gregata (0.4%). On stigmas of A. columbianum they were. Mertensia ciliata (9.6%), Ipomopsis aggregata (1%), and. Asteraceae (0.3%). Pollen of M. ciliata was ...
American Journal of Botany 86(6): 871–879. 1999.
EFFECTS
OF LOCAL DENSITY ON POLLINATION AND
DELPHINIUM NUTTALLIANUM AND ACONITUM COLUMBIANUM (RANUNCULACEAE)1
REPRODUCTION IN
MARIA BOSCH
AND
NICKOLAS M. WASER2
Department of Biology, University of California, Riverside, California 92521; and Rocky Mountain Biological Laboratory, Crested Butte, Colorado 81224 Plant populations vary in density both naturally and as a consequence of anthropogenic impacts. Density in turn can influence pollination by animals. For example, plants in dense populations might enjoy more frequent visitation if pollinators forage most efficiently in such populations. We explored effects of plant density on pollination and seed set in the larkspur Delphinium nuttallianum and monkshood Aconitum columbianum. At our site in the Colorado Rocky Mountains, flowers of D. nuttallianum are pollinated primarily by queen bumble bees, solitary bees, and hummingbirds, whereas those of A. columbianum are pollinated primarily by queen and worker bumble bees. We found that the quantity of pollination service to both species (pollinator visitation rate and pollen deposition) was at best weakly related to density. In contrast, seed set declined by approximately one-third in sparse populations relative to nearby dense populations. This decline may stem from the receipt of low-quality pollen, for example, inbred pollen. Alternatively, sparsity may indicate poor environmental conditions that lower seed set for reasons unrelated to pollination. Our results demonstrate the value of simultaneously exploring pollinator behavior, pollen receipt, and seed set in attempting to understand how the population context influences plant reproductive success. Key words: seed set.
Aconitum; Bombus; conservation biology; Delphinium; density; hummingbird; pollination; Ranunculaceae;
Natural populations are characterized by a number of ecological attributes that influence the intraspecific and interspecific interactions of their members and thus the dynamics of the population itself. Among these attributes are size and age structure, sex ratio (or ratio of sex expression), number of individuals in the population (‘‘population size’’), isolation of the population from others, and population density. All of these attributes can vary dramatically from population to population, due both to natural causes and increasingly to anthropogenic influences. An example of the latter is massive anthropogenic change in land use, in particular the division of continuous natural habitats into disjunct fragments (Vitousek, 1994). Pollination by animals is one of the important interspecific interactions that we can expect to vary with the population context of each given plant and flower. This is because the animals visit flowers while foraging for resources, because they tend to forage in ways that are efficient economically, and because efficiency will be influenced by attributes of the plant population (Pyke, 1984; Ingvarsson and Lundberg, 1995; Waser et al., 1996; Waser and Price, 1998, and references therein). Indeed, a growing number of studies examines how pollination varies with plant size and size structure (Thomson, 1988; Campbell and Waser, 1989; Klinkhamer, de Jong, and de Bruyn, 1989), sex ratio (Brunet and
Charlesworth, 1995; Cunningham, 1995; Groom, 1998), population size (Sih and Baltus, 1987; Krannitz and Maun, 1991; Byers, 1995; Groom, 1998), isolation (Aker, 1982; Sih and Baltus, 1987; Klinkhamer, de Jong, and de Bruyn, 1989; Kwak et al., 1991; Groom, 1998), and density (Thomson, 1981; Schmitt, 1983; Kwak, 1987; Lyons and Mully, 1992; Van Treuren et al., 1993; Dreisig, 1995). In this paper we report on pollination service and female reproductive success in two herbaceous perennial plant species, Delphinium nuttallianum and Aconitum columbianum. We chose population density as a focus, based on Kunin’s (1997a, b) conclusion that density often has strong effects on pollination, and because density is one attribute of populations likely to be affected by anthropogenic change such as habitat fragmentation (Bierregaard et al., 1992). In pairs of plots characterized by differing densities of conspecific plants, we recorded the identity and visitation rate of pollinators, the numbers and identity of pollen grains deposited on stigmas, and the numbers and proportions of ovules matured into seeds. In both species we detected only slight effects of density on pollinator visitation and pollen receipt, but strong effects on seed set. MATERIALS AND METHODS Plant species—Delphinium nuttallianum Pritz. (5 D. nelsonii Greene; Weber and Wittmann, 1996) and Aconitum columbianum Nutt., respectively Nelson’s larkspur and monkshood (Ranunculaceae), are montane herbaceous perennials with widespread distributions in the western United States. The two species are close taxonomically as members of tribe Delphineae (Bosch et al., 1997). Both species produce deep blue to purple complex, zygomorphic, protandrous flowers on a racemous inflorescence. Delphinium nuttallianum is smaller (10–25 cm inflorescence height) than A. columbianum (30–150 cm) and produces
1 Manuscript received 27 May 1998; revision accepted 5 November 1998 The authors thank P. Aigner and C. Koehler for field assistance and S. Cunningham, C. Koehler, G. Muenchow, M. Price, J. Smith, and C. F. Williams for comments. Support for MB was provided through a postdoctoral FPI fellowship from the Ministerio de Educacio´n y Cultura, Spain. 2 Author for correspondence.
