Here we explore variation in pollen pigmentation among C. americana populations and then elucidate abiotic factors that may drive this variation. Specifically we ask, to what degree does pollen pigmentation vary within and among populations and does it show geographic variation? Does pollen color have a genetic basis? Do abiotic factors correspond with geographic variation in pollen color and does pollen color covary directly with abiotic factors? And is the performance of light and dark pollen differentially affected by varied temperature and UV?

The eastern North American herb Campanula americana is an ideal system in which to explore geographic variation in pollen color. Pollen is known to vary from tan to deep purple in C. americana (Lau & Galloway, 2004 ). The species is widespread in eastern North America, and thus populations experience highly varied abiotic conditions across the range (Prendeville et al ., 2013 ). The mode of pollen presentation in C. americana is well suited for testing the effects of abiotic conditions on pollen performance. Anthers dehisce in the bud stage of the flower, depositing pollen in pollen‐collecting hairs along the style. As a result, pollen is unprotected by an anther wall or by petal tissue once the flower is open. Thus, exposure to potentially stressful conditions such as extreme temperatures or UV irradiance may be elevated compared with species for which pollen is protected within petal or anther tissue. Anthocyanin compounds (delphinidins) provide color to the petals of C. americana (Buckles, 1975 ). While the exact compounds are not known from the pollen, it is assumed that these are anthocyanic because other potential pigments (betalains) are not known in the Campanulaceae, and betalains are often yellow‐red (Tanaka et al ., 2008 ).

Pollen performance can be strongly affected by abiotic conditions. Exposure to high heat (Pressman, 2002 ; Kakani, 2005 ; Rang et al ., 2011 ), ultraviolet (UV) irradiance (Feng et al ., 2000 ), and drought (Fang et al ., 2010 ) often reduce pollen viability. However, the pigmentation of pollen may influence its resistance to abiotic stress. Pollen is pigmented with flavonoid compounds in many taxa (Mo et al ., 1992 ; Lunau, 2000 ), and anthocyanins have been detected in the pollen of some species (di Paola‐Naranjo et al ., 2004 ; Leja et al ., 2007 ). When disrupted, chalcone synthase, the first enzyme in the anthocyanin biosynthetic pathway, yields pollen‐sterile individuals in both Petunia and Maize (Coe et al ., 1981 ; Mo et al ., 1992 ; Napoli et al ., 1999 ). Addition of the flavonoid kaempferol rescues pollen fertility, and a dose‐dependent increase in pollen germination with increasing flavonol concentrations (Mo et al ., 1992 ) suggests that gradations in flavonol concentration in pollen could lead to differential pollen viability. Flavonols and pigmented anthocyanins (cyanidin, delphinidin, pelargonidin) share common precursors (dihydroflavonols), and thus highly pigmented pollen may have elevated flavonol concentrations if flavonols and anthocyanins are jointly regulated (Winkel‐Shirley, 2002 ; Koes et al ., 2005 ). To date, however, there is no evidence from wild species that pollen color contributes to abiotic stress resistance.

The functional roles of pollen‐color variation with respect to abiotic conditions and whether natural selection via the abiotic environment contribute to local adaptation of pollen‐color phenotypes are little understood. However, a notable body of work exists for Nigella degenii (Ranunculaceae) where plants with dark pollen are more likely to inhabit north‐ and east‐facing slopes, which are expected to experience reduced drought compared with south‐ and west‐facing slopes (Jorgensen & Andersson, 2006 ). Additionally, pollen color has a genetic basis, suggesting the potential for a response to natural selection (Jorgensen et al ., 2006 ). In experimental drought conditions, plants with dark pollen display reduced survival relative to light morphs (Andersson & Jorgensen, 2006 ). This work suggests a functional relationship between individuals with different pollen‐color morphs and abiotic conditions, but whether the performance of pollen itself under varied abiotic conditions depends on color has not been tested.

Abiotic factors are becoming increasingly recognized agents of natural selection on floral traits (Strauss & Whittall, 2006 ; Narbona et al ., 2017 ). For example, drought, ambient light conditions and temperature contribute to geographic variation in petal color phenotypes within multiple taxa (e.g. Schemske & Bierzychudek, 2007 ; Arista et al ., 2013 ; Koski & Ashman, 2015 ; del Valle et al ., 2015 ; Berardi et al ., 2016 ), and abiotic selection underlies diversification in petal‐color patterning phenotypes among species (Koski & Ashman, 2016 ). Such abiotic selection acts on petal phenotypes indirectly through selection on whole‐plant biochemistry (Wessinger & Rausher, 2012 ), or directly through the effects of petal color on the pollen and ovule environment (Lacey et al ., 2010 ; Koski & Ashman, 2015 ).

