Plant P1. Syst. Evol. 177:81-91 (1991) Systematics and Evolution © Springer-Verlag 1991 Printed in Austria Floral scent and intrafloral scent differentiation in Moneses and Pyrola (Pyrolaceae) JETTE T. KNUDSEN and LARS TOLLSTEN Received January 23, 1991 Key words: Angiosperms, Pyrolaceae, Pyrola, Moneses. - Floral scent, intrafloral scent differentiation, orientation cues, poricidal anthers, pollen flowers, buzz pollination. Abstract: Floral scent was collected by headspace methods from intact flowers, petals, and stamens of four species of Pyrolaceae. The scent samples were analyzed by coupled gas chromatography-mass spectrometry (GC-MS). The floral scent in PyroIa spp. is differ- entiated into a characteristic petal scent-phenyl propanoids and a characteristic stamen scent-methoxy benzenes. In Moneses the scent is characterized by isoprenoids and ben- zenoids, with a larger proportion of benzenoids in the stamens compared to the petals. Specific anther scents may promote foraging efficiency in buzz-pollinated species and enhance flower fidelity. Variation in floral scent composition is consistent with the tax- onomic relationships among the genera and species examined. Most plants reproduce through pollination and the selective forces for improvement of a plants reproductive success, through enhanced pollination success, act strongly on floral characters (e.g., GRANT 1949, KEVAN 1984). GRANT (1949) found that 40 % of the taxonomic characters used to distinguish between closely related species of bee-pollinated plants pertained to floral parts as compared to 15% in promis- cuous, entomophilous plants, indicating that the flower constancy of bees has had a strong effect on the evolution of floral traits in bee-pollinated plants. Floral scents together with visual cues are important in attracting pollinators. Floral scents may function both at long distances as attraction cues to pollinators and at short distances as orientation cues among closely spaced flowers or between different parts within a single flower (e.g., PROCTOR & YEO 1973, FAEGRI & VAN DER PIJL 1979, WILLIAMS 1983, KEVAN 1984). Floral scents were not considered in GRANT'S (1949) review, but may provide additional, useful characters since bumble bees and honey bees, as well as many other flower visitors, can discriminate qualitatively and quantitatively between scents (e.g., VON FRISCH 1919, KUOLER 1932, LEX 1954, AUFSESS 1960). Scent variation between different parts within flowers and among different flowers within an inflorescence has been found (e.g., LEX 1954, PORSCH 1954, AUFSESS 1960, CAMPBELL & al. 1986), but chemically characterized in a few species only (e.g., NILSSON 1979, DOBSON & al. 1990). Variation in floral scents has only recently been used taxonomically to distinguish between closely related species of orchids (GREOO 1983, and review in WILLIAMS 82 J.T. KNUDSEN ~, L. TOLLSTEN: 1983). Differences in floral scents have also been recorded, e.g., among species of Rosa (Rosaceae, DOBSON & al. 1987), between different pollination morphs of Majorana syriaca L. (Lamiaceae, BEKER & al. 1989), Cimicifuga simplex WORMSK. ex DC. (Ranunculaceae, PELLMYR 1986, GROWTH & al. 1987), and Polemonium viscosum NUTT. (Polemoniaceae, GALEN & KEVAN 1983). However, in a study of seven taxa of Hypecoum (Papaveraceae)DAHL & al. (1990) found that floral scent composition only to a certain degree was correlated with morphological characters, and related this finding to the unspecialized pollination reported in the genus. We investigated the scent profiles of flowers and floral parts of Moneses uniflora (L.) A. GRAY, Pyrola rotundifolia L., P. norvegica G. KNABEN, and P. media Sw. (all Pyrolaceae). Our objectives were to: (1) Interpret interspecific and intrafloral scent differentiation in the context of pollination biology. (2) Evaluate floral scents as a possible taxonomic character. Material and methods The northern hemisphere genera Pyrola and Moneses have nectarless flowers offering large pollen rewards. The studied species have white petals contrasted by large, yellow, and poricidally dehiscent anthers. In a study of the pollination biology in Danish and Swedish populations, the flowers were found to be pollinated by queens and workers of Bombus spp., which harvested pollen by means of vibration (KNuDSEN & OLESEN, unpubl.). Scent samples of sympatric populations of Pyrola rotundifolia, P. media, and Moneses uniflora were collected in June-July 1988 and 1989 at lake Hornsj6n on the island of Oland in southeastern Sweden, and of P. norvegica in July 1989 near Abisko Scientific Research Station, northernmost Sweden. The samples were collected in the field by means of headspace adsorption: flowering ramets were enclosed in glass vessels and the air around the plants drawn through glass plugs containing 300+ 5 mg Porapak Q adsorbent (mesh size 50-80) with a battery- operated membrane pump. The airflow through the plugs was approximately 150 ml/min. Volatiles