871
872
AMERICAN JOURNAL
¯ 6 SD 5 3.8 6 2.1, N 5 644 fewer flowers (1–14 flower buds/plant, X ¯ 6 SD 5 9.5 6 7.4, N 5 450 plants). At plants; vs. 1–54 buds/plant, X our study site at the Rocky Mountain Biological Laboratory (RMBL) in west-central Colorado, D. nuttallianum usually flowers from late May to early July and A. columbianum usually flowers from early July to late August. The major pollinators of D. nuttallianum at the RMBL are queen bumble bees and hummingbirds; solitary bees and other insects also visit (Waser, 1978, 1982; Waser and Price, 1990). The major pollinators of A. columbianum are queen and worker bumble bees, although other insects and hummingbirds are sometimes seen (Watt, Hoch, and Mills, 1974; Brink, 1980; Pleasants and Zimmerman, 1990; Graham and Jones, 1996). Seed set in the plants depends on these animals. Thus in A. columbianum, seed set in flowers bagged to exclude all visitors is ¯ 6 SD 5 0.1 6 0.67 seeds, corresponding on average to nearly zero (X 0.4% of ovules set, N 5 92 flowers) and is dramatically and significantly ¯ 6 SD 5 30.6 6 9.25, below seed set in open-pollinated flowers (X 76.7%, N 5 99 flowers; two-tailed t 5 231.9, P , 0.001). Waser (1978) reported a similar result for D. nuttallianum. Plants of D. nuttallianum around the RMBL are known to be partially self-compatible. Three hand-pollination experiments conducted over 2 yr showed that flowers set ; 63% as many seeds on average when selfed as when outcrossed over a distance of 10 m (Price and Waser, 1979; Waser and Price, 1991). Even though flowers are protandous, therefore, some production of selfed seed is likely by transfer of pollen among flowers of the same plant (geitonogamy). Bosch (1999) found seed set after selfing that averaged 72% of seed set after outcrossing (range 19–117%) for 11 western Mediterranean species of Delphinium and Consolida, and Darwin (1876) reported a value of 59% for Delphinium consolida (5 Consolida regalis), suggesting that partial selfcompatibility is widespread in tribe Delphineae. By extension this suggests partial self-compatibility in A. columbianum, although we are unaware of direct experimental evidence for this species. Study site—We studied both species during the summer of 1997 at the RMBL, (2900 m elevation). In this area D. nuttallianum is found in habitats ranging from open dry subalpine meadows and open rocky slopes to more shaded areas at the boundary of aspen woodlands. Common plants flowering with D. nuttallianum include Androsace septentrionalis, Arabis drummondii, Castilleja miniata, Claytonia lanceolata, Erythronium grandiflorum, Hydrophyllum capitatum, H. fendleri, Lathyrus leucanthus, Lupinus argenteus, Mertensia fusiformis, Potentilla pulcherrima, Ribes cereum, R. montigenum, Sambucus racemosa, Senecio intergerrimus, Thalictrum fendleri, Taraxacum officinale, and Viola nuttallii. In contrast, A. columbianum is rarer at the RMBL and prefers more mesic meadows. It flowers in association with Anticlea elegans, Castilleja sulphurea, Delphinium barbeyi, Frasera speciosa, Geranium richardsonii, Helenium hoopsii, Helianthella quinquinervis, Heracleum lanatum, Linum lewisii, Lonicera involucrata, Mertsensia ciliata, Potentilla pulcherrima, and Veratrum tenuipetalum. Study design—The goal of the study was to determine whether natural variation in local density of conspecific plants influences pollination and reproduction. To this end we chose five separate sites at the RMBL representing the range of habitats for D. nuttallianum. Within each site we chose two areas separated by 10–40 m, one in which plants were as dense as could be found and one in which they were as sparse as could be found. Composition of the rest of the flora was qualitatively similar in paired sparse and dense areas. We established a 2 by 4 m study plot in each of these areas and counted all plants and flowers in each plot. ‘‘Dense’’ plots averaged 14.2 plants/m2 (range 7.1–16.2, SD 5 2.5, N 5 5 plots) and ‘‘sparse’’ plots averaged 1.7 plants/m2 (range 0.6–4.5, SD 5 1.6, N 5 5). The mean total number of flowers produced by plants in dense plots was slightly greater than in sparse plots, but ¯ 5 9.5, SD 5 4.0, N 5 37 plants; sparse: not significantly so (dense: X ¯ 5 8.1, SD 5 3.2, N 5 37; t 5 1.82, P 5 0.08). Within each plot we X
OF
BOTANY
[Vol. 86
also chose 7–9 focal plants, for a total of 74 focal plants across all plots, and counted all plants and flowers within a 3 m radius of each focal plant. Within the neighborhoods of focal plants, density expressed as flowers per square metre was highly correlated with density expressed as number of plants per square metre (r 5 0.95, N 5 74 focal plants, P , 0.001). In analogous fashion we chose three separate sites representing habitats of A. columbianum, established dense and sparse study plots separated by 5–15 m at each site, and counted plants and flowers. Once again, composition of the rest of the flora was similar in paired sparse and dense plots. ‘‘Dense’’ plots averaged 9.1. plants/m2 (range 7.0–10.2, SD 5 1.5, N 5 3 plots) and ‘‘sparse’’ plots averaged 1.5 plants/m2 (range 1.0–2.0, SD 5 0.5, N 5 3). Once again the mean total number of flowers produced by plants in dense plots was slightly greater than ¯ 5 21.6, SD 5 10.4, in sparse plots, but not significantly so (dense: X ¯ 5 19.3, SD 5 9.3, N 5 27; t 5 0.81, P 5 0.42). N 5 27; sparse: X Within each plot we again chose 7–9 focal plants, for a total of 54 focal plants, and counted all plants and flowers within a 3 m radius. Densities expressed as flowers per square metre and as plants per square metre were again highly correlated (r 5 0.98, N 5 54, P , 0.001). We censused pollinators in the ten D. nuttallianum study plots during a series of 30-min observations spread evenly between 12 June and 1 July 1997. Observations