Intraspecific floral color variation is common and, in many cases, molded by selection to attract pollinators (Rausher, 2008 ). Petal color polymorphisms have been used to dissect the agents and targets of natural selection (Clegg & Durbin, 2000 ), evaluate the evolution of development (Streisfeld et al ., 2013 ), and assess the contribution of neutral population genetic processes to phenotypic evolution (Schemske & Bierzychudek, 2007 ). While studies on variation of petal color abound, pollen color can also vary (e.g. Erythronium , Thomson & Thomson, 1989 ; Maize, Coe et al ., 1981 ; Linum , Wolfe, 2001 ; Campanula , Lau & Galloway, 2004 ; Nigella , Jorgensen & Andersson, 2005 ). However, few studies consider diversity in pollen pigmentation and its ecological role (however, see Jorgensen et al ., 2006 ), despite its potential importance as a visual cue for pollinators (Lau & Galloway, 2004 ), and the requirement of flavonoid (i.e. pigment) compounds for successful pollen germination and pollen tube growth (Mo et al ., 1992 ; Ylstra et al ., 1992 ). A detailed profiling of pollen‐color variation across the range of a species will help to identify the potential ecological contributors to such variation.

To assess whether pollen‐color phenotypes differed in germination potential under different UV treatments, we constructed a similar generalized mixed linear model (proc glimmix, Sas) as in the temperature experiment. We modeled the proportion of germination as a function of UV (present/absent), pollen‐color class, and their interaction with a beta distribution. We used population and experimental replicate as random effects in the model. The beta distribution provided a good fit to the data, generalized chi‐square / d.f. = 1.0.

To determine the effect of UV‐B on pollen germination, styles from two to three dark‐ and two to three light‐pollen individuals were collected from five populations (Table S1 ). Styles were cut in half and one half was exposed to UV for 8 h, while the other was protected from UV. Specifically, a pollen‐coated style was placed in a Petri dish within a growth chamber with four UV light bulbs (26W Repti Glo UVB 150; Rolf C. Hagen Corp., Mansfield, MA, USA) along with fluorescent lighting to provide nonUV wavelengths. One pollen sample from each individual was protected from UV with a filter placed over the Petri dish that blocks UV but transmits all other wavelengths of light (Rosco no. 3114, Stamford, CT, USA; see Koski & Ashman, 2015 ). The other pollen sample was similarly covered with a piece of Saran wrap (clingfilm) which transmits UV and other wavelengths as a control. The UV+ treatment generated a UV‐B (280–315 nm) environment of 2869 J m −2 d −1 (range 1671–4751; absolute irradiance measurements taken from Koski & Ashman, 2015 , which used roughly the same experimental design). In the field, C. americana populations experience an average of 4458 J m −2 d −1 during the summer months (range 3787–5045). Thus, pollen in the UV+ treatment in the growth chamber experienced c . 35% lower UV‐B than field conditions on average. After exposure, pollen was plated into 10% Brewbaker–Kwack solution, allowed to germinate, and fixed in the same manner as the temperature experiment. The UV experiment was repeated three times in the same Conviron growth chamber. We scored 60 dark and 50 light individuals for a total of 110 samples.

To assess whether dark and light pollen differed in germination proportion under different temperature treatments, we used a generalized mixed linear model (proc glimmix, Sas). We modeled the proportion of germination as a function of temperature, pollen‐color class, and their interaction with a beta distribution. We used population and experimental replicate as random effects in the model. A significant temperature × color interaction indicates that pollen‐color phenotypes respond differently to temperature. Upon finding a significant interaction term, we used the slice option of proc glimmix to assess whether dark and light phenotypes differed in pollen germination within each temperature treatment. Finally, we used a separate slice statement to address whether each pollen‐color class (light or dark) displayed a significant germination response to the temperature treatments. The beta distribution provided a good fit to the data, generalized chi‐square / d.f. = 1.0.

