in plant morphological evolution.

How petals change their spots: cis-regulatory re-wiring in Clarkia (Onagraceae).

A long-standing question in evolutionary developmental biology is how new traits evolve. Although most floral pigmentation studies have focused on how pigment intensity and composition diversify, few, if any, have explored how a pattern element can shift position. In the present study, we examine the genetic changes underlying shifts in the position of petal spots in Clarkia. Comparative transcriptome analyses were used to identify potential candidate genes responsible for spot formation. Co-segregation analyses in F2 individuals segregating for different spot positions, quantitative PCR, and pyrosequencing, were used to confirm the role of the candidate gene in determining spot position. Transient expression assays were used to identify the expression domain of different alleles. An R2R3Myb transcription factor (CgMyb1) activated spot formation, and different alleles of CgMyb1 were expressed in different domains, leading to spot formation in different petal locations. Reporter assays revealed that promoters from different alleles determine different locations of expression. The evolutionary shift in spot position is due to one or more cis-regulatory changes in the promoter of CgMyb1, indicating that shifts in pattern element position can be caused by changes in a single gene, and that cis-regulatory rewiring can be used to alter the relative position of an existing character.

Winning in style: Longer styles receive more pollen, but style length does not affect pollen attrition in wild Clarkia populations.

PREMISE OF THE STUDY: One proposed function of long styles is to intensify selection among male gametophytes relative to short styles. If so, given sufficient competition, longer styles will have higher rates of pollen tube attrition (failure to reach the style base) within the style than shorter ones. Alternatively, style length may influence pollen receipt, which itself may affect attrition rates. METHODS: We tested these predictions by collecting senescing styles from wild populations of two insect-pollinated Clarkia species. We examined the number of pollen grains adhering to the stigma, length of styles, and rates of attrition from the stigma surface to the stigma-style junction (SSJ), from the SSJ to the style base, and from the stigma surface to the style base. Multivariate analyses estimated the independent effects of pollen grains per stigma, the number of pollen tubes at the SSJ, and style length on attrition. KEY RESULTS: Style length was generally positively correlated with pollen receipt, and the number of pollen grains per stigma was positively correlated with all three attrition rates. In neither species was any attrition rate affected by style length independent of the number of pollen grains per stigma. CONCLUSIONS: Pollen attrition was mediated by style length, but the function of style length was primarily to increase the number of germinating pollen grains, which affected attrition rates either through stigma clogging or pollen-pollen interactions. Style length may have a direct effect on pollen receipt due to the stigma's position relative to pollinator body parts, but traits correlated with style length may also directly affect pollen receipt.

Experimental studies of adaptation in Clarkia xantiana. III. Phenotypic selection across a subspecies border.

Sister taxa with distinct phenotypes often occupy contrasting environments in parapatric ranges, yet we generally do not know whether trait divergence reflects spatially varying selection. We conducted a reciprocal transplant experiment to test whether selection favors "native phenotypes" in two subspecies of Clarkia xantiana (Onagraceae), an annual plant in California. For four quantitative traits that differ between subspecies, we estimated phenotypic selection in subspecies' exclusive ranges and their contact zone in two consecutive years. We predicted that in the arid, pollinator-scarce eastern region, selection favors phenotypes of the native subspecies parviflora: small leaves, slow leaf growth, early flowering, and diminutive flowers. In the wetter, pollinator-rich, western range of subspecies xantiana, we expected selection for opposite phenotypes. We investigated pollinator contributions to selection by comparing naturally pollinated and pollen-supplemented individuals. For reproductive traits and for subspecies xantiana, selection generally matched expectations. The contact zone sometimes showed distinctive selection, and in ssp. parviflora selection sometimes favored nonnative phenotypes. Pollinators influenced selection on flowering time but not on flower size. Little temporal variation in selection occurred, possibly because of plastic trait responses across years. Though there were exceptions and some causes of selection remain obscure, phenotypic differentiation between subspecies appears to reflect spatially variable selection.

Seasonal changes in physiological performance in wild Clarkia xantiana populations: Implications for the evolution of a compressed life cycle and self-fertilization.