of the pair of plots at a given site were made simultaneously in most cases by two observers. In all we observed each plot for nine observation periods, i.e., for 4.5 h, and over all ten plots we therefore observed for a total of 45 h. In each census we recorded the species identity of each pollinator, number of approaches to a plot, and number of plants and flowers visited per plot. Censuses for the six plots of A. columbianum at three sites were made in the same fashion between 21 July and 25 August 1997. We observed each plot for ten observation periods, i.e., for 5 h; and over all plots we observed for a total of 30 h, recording information on pollinators and their behavior as before. On each of the 74 focal plants of D. nuttallianum and 54 of A. columbianum mentioned above, we chose 2–5 individual flowers for measurements of pollen load and seed set. To minimize variation introduced by the timing of flower opening during the season, we always chose the earliest flower to open on the main inflorescence stem (the most basal flower), and the flowers most closely adjacent to it. We removed stigmas from these flowers 24–48 h after they had shed sepals, leaving the rest of the gynoecium intact. Sepals become brown and shrivelled over 1–2 d before being shed, and pollinators ignore such senescent flowers (personal observations). Furthermore, a delay of 26 h between pollination and removal of stigmas suffices for all pollen to germinate and fertilize ovules in D. nuttallianum (Waser and Price, 1991; N. Waser and M. Price, unpublished data). Thus we were confident that our removal of stigmas did not affect seed set in D. nuttallianum, and we assumed similar dynamics in A. columbianum. Stigmas of D. nuttallianum were collected between 13 June and 1 July and those of A. columbianum between 21 July and 16 August. We mounted stigmas in basic fuchsin gel on a microscope slide (Kearns and Inouye, 1993) and counted loads of conspecific and heterospecific pollen at 100–2503. We did not emasculate flowers, so self pollen was included in counts. Heterospecific pollen was identified by comparison to a pollen reference collection. Approximately 1 mo after pollination we counted numbers of good expanded seeds and of undeveloped ovules from flowers whose stigmas we had collected earlier. This allowed us to express seed set both as an absolute value and as a percentage of all ovules developed. Analysis—We used ANOVA (procedure GLM; SAS, 1990) to assess the effect of local conspecific density on the quantity of pollination service per 30-min census (number of pollinator approaches to a plot, number of plants and flowers visited per plot, number of approaches per plant, proportion of plants visited in a plot, and number of flowers visited per plant), pollen loads (total pollen load, conspecific pollen
June 1999]
BOSCH
AND
WASER—POPULATION
DENSITY, POLLINATION, AND REPRODUCTIVE SUCCESS
873
TABLE 1. ANOVAs of effects of density on pollinator approaches per plot, number of plants visited per plot, and number of flowers visited per plot, all per 30-min observation period. The total model r2 was $0.78 in each case. Significant effects (P , 0.05) are shown in boldface. Approaches Source
df
SS
D. nuttallianum Density Site Error
1 4 4
A. columbianum Density Site Error
1 2 2
Plants visited
F
P
df
2.47 0.85 0.93
10.59
0.031
1 4 4
91.26 39.09 2.17
84.11
0.012
1 2 2
SS
Flowers visited
F
P
df
77.64 55.13 29.55
10.51
0.032
1 4 4
145.43 167.49 63.47
9.16
0.039
6042.03 2213.69 953.45
23.21
0.071
1 2 2
34 625.61 10 759.96 5172.17
13.39
0.067
load, and heterospecific pollen load), and seed set (absolute number of seeds, percentage seed set, and total number of ovules per flower). Analyses of visitation measures were based on means across all replicate censuses in each plot, since we conservatively assumed that results of successive censuses might not be independent. Analyses of different aspects of pollen load and seed set were based on means across all focal plants in a given plot, to avoid pseudoreplication. In all analyses, local conspecific density was considered a categorical variable with two levels (sparse, dense), and site was used as a blocking factor. Thus the models had factors of density (fixed), site (random), and the interaction of density and site, with density tested over the interaction (Newman, Bergelson, and Grafen, 1997). Because these analyses are conservative in using plot means, we were unable to test site effects (see Sokal and Rohlf, 1995, p. 348). Type III sums of squares were used in all unbalanced analyses. Distributions of residuals from all models were approximately normal, without pronounced heteroscedasticity. Finally, we assessed dose-response relationships between pollen load and seed set for flowers, by using nonlinear regression (Procedure NLIN, Marquart estimation; SAS, 1990) to fit negative exponential functions of the form seeds 5 K(1 2 exp{2b pollen}) where K estimates the asymptotic maximum seed set and b estimates rate of approach to the asymptote. We began by fitting such a function separately to the data from dense and sparse plots for each species, to visualize any differences in pollen-seed relationships due to density. Next we fit a single function to the pooled data for each species and extracted residuals from the nonlinear regression. Residuals were further analyzed by ANOVA with factors of density, site, the interaction of density and site, and focal plant nested within density-site combination. Density was tested over the interaction of density and site as explained above (see Waser and Price [1991] for details and justification of this approach, which can be considered a nonlinear ANCOVA). We calculated r2 values for nonlinear regressions as 1 2 (residual SS/corrected total SS), as recommended by Kva˚lseth (1985).