Fixed and mounted pollen samples were scored for germination using a light microscope at 10×. A germinated grain was counted if the length of the pollen tube exceeded the diameter of the pollen grain. We scored germinated and ungerminated grains until at least 300 total grains were scored. For each sample, we calculated the proportion of pollen germination as the number of germinated grains divided by the total number of grains. In some cases, few grains were available on slides for scoring. We removed samples with fewer than 50 grains on the slide. We scored samples from 71 dark phenotypes and 86 light phenotypes, for a total of 157 samples across the temperature treatments (52 low, 53 medium and 52 high).

Each treatment was established in a separate Percival growth chamber. Relative humidity was 50% in each chamber and fluorescent lighting was the same for each (2.3 × 10 4 lx). Pollen was left in growth chambers for 8 h, at which point it was removed, placed into c . 30 μl of 10% Brewbaker–Kwack solution on a microscope coverslip within a Petri dish, and kept moist with a wet piece of filter paper. Pollen was allowed to germinate under fluorescent lights in a growth chamber set to 24°C for 15 h. Pollen germination was then arrested with a 10 μl drop of formalin‐acetic acid. Pollen samples on the coverslip were then inverted onto a microscope slide and sealed with clear nail polish until they were scored. As the temperature treatments were not independent of the growth chambers during a single experimental replicate, we repeated the experiment three times such that each temperature treatment was conducted in each of the three chambers.

We selected eight populations (Table S1 ) with diverse pollen colors to explore the response of pollen germination to abiotic factors. For each population, we selected two to three individuals with light pollen (white, tan, light purple) and two to three individuals with dark pollen (purple, dark purple) from the F 1 generation. We removed pollen‐bearing styles from flowers in the first day of the male phase (Fig 1 a). We cut styles into thirds and pollen from a single flower was exposed to each of three temperature treatments: low (13°C), medium (24°C), and high (38°C). Temperature treatments were guided by natural conditions during the flowering season. The median of the maximum temperatures experienced during the warmest quarter across populations is 31.2°C (range 27.8–34.0°C). The median of the average temperature across populations is 23.8°C (range 19.8–26.4°C). Low temperatures of 13°C are probably experienced by late‐flowering plants as their reproductive season continues into early fall.

Upon finding longitudinal patterns in pollen color, temperature, and UV (see the Results section), we assessed whether variation in pollen color among populations was associated with temperature and UV. We modeled mean pollen‐color score as a function of maximum temperature and UV using linear regressions. We used separate regressions for each predictor because the strong correlation between summer temperature and UV irradiance ( r = 0.95) resulted in high multicollinearity (Graham, 2003 ). We weighted the average pollen‐color score by the number of individuals scored in each population (Table S1 ). In addition, we modeled the proportion of individuals in a population with dark pollen as a function of maximum temperature and UV. Purple and deep‐purple pollen have similar spectral reflectance (‘dark’ phenotypes), while white, tan and light purple have similar reflectance spectra (‘light’ phenotypes, Fig. 1 b). We also use this binary categorization of pollen color that more closely reflects variation in reflectance spectra for experimental tests of the effect of temperature and UV on pollen color (see the ‘Pollen germination response to temperature’ and ‘Pollen germination response to UV’ subsections). To determine the relationship between the proportion of dark pollen morphs in a population and abiotic factors, we modeled the proportion of dark individuals in a population as a function of maximum temperature and UV using separate logistic regressions with a binomial distribution and a logit link (proc logistic, Sas). The response variable was (number of individuals with dark pollen/total individuals scored) in a given population.

We then assessed how maximum temperature, average UV irradiance, and average precipitation change across the latitudinal and longitudinal gradients of the 24 populations (Table S1 ). We chose these three variables because temperature, UV, and drought have all been associated with variation in plant pigmentation. The coordinates of each population were used to extract maximum temperature, average summer precipitation, and the sum of the monthly mean of UV irradiance for the warmest quarter of the year from the Worldclim (maximum temperature (bio5) and precipitation (bio18); Hijmans et al ., 2005 ) and giUV (UV (UV5); Beckmann et al ., 2014 ) databases. Conditions during the warmest quarter (bio18, UV5) reflect those experienced by pollen better than do average yearly conditions, as plants flower in mid‐to‐late summer. We used maximum temperature instead of average temperature because we were particularly interested in addressing whether pigmentation covaried with thermal stress. We used a multiple linear regression to model each abiotic variable (temperature, precipitation, UV) as a function of latitude and longitude. To compare the effects of latitude and longitude on each factor, we standardized regression coefficients by their standard deviations with the ‘lm.beta’ package in R.

