Partition of fragrance between calyx and corolla in Polemonium foliosissimum.
Many flowering plants have evolved chemicals attractive to pollinators (e.g., Dodson et al., 1969; Dodson, 1975; Barth, 1991) and to protect against insect herbivory and grazing animals (reviews in Green and Hedin, 1986; Smith, 1989). Polemonium viscosum (Polemoniaceae) has two fragrance morphs that are distributed according to pollinator availability (Galen and Kevan, 1980). The skunky fragrance morph of P. viscosum attracts flies, the prevalent pollinators at lower elevations (Galen and Newport, 1988), whereas the sweet fragrance morph, at higher elevations, attracts bees (Galen and Newport, 1987).
Casual field observations had suggested that Polemonium foliosissimum (Polemoniaceae), a subalpine perennial common in the central Rocky Mountains, produced a variety of fragrance volatiles similar to those found in P. viscosum. As a preliminary investigation in a program to characterize the volatiles and to determine whether they function in plant-insect interactions, we studied the distribution of volatiles on the reproductive structures of Polemonium foliosissimum. In this paper we present evidence that P. foliosissimum produces different concentrations of volatiles in the corolla (petals) than in the calyx (sepals) and that the flower offers a succession of fragrances during the reproductive cycle. Such a chemical adaptation opens several potential channels of communication between plant and insects.
Morphology and ecology. - The flowers of Polemonium foliosissimum are funnel-shaped, hypogynous and protandrous, with five blue petals and five contrasting yellow anthers. The petals reflect moderately in the ultraviolet; a UV "target" pattern in the throat of the corolla and a bright central UV reflectance may act as nectar guides (unpubl. data). The sepals and pedicel are covered with glandular trichomes which secrete a clear sticky fluid that tastes somewhat like pine tar. It is common to find small insects, such as aphids, Aedes mosquitoes and Empid flies, stuck in the secretions. The fragrances characteristic of P. foliosissimum are produced by the reproductive structures and are evident from earliest bud formation through fruit set. Foliage, both intact and crushed, has a leafy odor characteristic of green plants (pers. observ.).
Pollinator visitors to Polemonium foliosissimum include bumblebees (especially Bombus flavifrons and B. bifarius) and other solitary bees [especially the Western leafcutter bee (Megachile perihirta) and andrenids (Andrena salicifloris)]. Rove beetles (Staphylinidae sp.) are often found on P. foliosissimum corollae. Pests include green aphids and anthomyiid flies (Hylemya sp. and an unidentified large anthomyiid). Aphid infestations reduce seed set (pers. observ.), and an Hylemya larva destroys the entire seed production in the ovary where it matures.
We studied two populations of Polemonium foliosissimum in the West Elk Mountains and Ruby Range N of Gunnison, Colorado. The population at lower elevation (38 [degrees] 47 [minutes]N, 107 [degrees] 06[minutes]W, 2800 m elev.), hereafter referred to as the Swampy population, comprised plants in an open meadow surrounded by aspen (Populus tremuloides). The second study population occupied an open, S-facing slope on Ruby Mountain (38 [degrees] 53[minutes]N, 107 [degrees] 07[minutes]W, 3300 m elev.) near Lake Irwin (hereafter referred to as the Irwin population).
Fragrance at different stages of the reproductive cycle. - In order to evaluate the fragrance of floral structures during the growth cycle, we flagged cluster samples including 60 plants at Swampy and 47 plants at Irwin in June 1992. We judged bud fragrance, and, as the flowers developed, fragrance of blossoms and fruits on each of the flagged plants. (At Irwin, BD alone evaluated fruit fragrance.) Repeatability of odor determination was tested by evaluating blossom fragrance at Irwin twice, on different days. On the basis of our experience, we assigned one of four fragrances: "neutral" (no discernible odor), "sweet" [pleasant floral odor, similar to sweet clover (Melilotus)], "pungent" (similar to the western sagebrush, Seriphidium vaseyanum), or "skunk." To test whether different flowers on the same plant had similar fragrance, we smelled flowers on all panicles of each plant, and we blindfolded ourselves to records of odor determinations made on previous visits to the same plant. A Kruskal-Wallis test was performed for statistical comparison of fragrance at different stages of development using values neutral = 0, sweet = 1, pungent = 2, skunk = 3.