RESULTS Pollinator visitation—Delphinium nuttallianum plots were visited at a rate only one-tenth of that experienced by Aconitum columbianum plots (respective means of 0.9 vs. 9 approaches per plot per 30-min census). However, D. nuttallianum attracted a wider spectrum of pollinators. Of all approaches to D. nuttallianum plots, 56.9% were by queen bumble bees (17.9% by Bombus appositus, 17.1% by B. flavifrons, and 21.9% collectively by B. nevadensis, B. californicus, B. occidentalis, B. rufocinctus, and B. sylvicola), 28.5% were by halictid bees, and
SS
F
P
12.2% were by broad-tailed hummingbirds (Selasphorus platycercus). In contrast, A. columbianum plots were approached only by bumble bees (86.4% by B. flavifrons, mostly workers, and 14.3% by B. appositus, a mixture of queens and workers, with only single visits observed by B. occidentalis and B. sylvicola). Pollinators appeared to visit slightly fewer flowers per plant in dense than in sparse plots. The mean values for D. nuttallianum in dense and sparse plots were, respectively, 1.5 flowers per plant (SD 5 0.7, N 5 482 visits to plants in 100 foraging bouts) and 1.8 flowers per plant (SD 5 1.0, N 5 80 visits in 24 bouts), and those for A. columbianum in dense and sparse plots were, respectively, 2.4 flowers per plant (SD 5 1.5, N 5 2301 visits in 386 bouts) and 2.9 flowers per plant (SD 5 2.0, N 5 409 visits in 154 bouts). However, neither difference was significant by ANOVA (not shown). Overall, pollinators of D. nuttallianum visited 1.5 flowers per plant on average (SD 5 0.8, N 5 562 visits in 124 bouts), whereas pollinators of A. columbianum visited 2.5 flowers per plant on average (SD 5 1.6, N 5 2710 visits in 540 bouts; compare Pleasants and Zimmerman, 1990). Pollinators approached dense plots of D. nuttallianum significantly more often per 30-min census than sparse plots and visited more plants and flowers per plot (Table 1). However, the differences were of small magnitude (Fig. 1). On a per-plant basis local density of conspecifics caused no statistically detectable difference in final pollinator service (Table 2, Fig. 1). Thus plants and flowers in dense and sparse plots on average received very similar final numbers of pollinator visits. The pattern was similar for A. columbianum. Pollinators approached dense plots more often per census than sparse plots and visited more plants and flowers per plot, but only the difference in approaches was significant (Table 1, Fig. 1). On a perplant basis, plants in sparse plots actually received significantly more visits overall, but the difference was of small magnitude, and we detected no significant density effects on the proportion of plants visited per plot or number of flowers visited per plant (Table 2, Fig. 1). Pollen loads—The mean number of pollen grains of all species on stigmas was ;670 (range 5 12–2560) in D. nuttallianum and 235 (range 5 27–730) in A. columbianum. In both species the numbers were higher in dense plots, but not significantly so (Fig. 2, Table 3).
874
AMERICAN JOURNAL
OF
BOTANY
[Vol. 86
Fig. 1. Pollinator visitation per 30-min census (mean, 1 SE) in dense and sparse plots of D. nuttallianum (A, B) and A. columbianum (C, D). (A, C) show the number of approaches to a plot (‘‘Approach’’), number of plants and number of flowers visited per plot (‘‘Plants’’ and ‘‘Flowers,’’ respectively). (B, D) show the approaches per plant (‘‘Approach’’), proportion of plants visited in a plot (‘‘Plants’’), number of flowers visited per plant (‘‘Flowers’’). * significant differences (P , 0.05), using ANOVA (Tables 1, 2).