We addressed whether spatial patterns in pollen color exist using multiple linear regression. We calculated the mean pollen‐color score for each population ( n = 24), by averaging the individual scores. We then used a multiple linear regression to model pollen color as a function of latitude and longitude (R, ‘lm’), weighting each population average by the number of individuals scored in each population (Table S1 ). Evaluation of scatter plots did not suggest nonlinear terms of latitude or longitude.

We used midparent‐offspring regression to assess the degree to which genetic variation underlies pollen‐color variation. First, we averaged the quantitative score of pollen color (0, white; 1, tan; 2, light purple; 3, purple; 4, dark purple) between the maternal and paternal plants. We then modeled the pollen‐color score of the F 1 as a function of population, the mean parental pollen‐color score, and their interaction using ANCOVA (proc glm, Sas; SAS, Cary, NC, USA). A significant effect of mean parental pollen color indicates heritability of pollen color, and a significant effect of the population × pollen color interaction indicates that populations vary in their degree of heritability. We found that the interaction between population and midparent pollen color did not affect the pollen color of offspring (see the Results section), suggesting that heritability is of generally the same magnitude across populations. We used the slope of offspring pollen color on mean parental pollen color across all populations to estimate narrow‐sense heritability of pollen color in the species as a whole.

We outcrossed each plant from each of 24 populations with a randomly selected pollen donor from their respective populations to generate F 1 progeny with known parental pollen‐color phenotypes. Fruits were harvested when ripe. F 1 seed were planted in October 2016 in the same conditions as parental plants with a single offspring per family grown to flowering (Spring 2017). The progeny were scored for pollen color in the same manner as the parental generation to assess whether mean parental phenotype predicted offspring phenotype. Across populations, there were 352 F 1 progeny for which we measured both maternal and paternal pollen color (mean per population = 14.7).

(a) Pollen‐color variation in Campanula americana ranges from white to deep purple. Pollen is deposited in pollen‐collecting hairs along the style during the bud stage. Images above show pollen‐bearing styles removed from the flower, and images below depict pollen‐color variation in situ . (b) Average spectral reflectance of pollen‐color classes. For experimental tests of pollen germination response to abiotic variables, white, tan and light purple were grouped as ‘light’ pollen, and purple and dark purple were grouped as ‘dark’ pollen.

Upon flowering in spring 2016, we evaluated pollen color by scoring pollen visually as one of five pigment classes; white, tan, light purple, purple, and deep purple (Fig. 1 a). We scored 449 plants (mean per population = 18.7). To confirm spectral differences between pollen‐color classes, we quantified reflectance of pollen from two to five individual plants per class using a UV‐VIS spectrophotometer (Ocean Optics USB4000, Dunedin, FL, USA) with a DH‐2000 deuterium/halogen light source. We measured reflectance of pollen‐bearing styles that were completely covered in pollen such that reflection from the style was unlikely to influence the reflectance spectrum. Pollen‐bearing styles from multiple flowers (four to five) were aggregated to obtain spectral measurements because the diameter of the spectrophotometer probe is greater than that of a single pollen‐bearing style. For this reason, we scored pollen color qualitatively rather than characterizing the color of each plant with the spectrophotometer. Briefly, all color classes display a peak in the blue range around 460 nm, while major differences between color classes are pronounced between 500 and 600 nm, with white, tan, and light purple having higher reflectance than purple and dark purple in this range (Fig. 1 b). UV reflection (300–400 nm) from all pollen‐color classes is low (< 1.5%).

We scored pollen color in 24 populations ranging from southern Alabama to Minnesota (southern and northern extent), and Kansas to eastern Pennsylvania (western and eastern extent) (Supporting Information Table S1 ). In summer 2015, we collected ripe fruits from maternal plants in each population. Six seeds from each of 20–26 maternal families from each population were sown in metromix and turface (3 : 1) in November 2015 and left to germinate in growth chambers at 21 : 14°C (day : night) with light intensity of 2.3 × 10 4 lx. Seedlings were vernalized at 5°C with 12 h days for 45 d, and one seedling per family was transplanted into a conetainer and placed in the glasshouse at the University of Virginia with supplemental lighting to increase the day length to 16 h.