Determining the source of floral fragrance. - In order to determine which of the reproductive structures produced the fragrance volatiles, we selected two plants with typical floral fragrance, dissected 10 flowers from each, and sealed gynoeciae, androeciae, petals and sepals in separate odor-free film cannisters. After incubating the cannisters for 2 min in sunlight, we judged the fragrance of the contents. Then, in July (Swampy) and August (Irwin) 1993 we harvested five flowers from each plant in a cluster sample (n = 57 at Swampy, n = 30 at Irwin), separated corollae from calyces, and sealed the corollae in one odor-free film cannister, the calyces in another. Androeciae generally accompanied the corollae, and gynoeciae the calyces. We left the cannisters in sunlight for 2 min, then judged the fragrances. A Mann-Whitney test was performed for statistical comparison of the fragrances of corollae and calyces using values for fragrances as above.
Gas chromatography. - To compare the chemical volatile composition of corollae and calyces, volatile components were collected by headspace sampling (air flow over specimens into a volatile adsorbent, see, e.g., Hills and Schutzman, 1990, and below), and the samples were analyzed by gas chromatography (GC), using the following procedure. One hundred corollae pooled from flowers of different plants at peak bloom were sealed in one 250-ml elution flask, and calyces from the same flowers were sealed in a second 250-ml elution flask. The flasks were transported at ambient temperature to the laboratory (1 h transport time); 1 [[micro]liter] of geraniol was added to each flask to serve as an internal standard. Room air was suctioned for 6 h through the headspace collection system comprising, in series, an activated charcoal filter, the flask containing the specimen, and. a volatile trap containing Tenax GC and activated charcoal (see Hills and Schutzman, 1990, for general procedure). After headspace collection, traps were eluted with HPLC grade pentane. The first 1 ml of eluent was collected, and 0.2 ml of the eluent was evaporated at room temperature in room air to a volume of 0.01 ml in order to concentrate volatiles in the sample with boiling points higher than pentane. 3 [[micro]liter] of concentrate was injected into a 30 m SPB1 capillary column at a starting temperature of 40 C. Column temperature was held at 40 C for 4 min, then ramped to 200 C at 10 C/min, then held at 200 C for 5 min. Helium at 20 ml/min served as the carrier gas, and [N.sub.2] makeup at 40 ml/min swept the sample through an FID detector. Injector and detector temperatures were 250 C. Chart speed was 1 cm/min. Controls included blank runs of solvent, samples without the geraniol standard, and standards of some known floral volatiles, including alpha and beta pinene, limonene, cineole and geraniol. GC peak areas from calyces and corollae were standardized by calculating the ratio of those areas to the area of the geraniol peak.
Correlating fragrance with GC peaks. - GC peaks were correlated with components of the fragrance bouquet by timing the arrival of various fragrances at the GC detector, observer's (BD's) nose at the detector outlet. Since the GC column lacked a dual port at the detector, fragrance arrival times were matched against peaks on a chromatogram of the same specimen obtained on a separate run.
Fragrance at different stages of the reproductive cycle. - There was a similar pattern of fragrances in both populations over the reproductive cycle: most buds were pungent or skunky, most blossoms were sweet, and fruits reverted to a pungent or skunky fragrance [ILLUSTRATION FOR FIGURE 1 OMITTED]. It should be noted that during the season five plants at Swampy and one at Irwin were lost because of browsing, probably by mule deer (Odocoeleus hemionus).