PREMISE OF THE STUDY: One explanation for the evolution of selfing, the drought escape hypothesis, proposes that self-fertilization may evolve under conditions of intensifying seasonal drought as part of a suite of traits that enable plants to accelerate the completion of their life cycle, thereby escaping late-season drought. Here, we test two fundamental assumptions of this hypothesis in Clarkia xantiana: (1) that a seasonal decline in precipitation causes an increase in drought stress and (2) that this results in changes in physiological performance, reflecting these deteriorating conditions. METHODS: We examined seasonal and interannual variation in abiotic environmental conditions (estimated by ambient temperature, relative humidity, predawn leaf water potentials, and carbon isotope ratios) and physiological traits (photosynthesis, conductance, transpiration, instantaneous water-use efficiency, ascorbate peroxidase and glutathione reductase activities, quantum yield of photosystem II, PSII potential efficiency) in field populations of C. xantiana in 2009 and 2010. KEY RESULTS: In both years, plants experienced intensifying drought across the growing season. Gas exchange rates decreased over the growing season and were lower in 2009 (a relatively dry year) than in 2010, suggesting that the temporal changes from early to late spring were directly linked to the deteriorating environmental conditions. CONCLUSIONS: Seasonal declines in transpiration rate may have increased survival by protecting plants from desiccation. Concomitant declines in photosynthetic rate likely reduced the availability of resources for seed production late in the season. Thus, the physiological patterns observed are consistent with the conditions required for the drought escape hypothesis.

Novel adaptation to hawkmoth pollinators in Clarkia reduces efficiency, not attraction of diurnal visitors.

BACKGROUND AND AIMS: Plant populations experiencing divergent pollination environments may be under selection to modify floral traits in ways that increase both attractiveness to and efficiency of novel pollinators. These changes may come at the cost of reducing overall effectiveness of other pollinators. The goal of this study was to examine differences in attractiveness and efficiency between Clarkia concinna and C. breweri, sister species of annual plants with parapatric distributions. METHODS: An assessment was made as to whether observed differences in visitors between natural populations are driven by differences in floral traits or differences in the local pollination environment. Differences in floral attractiveness were quantified by setting out arrays of both species in the geographical range of each species and exposing both species to nocturnal hawkmoths (Hyles lineata) in flight cages. Differences in visitor efficiency were estimated by measuring stigma-visitor contact frequency and pollen loads for diurnal visitors, and pollen deposition on stigmas for hawkmoths. KEY RESULTS: The composition of visitors to arrayed plants was similar between plant species at any particular site, but highly divergent among sites, and reflected differences in visitors to natural populations. Diurnal insects visited both species, but were more common at C. concinna populations. Hummingbirds and hawkmoths were only observed visiting within the range of C. breweri. Despite attracting similar species when artificially presented together, C. concinna and C. breweri showed large differences in pollinator efficiency. All visitors except hawkmoths pollinated C. concinna more efficiently. CONCLUSIONS: Differences in the available pollinator community may play a larger role than differences in floral traits in determining visitors to natural populations of C. concinna and C. breweri. However, floral traits mediate differences in pollinator efficiency. Increased effectiveness of the novel hawkmoth pollinator on C. breweri comes at relatively little cost in attractiveness to other visitors, but at large cost in their efficiency as pollinators.

Experimental studies of adaptation in Clarkia xantiana. I. Sources of trait variation across a subspecies border.

Both genetic differentiation and phenotypic plasticity might be expected to affect the location of geographic range limits. Co-gradient variation (CoGV), plasticity that is congruent with genetic differentiation, may enhance performance at range margins, whereas its opposite, counter-gradient variation (CnGV) may hinder performance. Here we report findings of reciprocal transplant experiments intended to tease apart the roles of differentiation and plasticity in producing phenotypic variation across a geographic border between two plant subspecies. Clarkia xantiana ssp. xantiana and C. xantiana ssp. parviflora are California-endemic annuals that replace each other along a west-east gradient of declining precipitation. We analyzed variation in floral traits, phenological traits, and vegetative morphological and developmental traits by sowing seeds of 18 populations (six of ssp. xantiana and 12 of ssp. parviflora) at three sites (one in each subspecies' exclusive range and one in the subspecies' contact zone), in two growing seasons (an exceptionally wet El Niño winter and a much drier La Niña winter). Significant genetic differences between subspecies appeared in 11 of 12 traits, and differences were of the same sign as in nature. These findings are consistent with the hypothesis that selection is responsible for subspecies differences. Geographic variation within subspecies over part of the spatial gradient mirrored between-subspecies differences present at a larger scale. All traits showed significant plasticity in response to spatial and temporal environmental variation. Plasticity patterns ranged from spatial and temporal CoGV (e.g., in node of first flower), to spatial CnGV (e.g., in flowering time), to patterns that were neither CoGV nor CnGV (the majority of traits). Instances of CoGV may reflect adaptive plasticity and may serve to increase performance under year-to-year environmental variation and at sites near the subspecies border. However, the presence of spatial CnGV in some critical traits suggests that subspecies ranges may also be constrained by patterns of plasticity.