Campanula americana L. (= Campanulastrum americanum Small) is an annual to biennial herb inhabiting forest margins in eastern North America. The flowers are protandrous with secondary pollen presentation whereby anthers dehisce in the bud stage, depositing pollen on pollen‐collecting hairs along the style. During the first day of male floral phase, pollen‐collecting hairs retract and pollen is removed by bees (Evanhoe & Galloway, 2002 ). It is bee‐pollinated by bumblebees and solitary Megachilidae and Halictidae (Lau & Galloway, 2004 ; Koski et al ., 2017 ). Flowers transition to the female phase with the opening of three stigmatic lobes. Pollen color varies within and among three populations in southwest Virginia within Giles County, ranging from tan to deep purple (Lau & Galloway, 2004 ). The presence of tan, but not purple, pollen on male phase flowers leads to a preference towards pollen‐bearing flowers by Halictid bees (Lau & Galloway, 2004 ). Anther color does not vary. C. americana is divided into three genetically distinct lineages (Barnard‐Kubow et al ., 2015 ), and for this study we work with populations from only the western lineage which displays the widest geographic and environmental range (> 75% of total species range area), although all populations studied to date, including those in the Appalachian lineage, display variation in pollen color.

Temperature influenced pollen germination (Table 2 a; Fig. 5 a). Specifically, pollen germination was reduced in the high‐temperature treatment (23% germination) relative to both the low‐ (37% germination) and medium‐temperature treatments (42% germination). While light and dark pollen did not differ in germination across all treatments (Table 2 a), light and dark pollen responded differently to the temperature treatments (Table 2 a; Fig. 5 a). The pollen‐color phenotypes displayed differential responses only in the high‐temperature treatment, with dark pollen grains germinating 85% more frequently than light pollen grains (Table 2 a). The germination of light pollen was reduced by high temperatures, but dark pollen was not (Table 2 a).

Temperature and UV irradiance experienced during the flowering season were predicted by latitude and longitude, and the effect of latitude was stronger than that of longitude for both (Table 1 ). After accounting for the effects of latitude, longitude explains 82% of the residual variation in temperature ( P < 0.0001), and 47% of the residual variation in UV ( P < 0.001) (Fig. S1 ). Temperature and UV irradiance increase from the eastern populations to the western populations (Fig. S1 ). This corresponds with the east‐to‐west increase in pollen pigmentation. Precipitation did not vary predictably with latitude or longitude.

In the ANCOVA predicting pollen color of F 1 progeny by population and mean parental pollen color, population did not have a significant effect ( F 23,304 = 1.00, P = 0.47); however, mean parental pollen‐color score predicted offspring pollen color across populations ( F 1 , 304 = 17.9, P < 0.0001). There was not a significant interaction between population and mean parental pollen color ( F 23,304 = 0.88, P = 0.62). Across all populations, mean parental pollen color predicted F 1 pollen color ( t = 10.74, slope = 0.57, P < 0.0001; Fig. 3 ). The range‐wide narrow‐sense heritability estimate is 0.57 ± 0.053 SD. The population average pollen color of the parental and F 1 generations are tightly correlated ( r = 0.91, P < 0.0001).

Discussion

Campanula americana exhibits a strong longitudinal cline in a heritable pollen pigmentation phenotype. Specifically, western populations have darker purple pollen than eastern populations, where white to light‐purple pollen is more common. Accounting for latitudinal variation in abiotic factors, western populations also experience more extreme high temperatures and elevated UV‐B irradiance. Experimental manipulation of temperature and UV revealed that dark pollen phenotypes outperform light pollen under higher temperature, but not elevated UV. And dark pollen is more common in natural populations with warmer temperatures. Together, observational and experimental results suggest that differential heat tolerance among pollen color variants could contribute to geographic variation in pollen pigmentation.