Repeat fragrance test at Irwin showed 96% correspondence. In the Swampy population, there was significant variation between buds, blossoms and fruits (P [less than] 0.001; average ranks: buds 113.5 (n = 63), blossoms 60.0 (n = 62), fruits 101.5 (n = 57), compared to overall average 91.5; Kruskal-Wallis test). In the Irwin population, also, there was significant variation in fragrance at the different stages of the life cycle (P [less than] 0.001; average ranks: buds 94.1 (n = 48), blossoms 32.4 (n = 47), fruits 87.6 (n = 47), compared to overall average 71.5; Kruskal-Wallis test).
Source of fragrances. - In the plants selected for complete floral dissection, gynoeciae and androeciae both had a faint odor, reminiscent of pumpkin flesh, which was distinct in quality and amplitude from the stronger fragrances of petals and sepals; petals smelled sweet and sepals pungent. There was a significant difference in fragrance between calyces and corollae in the wider samples of both study populations (P [less than] 0.001 in both populations; Mann-Whitney test; two-tailed). The corollae were usually sweet, and the calyces were skunky or pungent (Table 1).
Gas chromatography. - Gas chromatograms revealed peaks characteristic of the calyces and other peaks with greater areas in the corollae than in the calyces [ILLUSTRATION FOR FIGURE 2 OMITTED]. Standardized areas of calyx peaks 2, 3, 4, 6 and 19 exceeded the standardized areas of the respective corolla peaks by more than 10:1, and standardized calyx peak 20 area exceeded corolla peak 20 area by 5:1 (Table 2). Peak 3 was identified as alpha pinene by comparison with alpha pinene standards run on the GC and by GC mass spectrometry (data not presented). Peak 4 was similarly identified as beta pinene. Peak 8 was found in the corolla sample but not in the calyces. Its area was quite small, but it was of interest because of possible correlation with the sweet fragrance characteristic of the petals (see below). Corolla peaks 5, 11, 12 and 13 had standardized peak areas more than twice those found in the respective calyceal peaks.
TABLE 1. - Results of fragrance determinations for corollae and calcyes in each population. Tables indicate number of plants in each population and proportion of plants in each population having corollae and calyces of a given fragrance. N = neutral; SW = sweet; P = pungent; SK = skunk. The data were obtained during the summer of 1993. (A) Swampy population, N = 57 plants. (B) Irwin population, N = 30 plants
Table 1A Swampy
Structure N SW P SK Total
corollae 2 50 5 0 57 0.04 0.88 0.09 0.00 calyces 0 0 4 53 57 0.00 0.00 0.07 0.93
Table 1B Irwin
Structure N SW P SK Total
corollae 0 30 0 0 30 0.00 1.00 0.00 0.00 calyces 0 0 7 23 30 0.00 0.00 0.23 0.77
Correlation of fragrance with GC peaks. - There were a number of distinct fragrances separated by the GC column [ILLUSTRATION FOR FIGURE 2 OMITTED]. Of particular note, the skunk odor was detected in the sample of calyces but not in the corolla sample, whereas the sweet fragrance was detected in the corolla sample, but not in the calyces. Other fragrances were common to both corolla and calyx. Identifying the skunk and sweet fragrances with particular peaks is problematic: the skunk odor may be associated with peak 1 or a nearby smaller peak, whereas the sweet fragrance may be associated with peak 8 or a nearby peak.
Various data from field observations, gas chromatography and odor of fragrances at the GC column outlet all indicate that the calyces and corollae of Polemonium foliosissimum synthesize different concentrations of certain volatiles, and the partitioning of volatiles between the corollae and calyces results in a succession of fragrances during the reproductive cycle. During the bud and fruit stages the flowers are covered by sepals, hence smell pungent or skunky. During the bloom the petals are exposed and release their sweet floral volatiles.