Conspecific pollen loads were similar to total pollen loads, and their analysis yields statistical patterns similar to those just described (Table 3). In short, we detected no significant effect of density on conspecific pollen receipt. Heterospecific pollen comprised, on average, 4.4% of all pollen on stigmas of D. nuttallianum and 11.6% on stigmas of A. columbianum. Conspecific density had no detectable effect on receipt of heterospecific pollen for either species (Fig. 2, Table 3). The most common heterospecific pollens on stigmas of D. nuttallianum were Hydrophyllum sp. (0.9% of all pollen), Castilleja miniata
(0.8%), Mertensia fusiformis (0.6%), and Ipomopsis aggregata (0.4%). On stigmas of A. columbianum they were Mertensia ciliata (9.6%), Ipomopsis aggregata (1%), and Asteraceae (0.3%). Pollen of M. ciliata was especially common, appearing on 68% of all A. columbianum stigmas. Because Delphinium barbeyi was in flower at the same time as A. columbianum, and we observed bumble bees consecutively visiting flowers of the two species, and because pollen grains of these two species are indistinguishable with light microscopy, some of the pollen scored as conspecific for A. columbianum may have been D. barbeyi.
TABLE 2. ANOVAs of effects of density on pollinator approaches per plant, proportion of plants visited in a plot, and number of flowers visited per plant, all per 30-min observation period. The total model r2 was $0.50 in each case. Conventions follow Table 1. Approaches Source
df
SS
D. nuttallianum Density Site Error
1 4 4
0.0004 0.0004 0.0007
A. columbianum Density Site Error
1 2 2
0.0738 0.0378 0.0044
F
Plants visited
Flowers visited
P
df
SS
F
P
df
SS
F
P
2.18
0.214
1 4 4
0.0003 0.0064 0.0068
0.18
0.690
1 4 4
0.0033 0.0325 0.0212
0.62
0.474
33.77
0.028
1 2 2
0.0036 0.8589 0.0122
0.59
0.523
1 2 2
0.1231 5.3168 0.5263
0.47
0.565
June 1999]
BOSCH
AND
WASER—POPULATION
DENSITY, POLLINATION, AND REPRODUCTIVE SUCCESS
875
Fig. 2. Pollen deposition and seed and ovule production (mean and SE) in dense and sparse plots of D. nuttallianum (A, B) and A. columbianum (C, D). (A, C) show total pollen loads on stigmas (‘‘Total’’), conspecific pollen loads (‘‘Consp.’’), and heterospecific pollen loads (‘‘Heterosp.’’). (B, D) show the absolute number of seeds set (‘‘Seeds’’), the percentage of ovules set as seeds (‘‘% seeds’’), and total ovule production per flower (‘‘Ovules’’). * significant differences (P , 0.05), using ANOVA (Tables 3, 4).
Seed set—Total average seed set per flower was 33.7 in D. nuttallianum and 24.8 in A. columbianum. The two species, respectively, produced 51.8 (range 5 27–96) and 37.6 (range 5 19–77) ovules per flower, with no statistical difference between dense and sparse plots (Table 4). In contrast, seed set per flower in sparse plots, expressed either as an absolute number or percentage of ovules filled, was only 60–75% as high as in dense plots for both species (Fig. 2).
fit by negative exponential functions (Fig. 3). There was substantial scatter, so that r2 values were small, as reported for other species (e.g., Kohn and Waser, 1985; Waser and Price, 1991; Mitchell, 1997). In both D. nuttallianum and A. columbianum the asymptotic seed sets in sparse plots were ;65% of those in dense plots (Fig. 3). This agrees well with differences noted immediately above in mean seed set and indicates that plants produced more seeds in dense than in sparse plots for the same amount of pollen, over most of the natural range in pollen loads. Significant overall density effects in ANOVA of residuals from pooled negative exponential regressions (Table 5) confirm this impression. Density effects on re-
Relationship between pollen load and seed set— Dose-response relationships between total conspecific pollen load per flower and seed set were reasonably well
TABLE 3. ANOVA of effects of density on total stigma pollen loads, conspecific pollen loads, and heterospecific pollen loads. The total model r2 was $0.55 in each case. Conventions follow Table 1. Total pollen Source
Conspecific
df
SS
F
P
df
SS
D. nuttallianum Density Site Error
1 4 4
82 795.80 84 504.52 96 462.53
3.43
0.137
1 4 4
80 804.46 84 492.97 86 696.23
A. columbianum Density Site Error
1 2 2
5376.86 3730.60 1194.82
9.00
0.095
1 2 2
2231.47 1617.96 429.72
Heterospecific F
P
df
SS
F
P
3.73
0.126
1 4 4
12.12 606.96 515.39
0.09
0.774
10.39
0.084
1 2 2
680.60 441.00 781.10
1.74
0.318
876
AMERICAN JOURNAL
OF
BOTANY
[Vol. 86
TABLE 4. ANOVAs of effects of density on numbers of seeds set, proportional seed set, and total ovules per flower. The total model r2 was $0.56 in each case. Conventions follow Table 1. Number of seeds
% of seeds
Total ovules
Source
df
SS
F
P
df
SS
F
P
df
SS
F
P
D. nuttallianum Density Site Error
1 4 4
755.25 68.35 119.51
25.28
0.007
1 4 4
2608.71 223.99 179.46
58.15
0.002
1 4 4
31.93 54.72 67.71
1.89
0.242
A. columbianum Density Site Error
1 2 2
128.07 23.30 2.65
96.62
0.010
1 2 2
431.90 176.82 10.18
84.81
0.012
1 2 2
39.49 8.85 11.80
6.69
0.123
siduals for both species also varied from site to site, for unknown reasons. Finally, we were unsurprised to detect variation in residuals among focal plants of both species, because individual plants are likely to vary in nutrient status and other factors that influence their translation of pollen received into seed set.