Variation in pollen color among populations was concordant across two generations in a common environment, and parental pollen color was a strong predictor of offspring pollen color in C. americana. Both lines of evidence indicate that pollen color in C. americana has a heritable basis and thus has the potential to respond to natural selection. While the variation among populations in plants grown from field‐collected seed may be influenced by maternal environmental effects, the consistent pattern of population‐level variation in the F 1 generation confirms strong genetic structure to pollen coloration. The range‐wide narrow‐sense heritability estimate for pollen color is 0.57 (± 0.053 SD). In Nigella degenii, controlled crosses indicate that a single locus controls dimorphic pollen color, with dark pollen dominant over light pollen (Andersson & Jorgensen, 2005). However, to date heritability of pollen color has not been estimated for any taxa. While we binned pollen color for the purpose of this study, the variation in color in C. americana is continuous from white to deep purple. Therefore, the genetic control of pollen color is likely to be related to regulation of genes in the anthocyanin biosynthetic pathway (e.g. Streisfeld & Rausher, 2009). This heritable basis of pollen color suggests the potential for response to natural selection.

The evaluation of color phenotypes across a large number of populations representing a wide geographic range in C. americana revealed a strong longitudinal pattern, with darker pollen in the western portion of the range. For example, in two of the western‐most populations (MN 118 and KS 60), the proportions of deep‐purple pollen were 52% and 58%, respectively, while white pollen was not present in either population. Conversely, the easternmost populations (OH 64 and PA 27) did not have deep‐purple pollen, but had white‐pollen frequencies of 22% and 27%, respectively. At any given latitude, populations in the west of the range experience stronger UV irradiance and higher temperatures during the flowering season. After accounting for the effect of latitude, maximum temperature explained 44% of the average pollen‐color variation among populations (P = 0.0004), while residual UV explained 43% (P = 0.0003). Tests of the direct relationship between pollen color and both maximum temperature and UV show that temperature is the most likely abiotic factor to contribute to pollen‐color variation. Similar longitudinal but not latitudinal variation has been observed for petal pigmentation in Calceolaria uniflora (Mascó et al., 2004) and body color in Drosophila americana (Wittkopp et al., 2011). While the drivers are not completely understood in those systems, correlative results in C. americana suggest that pollen color may be locally adapted to temperature, which has been shown to elevate flavonoid production (Winkel‐Shirley, 2002).

Experimental data show that dark pollen is more resilient to heat stress than is light pollen. At high temperature (38°C), dark pollen displayed 85% greater germination than did light pollen, and only light pollen germination was reduced by high heat. The observed longitudinal cline in pollen color, and the direct relationship between the frequency of dark morphs and maximum temperatures are consistent with the hypothesis that selection favors dark pollen in western populations that experience higher summer temperatures. Studies have shown that high heat can reduce pollen germination (Koti, 2005; Prasad et al., 2006), but this is the first to show that the negative effects of high temperature can depend on pollen pigmentation. Interestingly, there is no advantage for light pollen under any temperature treatment, suggesting that its maintenance is unlikely as a result of selection by the thermal environment. Other factors such as pollinator preference or cost of flavonoid production in darker pollen could explain the maintenance of light pollen in natural populations. While Lau & Galloway (2004) showed that one pollinator type (halictid bees) preferred light pollen over the absence of pollen, explicit tests of the color preferences and effectiveness of common pollinators may help to explain the maintenance of light pollen.

The high temperature used in our growth chamber study (38°C) is c. 4°C higher than the highest maximum temperature in the field populations recorded in BIOCLIM. The BIOCLIM database integrates measurements made between 1960 and 2000, so we expect that contemporary field temperatures are even higher. Additionally, plants often occur in full sun, and incident radiation may heat them beyond air temperature, probably resulting in temperatures that reach our experimental treatment high of 38°C. Our approach evaluated the effect of pollen color within populations, sampling from populations in the east and west of C. americana’s range. Thus, the difference in pollen color performance does not reflect local adaptation at the whole‐plant level but some characteristic of the pollen itself. In C. americana there is weak correlation between petal and pollen color within and among populations (e.g. Fig. 1; M. Koski & L. Galloway, unpublished), so plant‐wide variation in pigmentation is unlikely to affect results. Finally, this experiment also confirmed that temperature can act as an agent of selection on pollen color directly, as the pollen‐bearing styles were removed from plants during exposure to the temperature treatments.

