This study was conducted during 2001 at montane and subalpine sites in the Kananaskis Valley, 60 km west of Calgary, Alberta, Canada. Four legume species, Hedysarum boreale Nutt. var. mackenzii (Richards.) C. L. Hichc., Hedysarum sulphurescens Rhdb., Oxytropis sericea Nutt. and Astragalus striatus Nutt. were studied at an open, montane grassland near Barrier Lake (51°02′ N, 115°02′ W, 1480 m elevation). Astragalus americanus (Hook.) M. E. Jones was studied at a forest edge (at 1800 m elevation), and Oxytropis splendens Dougl. ex Hook. at an open meadow (at 1880 m elevation) in the subalpine near Ribbon Creek (50°56′ N, 115°09′ W).
We measured reproductive phenology and natural seed production for three inflorescences on each of 30 plants of H. boreale (five inflorescences), H. sulphurescens and O. sericea, and 25 plants of A. striatus, A. americanus and O. splendens, which were selected randomly within a c. 20 × 20-m2 area at each site at the beginning of the flowering season and marked with numbered tags. The numbers of floral buds, open flowers (i.e. without wilted petals) and developing fruits of each inflorescence were recorded at 1–3 day intervals throughout the flowering and fruiting periods. Once fruit matured, they were harvested, and the number of mature seeds was counted for each fruit.
In addition to measuring floral phenology, we estimated average floral longevity (L) for each species based on the average number of flowers produced per inflorescence (N), the average duration of flowering by an inflorescence (T) and the average display size (D). If flowers open at a fixed rate, r, then the duration of flowering by an inflorescence equals the time required for all flowers to open (N/r) plus the longevity of the last flower to open, so that T = N/r + L. We did not measure the rate of flower opening, but it can be estimated from the average display size. In particular, if flowers open at a fixed rate, then display size will equilibrate at D = rL during peak flowering, so that r = D/L. Therefore, floral longevity can be estimated by L =T/[1 + (N/D)].
Self-compatibility and the ability of flowers to set seed autonomously were assessed for 20 plants of each species (except 10 plants for O. splendens). Two inflorescences on each plant were bagged with fine mesh before flowering. Flowers that opened subsequently on one inflorescence were hand self-pollinated, whereas the other inflorescence was not disturbed. Mature fruits were harvested and seeds were counted for each fruit.
During flowering we measured aspects of floral morphology, ovule number and nectar production. Floral-tube length and dry mass (after 48 h at 60 °C) were measured for one flower sampled from each of 42–50 randomly selected plants of each species. At the same time, we counted ovules from three to five flowers on 20 randomly sampled inflorescences. To measure floral nectar we caged 25 (O. sericea) or 20 (other species) randomly selected plants on sunny, calm days between 700 and 800 h before bumble-bees became active to exclude visits. During the subsequent hour we measured nectar volume (V: µl) and sugar concentration (C: mass of solute/mass of solution) for three (H. boreale), four (A. striatus) or five (other species) flowers with calibrated capillary tubes and a Bellingham and Stanley refractometer (Tunbridge Wells, UK). Therefore, our measure of nectar availability estimates the reward that pollinators encountered during a flower's first visit of the day. We calculated the sugar content of nectar (mg) as ρCV, where ρ is the density of a sucrose solution with concentration C (CRC 2000).
We consider two measures of pollen removal: removal during a single bumble-bee visit, and total removal during the first 24 h after anthesis. For both measures, pollen removal was calculated from pollen counts for visited or exposed flowers (Pv) and unvisited flowers (or flower buds, P0) within the same inflorescence as (P0 – Pv)/P0. Pollen removal per visit was measured for inflorescences on which we removed previously open flowers and then caged the inflorescence to exclude pollinator visits until new flowers opened. On calm days, inflorescences with more than three open flowers were exposed to bumble-bee visits. After a bee visited an inflorescence, we removed unvisited flowers (or fully developed flower buds) and flowers that received a single visit and stored them separately in vials containing 70% ethanol. Fourteen inflorescences were replicated for each species (seven inflorescences for O. splendens). Pollen removal during 24 h was measured on consecutive sunny, calm days during peak flowering. During early morning, we recorded the positions of three randomly selected flowers on 20 previously bagged inflorescences. Twenty-four hours later, we stored the anthers and any pollen washed from the keel petals of these flowers in vials containing 70% ethanol. Anthers from the three flowers from an inflorescence were stored together, because too few pollen grains remained in individual flowers to allow electronic counting. At the same time, we also collected fully developed flower buds from the selected inflorescences for measurement of pollen production. Pollen was counted electronically with an Elzone 5380 particle analyser (Micromeritics, Norcross, GA) after the vials containing pollen had been sonicated for 15 min to dislodge pollen from the anthers (see Harder 1990a for details).
As a measure of pollinator activity, we considered the ratio of pollen removal during 24 h to pollen removal per visit. If pollinators remove roughly the same number of pollen grains per visit, at least for the first few visits, then this ratio represents the mean number of pollen-removing visits during a flower's first day.
We recorded the number of flowers visited per inflorescence on three to ten consecutive inflorescences by 31–49 bumble-bees for each plant species (20 bees for O. splendens). Bumble-bee visitation was observed during 2 or 3 calm days (mainly from 10.00 to 15.00 hours) during peak flowering for each species.
Our comparison of the reproductive ecology of these six legume species involved two perspectives: species comparisons with individual plants as the unit of replication, and tests of association between species means. Most species comparisons involved single-factor anova (Neter et al. 1996), with species as a fixed factor (SAS release 8·2, proc glm; SAS Institute Inc. 2001). For variables that were measured from several flowers per plant, or for several inflorescences per bee, we used the plant or bee mean as an individual observation. The analysis of total seed production by plants from which pollinators had been excluded involved a two-factor, repeated-measures anova (SAS, proc mixed), with species as a fixed, between-plant factor and pollination treatment (autonomous vs hand self-pollination) as a fixed, within-plant factor. This analysis used restricted maximum likelihood (Jennrich & Schluchter 1986) to characterize the covariance between responses to different pollination treatments by individual plants. A model of heterogeneous compound symmetry was more appropriate than one of independent responses (G3 = 20·31, P < 0·001). Denominator degrees of freedom for F-tests of this analysis were calculated by Kenward & Roger's (1997) technique. We used Tukey's multiple comparisons to distinguish species and/or treatment means that differed significantly (α = 0·05: Neter et al. 1996).
We analysed associations between species means with either Spearman's correlation or linear regression (Neter et al. 1996). Correlation analysis was used for floral traits for which there was no obvious causal relation between variables. Regression analysis was used to analyse response variables (nectar-sugar production, pollinator visitation, pollen removal, fruit and seed set) and employed a stepwise procedure to select independent variables that explained significant proportions of the variation in the dependent variable. Variables were suitably transformed to assure a linear relation between dependent and independent variables. Only the analysis of the mean ratio of pollen removal during 24 h to removal during a flower's first visit (A) found more than one independent variable to influence the dependent variable significantly (see below). To illustrate the independent influences of both variables, we present adjusted values of the removal ratio. Adjustment involved adding the residual (Ai − Âi) for each observation to the ratio predicted by the regression equation for the observed value of one independent variable and the overall mean of the other independent variable.