Fig. 3. Dose–response relationships between total pollen loads and seed set per flower in dense plots (filled circles, thick line) and sparse plots (open circles, thin line) in D. nuttallianum (A) and A. columbianum (B). The equations were: (A) dense, y 5 42.9(1 2 exp{20.008x}), r2 5 0.03, N 5 158 flowers; (A) sparse y 5 29.8(1 2 exp{20.005x}), r2 5 0.11, N 5 141; (B) dense y 5 32.7(1 2 exp{20.011x}), r2 5 0.08, N 5 135; (B) sparse y 5 21.2(1 2 exp{20.022x}), r2 5 0.04, N 5 133.
DISCUSSION Our study species differ from each other in several features that should influence their pollination. For example, D. nuttallianum begins flowering soon after snowmelt around the RMBL. During this period broad-tailed hummingbirds are arriving and beginning to breed, whereas bumble bee queens are emerging from winter hibernation and establishing nests. Hummingbirds are important pollinators of D. nuttallianum early in its flowering period, but later switch to other plant species with greater nectar rewards as these come into bloom (Waser, 1978). Queen bumble bees dominate as pollinators later in the season (Waser and Price, 1981). The overall diversity of visitors to D. nuttallianum is high, perhaps because the diversity of flowering plant species is relatively low early in the summer. In contrast, A. columbianum flowers later in the summer and attracts mostly worker bumble bees, especially Bombus flavifrons, a species with an intermediate proboscis length (see also Graham and Jones, 1996). The total diversity of visitors is much lower than for D. nuttallianum. Perhaps because of the different identities of visitor species and different morphologies of flowers, or perhaps because of differences in total pollen production between the species, pollen loads on stigmas of D. nuttallianum were substantially higher, on average, than those on stigmas of A. columbianum, even though we observed many fewer visits to the former species. In spite of these differences, natural extremes in conspecific density had remarkably similar effects on pollination and fecundity of the two species. Pollinators approached dense populations at a higher rate than sparse populations and visited more plants and flowers. However, these differences disappear for the most part when visitation rate is expressed on a per-plant or per-flower basis. Thus we detected no statistically significant differences between dense and sparse plots in the rate of visitation to individual plants of D. nuttallianum, in rate of visitation to flowers of either species, or in conspecific or heterospecific pollen loads on stigmas of either species. In contrast to these direct assessments of pollination services, seed set responded strongly to population density. Even though plants in dense and sparse plots provisioned their flowers with equivalent numbers of ovules, only about two-thirds as many of these ovules were matured as seeds in sparse plots of both species. Our study adds to the evidence for reduced fecundity in plant populations at low density, which has implications for man-
June 1999]
BOSCH
AND
WASER—POPULATION
DENSITY, POLLINATION, AND REPRODUCTIVE SUCCESS
877
TABLE 5. ANOVA of residuals from pollen loads and seed set relationships. Density was tested over Density 3 Site. The factor ‘‘Plant’’ refers to focal plant nested within a density–site combination. The total model r2 was 0.60 for D. nuttallianum and 0.67 for A. columbianum. Conventions follow Table 1. D. nuttallianum
A. columbianum
Source
df
SS
F
P
df
SS
F
P
Density Site Density 3 Site Plant Error
1 4 4 64 225
14 013.32 419.98 3569.54 30 569.27 32 558.69
15.70 0.73 6.17 3.30
0.017 0.575 0.0001 0.0001
1 2 2 48 214
3921.11 338.49 398.01 22 233.31 13 476.37
19.70 2.69 3.16 7.36
0.047 0.070 0.044 0.0001
agement of endangered species (Kearns, Inouye, and Waser, 1998). Density responses of D. nuttallianum and A. columbianum are comparable to those reported for other species in several regards. Pollinator approaches to populations usually increase as local density increases (Klinkhamer, de Jong, and de Bruyn, 1989; Feinsinger, Tiebout, and Young, 1991; Dreisig, 1995; but see Brody and Mitchell, 1997), and our species were no exception. An increase in approach rate sometimes translates into increased perflower visitation in dense populations (Schaal, 1978; Thomson, 1981; Kunin, 1997a, and references therein, 1997b). But in other cases, ours included, approach rate increases in proportion to density so that per-flower visitation is comparable across densities (Schmitt, 1983; Brody and Mitchell, 1997). And approach rate sometimes increases more slowly with density or increases nonlinearly, so that per-flower visitation actually peaks at intermediate or low densities (Kwak, 1987, 1994; Jennersten and Nilsson, 1993). Thus, the translation of local plant density into per-flower visitation seems to be idiosyncratic to the particular plant and pollinator system, a conclusion similar to that derived from studies of pollinator responses to the number of flowers per plant (e.g., Brody and Mitchell, 1997, and references therein). Density effects on receipt of pollen by stigmas have been less widely studied, but in contrast to our results, conspecific pollen loads usually increase with density and heterospecific loads usually decrease (Thomson, 1981; Kunin, 1993, 1997a, and references therein; Kwak, 1994; Smith, 1998). Finally, a number of workers have reported higher seed or fruit set in dense plots (Kunin, 1992, 1997a, b; Jennersten and Nilsson, 1993; Van Treuren et al., 1994; Roll et al., 1997), as we also found. However, we are unaware of any other report such as ours of a density effect on fecundity in the absence of a density effect on per-flower visitation by pollinators. Several possibilities present themselves to explain differences in seed set despite apparent similarity in pollination per flower. One hypothesis involves a difference not in the quantity of pollination services, as measured by per-flower visitation rate and pollen receipt, but in the quality of services (sensu Waser and Price, 1991; Kunin, 1993; Aizen and Feinsinger, 1994). The quality of services could vary as a function of the local population context for a number of reasons. One attribute of a local population is the availability of pollen relative to receptive stigmas, i.e., the ratio of sex expression. Cunningham (1995) showed that fruit set in a tropical palm increased with the ratio of male to female flowers. However, such a process seems unlikely