Elevated heat tolerance could be a product of increased anthocyanin content in darker pollen grains. In Campanulaceae, anthocyanins (e.g. delphinidin) underlie the coloration of blue‐purple petals (Buckles, 1975), and it is likely that anthocyanins underlie pollen coloration as well. The gradation of pollen color in C. americana could thus arise from differential regulation of the anthocyanin biosynthetic pathway. For example, white pollen morphs may have inactive or down‐regulated structural genes in the pathway, halting the production of purple pigments. Our data are in line with those showing that flavonoid pigments are important under heat stress. For example, nonpigmented mutants of Ipomoea purpurea have reduced male and female fertilization success compared with pigmented individuals when reared under high but not under low temperatures (Coberly & Rausher, 2003). Given the current results, an interesting next step would be to understand the regulation of the flavonoid biosynthetic pathway in C. americana. This could provide some insight into the mechanisms by which the array of pollen colors is achieved and how biochemistry may drive differential heat tolerance of the different color morphs.

While the presence of UV irradiance reduced pollen germination, geographic variation in UV is less likely than temperature to contribute to the longitudinal cline in pollen color. First, pollen pigmentation does not covary directly with UV irradiance. Second, both dark and light pollen germination were similarly reduced upon UV exposure. Our results join other studies confirming that UV exposure reduces pollen germination (Koti, 2005; Koski & Ashman, 2015). The UV environment created in growth chamber conditions was c. 35% lower than the average UV experienced in the field. Higher UV conditions or studies in the field could provide a more detailed profiling of the response of light and dark pollen to UV. An interesting next step in C. americana will be to test the interactive effects of elevated temperature and UV on pollen germination. In some soybean cultivars, elevated UV and temperature together reduce pollen germination relative to either treatment alone (Koti, 2005). Thus, while the growth chamber experiment in C. americana does not support the idea that longitudinal patterns in UV exposure contribute to clinal variation in pollen pigmentation alone, the effects of UV could be more pronounced when combined with high temperatures under field conditions.

If heat tolerance is the driver of pollen‐color variation, we would expect a latitudinal cline. Three factors could obscure this expected cline. First, population‐genetic structure may affect pollen‐color variation. However, C. americana's northward postglacial migration (Barnard‐Kubow et al., 2015) would be expected to result in a latitudinal gradient in color. There is also limited evidence of neutral genetic processes shaping petal or pollen‐color variation among populations in other systems (Jorgensen et al., 2006; Schemske & Bierzychudek, 2007; Hopkins et al., 2012). Second, pollinator preference has shaped the evolution of petal color (Rausher, 2008), and may similarly influence pollen color. However, work in Campanula and Nigella suggests that pollinators may not have strong, consistent pollen‐color preferences (Lau & Galloway, 2004; Jorgensen et al., 2006), and there are no geographic patterns in pollinator community composition in C. americana (Koski et al., 2017). Finally, geographic variation in flowering phenology could underlie the lack of a latitudinal cline in pollen color. Southern populations flower earlier (mid‐to‐late June) than northern populations (mid‐July; M. H. Koski, pers. obs.). Thus, at the time of peak flowering, disparities in temperatures between northern and southern populations may be less pronounced than suggested by season‐long data. Field studies of peak flowering times could elucidate whether sequential flowering from south to north results in latitude being a less important axis of temperature variation among populations than expected. Despite the lack of a latitudinal cline, the frequency of dark‐pollen morphs is elevated in populations that experience more extreme high temperatures, supporting the idea that the thermal environment plays a role in shaping geographic patterns of pollen‐color variation.

Global climate change is expected to influence plant reproduction in a variety of ways (Hedhly et al., 2009), and our work sheds light on how elevated temperatures differentially affect male fertility depending on pollen pigmentation. The extent to which intraspecific pollen‐color variation exists across flowering plants is difficult to gauge, as few studies focus on pollen coloration. Given the species with known intraspecific variation, however, the occurrence is likely to be taxonomically widespread (Lilliaceae, Thomson & Thomson, 1989; Ranunculaceae, Jorgensen & Andersson, 2005; Caryophyllaceae, E. Bruns, pers. comm.; Campanulaceae, Lau & Galloway, 2004; Poaceae, Coe et al., 1981; Linaceae, Wolfe, 2001). Our study provides support for thermal stress tolerance as a novel ecological role of pollen pigmentation which may contribute to geographic structuring of phenotypic variation in C. americana. Characterization of pollen‐color variation and experimental tests of the functional role of pollen color in other systems will help to elucidate the degree to which adaptation and neutral genetic processes contribute to pollen‐color diversity.