from P. rotundifolia, P. media, and P. norvegicawere collected for 12 h, and from M. uniflora for 24 h. For each specimen a blank sample was collected from a nearby empty glass vessel. In all Pyrola spp. scent samples were collected on individuals with five to 13 open flowers, and in M. uniflora from the single flower. Freshly cut-off petals and stamens of all four species were placed in separate containers in which the influent air was cleaned by passing through a glass plug with Porapak Q. The volatiles in the effluent air were trapped in another glass plug containing 200 • 5 mg Porapak Q. The air-flow through the glass containers was c. 150 ml/min. Volatiles from petals and stamens were collected for 6 h from five, ten or 25 petals and from 50 or 100 stamens from five or ten flowers. Compounds found in petals and/or stamens, but not in intact flowers, were excluded from this study, as it was not possible to judge if they originated from cell sap in the wound area or were parts of the actual floral scent profile. The plugs with adsorbed scent were eluted with 2 ml distilled pentane into glass vials, which were stored at - 18 °C until the samples were analyzed. Before analysis the samples were concentrated to 10 - 50 pl at ambient temperature (c. 20 °C). The samples were analyzed on a Varian 3400 Gas Chromatograph (GC) connected to a Finnigan Ion Trap Detector (MS). A fused silica GC-column was used; 25 m long, inner diameter 0.25 ram, coated with Superox FA as stationary phase. Injector temperature was 220 °C, and the GC was pro- grammed as follows: four minutes at 50 °C, increased by 8 °C per minute to 220 °C, then isothermal for 15 minutes. A 2111 sample was injected each time. Identification of mass spectra were made by comparison with computer library spectra and other published spectra, Floral scent in Pyrola and Moneses 83 and confirmed by comparison with spectra and GC-retention times of authentic reference compounds. Neutral red staining can be used as an indicator of scent producing tissue, as osmophoric tissue often contains lipids that are readily stained by neutral red (VOGEL 1963, STERN & al. 1986). Flowers of the above species were stained with neutral red in tap water solutions for 1 -5 h and then rinsed in tap water. Floral parts of M. uniflora were tested by blind-folded people for differences in floral scents. Basal, middle, and distal portions of petals, styles and stamens were put in separate glasses. After 10 h a blind test was conducted to find out which parts produced scent, and which part of the petal had the strongest scent. Results The fragrance of flowers and floral parts of Moneses un~ora was dominated by isoprenoid compounds. In intact flowers, 6-methyl-5-hepten-2-one and benzalde- hyde were found in larger relative amounts compared to the content in petals and stamens (Table 1 and Fig. 1). In intact flowers citronellol occurred in the largest amount in five samples, nerol in three, and geraniol in one sample. The petal scent originated almost exclusively from isoprenoids (99.3%), and citronellol was always found in the largest amount. Citronellol, too, was on average found in the largest amount in stamens, although the amount of methyl geranate was largest in three samples. Furthermore, methyl geranate was found in a 4-fold higher relative amount in the stamens than in the petals. The stamens too had an almost 100-fold higher relative amount of benzenoids than the petals, originating mainly from a higher total content of 4-methoxy methyl benzoate and methyl benzoate. All Pyrola spp. were characterized by benzenoids in the anthers and phenyl propanoids in the petals, especially methoxy benzenes and derivatives of cinnamic acid were common (Table 1 and Fig. 1). In P. rotundifolia the compound found in the largest amount was 1,4-dimethoxy benzene in all samples of intact flowers and stamens, whereas in petals it was cinnamic alcohol. In P. norvegica benzaldehyde and cinnamic alcohol always were the largest compounds found in intact flowers and petals, respectively. In stamens 1,4-dime- thoxy benzene on average was the largest compound, although 1,3,5-trimethoxy benzene was largest in one sample. In intact flowers of P. media 1,4-dimethoxy benzene and benzaldehyde occurred each in the largest amount in three samples. In the petals cinnamic aldehyde and in the stamens 1,4-dimethoxy benzene on average was the largest compound, al- though benzaldehyde was largest in one sample of petals and stamens, respectively. In the genus Pyrola, phenylacetaldehyde and benzyl alcohol were exclusively found in P. rotundifolia, and 3-phenyl propanol acetate exclusively in P. norvegica. The two trimethoxy benzenes and cinnamic acetate were missing in P. media, as was 6-methyl-5-hepten-2-one in P. norvegica. Benzaldehyde was always the ben- zenoid apart from methoxy benzenes found in the largest amount in intact flowers, whereas in petals and stamens benzaldehyde occurred only in small amounts except in P.