The greater proportion of sweet corollae at Irwin than at Swampy may reflect a pollinator-attracting mechanism similar to that described for Polemonium viscosum (Galen and Kevan, 1980; Galen and Newport, 1987, 1988); i.e., the population at higher elevation may attract bees, while the population at lower elevation may include more individuals with fragrance relatively attractive to flies. It is possible, however, that contamination of some of the cannister specimens affected the fragrance determinations: many of the calyces were exceptionally skunky in odor and the sticky exudate from their trichomes clung to fingers. Although we took precautions to avoid contamination, skunky or pungent volatiles may have been transferred to some of the corollae when the flowers were dissected, masking the sweet floral scent of those corollae. Similarly, in field evaluation of odor succession, high concentrations of pungent and skunky volatiles from the calyces may have overwhelmed relatively faint sweet volatiles in some flowers, giving an overall impression that the blossom itself was pungent or skunky.
TABLE 2. - GC Peak areas for corollae and calyces collected at Irwin in August 1994. Peak numbers refer to peaks indicated on Figure 2. Pet/Ger = ratio of petal peak area to area of geraniol standard (peak 17). Sep/Ger = ratio of sepal peak area to area of geraniol standard
Peak Petal area Sepal area Pet/Ger Sep/Ger
1 100 663 0.001 0.002 2 100 4371 0.001 0.011 3 608 26,098 0.004 0.065 4 1385 67,023 0.009 0.168 5 1361 1044 0.009 0.003 6 100 6059 0.001 0.015 7 860 1417 0.006 0.004 8 380 120 0.002 0.000 9 806 2169 0.005 0.005 10 100 2896 0.001 0.007
11 892 794 0.006 0.002 12 1772 2038 0.012 0.005 13 1787 2592 0.012 0.006 14 2291 5869 0.015 0.015 15 6185 10,822 0.040 0.027 16 9793 18,186 0.064 0.045 17 153,727 399,907 1.000 1.000 18 910 1180 0.006 0.003 19 1316 39,878 0.009 0.100 20 869 13,542 0.006 0.034 21 64,392 125,129 0.419 0.313
On the basis of evidence from fragrances separated by the GC column, the field fragrance of the corolla probably results from a bouquet, including fruity fragrances, a rose fragrance, and, especially, a strong sweet fragrance subjectively similar to the fragrance of sweet clover (Melilotis). The pungent fragrance of some calyces probably results from a bouquet including alpha pinene, beta pinene, and other terpenes. We suspect that a thiol produces the skunk odor.
Our findings are similar to those reported by Dobson et al. (1990), who found a partitioning of volatiles between floral structures in Rosa rugosa. In R. rugosa, petal volatiles are dominated by terpenoid and benzenoid alcohols, sepal volatiles are dominated by sesquiterpenes, and pollen and anther volatiles are dominated by fatty acid derivatives.
The distribution of volatile compounds in calyx and corolla and the succession of odors during the reproductive cycle may help protect the flower from herbivores, whether insects or grazing animals, during the bud and fruit stages, while attracting efficient pollinators, such as bumblebees and other solitary bees, during the bloom. Alternatively, other insects, such as Hylemya sp., which oviposit in developing buds, may exploit the volatiles to identify plants at the proper stage of development for oviposition. Some terpenes, including alpha and beta pinene (present in highest concentrations on Polemonium foliosissimum calyces); are toxic to certain insect pests (see Raffa, 1986, and Gregory et al., 1986, for examples). However, terpenes play such a variety of roles in insect/plant interactions that one cannot generalize. A. K. Brody (pers. comm.) finds no evident protection in P. foliosissimum against Hylemya.
Acknowledgments. - The authors thank Rick Jagger in the Chemistry Department at Western State College, Jim Belliveau, Department of Chemistry, Providence College, and Don Dick in the Analytical Laboratory at Colorado State University for lab facilities and assistance. Thanks to Alison Brody and Mac Given for insightful suggestions and comments on the manuscript. This research was supported, in part, by a CAFR grant from Providence College.
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|Author:||Dorsett, Bob; Pike, Alger|
|Publication:||The American Midland Naturalist|
|Date:||Oct 1, 1995|
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