to explain our results. Optimal foraging theory (Charnov, 1976; Pyke, 1984) predicts that pollinators will visit more flowers per plant on average in sparse plots, and pollinators indeed tended to do so with our study species, in agreement with another report for D. nuttallianum (Cibula and Zimmerman, 1984) and reports from other systems (e.g., Heinrich, 1979). Furthermore, bumble bees tend to visit flowers near the bottom of a D. nuttallianum or A. columbianum inflorescence first and then to move upward (Pyke, 1978). Upper flowers are staminate, so a bumble bee visiting more flowers per plant in sparse plots should tend to visit a greater proportion that are shedding pollen, which might increase fecundity by analogy to Cunningham’s (1995) result. However, this argues that flowers in sparse plots should receive more pollen and have higher seed set than flowers in dense plots, neither of which occurred in our study. Another candidate for a difference in quality of pollination service involves a greater frequency of self and other inbred pollinations in sparse plots. As just explained, pollinators tend to visit more flowers per plant in such plots, and so may cause more geitonogamy (compare Watkins and Levin, 1990, and references therein; Van Treuren et al., 1993, 1994; Karron et al., 1995). Although we did not measure this in our study, some workers also report that pollinators fly more often from plants to their nearest neighbors in sparse populations (Schaal, 1978). Local kinship structure is a common feature of natural plant populations, including those of D. nuttallianum (Waser, 1987; Waser and Price, 1994), so that neighbors tend to be related. If the frequency of flights between neighbors does increase in sparse populations of D. nuttallianum and A. columbianum, and if neighbors are no less closely related in sparse than in dense populations (but see Watkins and Levin, 1990), the outcome would be a greater incidence of pollinations representing biparental inbreeding. Seed set declines under self-pollination in D. nuttallianum and some related species (see Materials and Methods) and also declines in D. nuttallianum when near neighbors are crossed (Waser and Price, 1991). Thus self-pollination and other forms of pollination among relatives might contribute to reduced seed set in sparse populations of D. nuttallianum and A. columbianum. A second, very different approach to explain density effects on fecundity is to invoke a mechanism altogether unrelated to pollination services. Local population density in nature might reflect differences in environmental quality, for example in availability of water, soil nutrients, or sunlight. Although plants of neither species provisioned their flowers with more ovules in dense than in
878
AMERICAN JOURNAL
sparse plots, plants in dense plots may nonetheless have had more resources available to fill pollinated ovules into seeds. Consistent with this idea is the observation that plants of both species produced slightly more flowers on average in dense plots. This ‘‘resource limitation’’ hypothesis and the hypothesis of reduced pollen quality are not mutually exclusive, nor necessarily exhaustive of all possibilities. An unexpected diversity of outcomes has emerged from our examination of different facets of pollination and reproduction. If we had limited ourselves to any single facet or subset of them, our conclusions might have differed fundamentally from those presented here. Thus a focus only on pollinator visitation to plots or on seed set would have suggested benefits of density, whereas a focus only on per-flower visitation or pollen receipt would have suggested no density effect. The truth appears to be more complex and subtle: a strong density effect on fecundity that is not mediated through changes in the quantity of pollination services. We suspect that such complexity often characterizes plant reproductive ecology, and this argues that ecologists and conservation biologists interested in effects of density and other population attributes will be rewarded if they simultaneously attend to multiple facets of pollination and reproduction. Nonmanipulative approaches in ecology have certain intrinsic strengths. By examining natural variation in local conspecific density we ensured that our results reflect real patterns experienced by plants and are not artifacts of some contrived situation. Whereas it reveals natural patterns, however, the approach used here cannot clarify the mechanism of density effects in D. nuttallianum and A. columbianum. To go further we must reach into the ecological toolbox for an approach, the manipulative field experiment, that remains surprisingly rare in pollination ecology. Experimental manipulation of the density of potted plants could indicate whether seed set differences remain even when edaphic conditions are controlled, and the emasculation of flowers on some plants could indicate whether geitonogamy depresses seed set more strongly in sparse than in dense artificial populations. In the future we hope to use such experiments to elucidate the mechanisms of density effects on fecundity in D. nuttallianum and A. columbianum. LITERATURE CITED AIZEN, M. A., AND P. FEINSINGER. 1994. Habitat fragmentation, native insect pollinators, and feral honey bees in Argentine ‘‘chaco serrano.’’ Ecological Applications 4: 378–392. AKER, C. L. 1982. Spatial and temporal dispersion patterns of pollinators and their relationship to the flowering strategy of Yucca whipplei (Agavaceae). Oecologia 54: 243–252. BIERREGAARD, R. O., T. E. LOVEJOY, V. KAPOS, A. A. D. SANTOS, AND R. W. HUTCHINGS. 1992. The biological dynamics of tropical rainforest fragments: a prospective comparison of fragments and continuous forest. BioScience 42: 859–866. BOSCH, M. 1999. Bı`ologia de la reproduccio´ de la tribu Delphineae a la Mediterra`nia occidental. Arxius de la Seccio´ de Cie`ncies, 120. Institut d’Estudis Catalans, Barcelona. ———, J. SIMON, C. BLANCHE´, AND J. MOLERO. 1997. Pollination ecology in tribe Delphineae (Ranunculaceae) in W Mediterranean area: floral visitors and pollinator behaviour. Lagascalia 19: 545–562. BRINK, D. E. 1980. Reproduction and variation in Aconitum columbianum (Ranunculaceae) with emphasis on California populations. American Journal of Botany 67: 263–273.
OF
BOTANY
[Vol. 86
BRODY, A. K., AND R. J. MITCHELL. 1997. Effects of experimental manipulation of inflorescence size on pollination and pre-dispersal seed predation in the hummingbird-pollinated plant Ipomopsis aggregata. Oecologia 110: 86–93. BRUNET, J., AND D. CHARLESWORTH. 1995. Floral sex allocation in sequentially blooming plants. Evolution 49: 70–79. BYERS, D. L. 1995. Pollen quantity and quality as explanations for low seed set in small populations exemplified by Eupatorium (Asteraceae). American Journal of Botany 82: 1000–1006. CAMPBELL, D. R., AND N. M. WASER. 1989. Variation in pollen flow within and among populations of Ipomopsis aggregata. Evolution 43: 1444–1455. CHARNOV, E. L. 1976. Optimal Foraging, the marginal value theorem. Theoretical Population Biology 9: 129–136. CIBULA, D. A., AND M. ZIMMERMAN. 1984. The effect of plant density on departure decisions: testing the marginal value theorem using bumblebees and Delphinium nelsonii. Oikos 43: 154–158. CUNNINGHAM, S. A. 1995. Ecological constraints on fruit initiation by Calyptrogyne ghiesbreghtiana (Arecaceae): floral herbivory, pollen availability, and visitation by pollinating bats. American Journal of Botany 82: 1527–1536. DARWIN, C. 1876. The effects of cross and self fertilisation in the vegetable kingdom. Murray, London. DREISIG, H. 1995. Ideal free distributions of nectar foraging bumblebees. Oikos 72: 161–172. FEINSINGER, P., H. M. TIEBOUT III, AND B. E. YOUNG. 1991. Do tropical bird-pollinated plants exhibit density-dependent interactions? Field experiments. Ecology 72: 1953–1963. GRAHAM, L., AND K. N. JONES. 1996. Resource partitioning and perflower foraging efficiency in two bumble bee species. American Midland Naturalist 136: 401–406. GROOM, M. J. 1998. Allee effects limit population viability of an annual plant. American Naturalist 151: 487–496. HEINRICH, B. 1979. Resource heterogeneity and patterns of movement in foraging bumblebees. Oecologia 40: 235–245. INGVARSSON, P. K., AND S. LUNDBERG. 1995. Pollinator functional response and plant population dynamics: pollinators as a limiting resource. Evolutionary Ecology 9: 421–428. JENNERSTEN, O., AND S. G. NILSSON. 1993. Insect flower visitation frequency and seed production in relation to patch size of Viscaria vulgaris (Caryophyllaceae). Oikos 68: 283–292. KARRON, J. D, N. N. THUMSER, R. TUCKER, AND A. J. HESSENAUER. 1995. The influence of population density on outcrossing rates in Mimulus ringens. Heredity 75: 175–180. KEARNS, C. A., AND D. W. INOUYE. 1993. Techniques for pollination biologists. University Press of Colorado, Niwot, CO. ———, ———, AND N. M. WASER. In press. Endangered mutualisms: the conservation of plant-pollinator interactions. Annual Review of Ecology and Systematics 28: 83–112. KLINKHAMER, P. G. L., T. J. DE JONG, AND G. L. DE BRUYN. 1989. Plant size and pollinator visitation in Cynoglossum officinale. Oikos 54: 201–204. KOHN, J. R., AND N. M. WASER. 1985. The effect of Delphinium nelsonii pollen on seed set in Ipomopsis agreggata, a competitor for hummingbird pollination. American Journal of Botany 72: 1144– 1148. KRANNITZ, P. G., AND M. A. MAUN. 1991. An experimental study of floral display size and reproductive success in Viburnum opulus: importance of grouping. Canadian Journal of Botany 69: 394–399. KUNIN, W. E. 1992. Density and reproductive success in wild populations of Diplotaxis erucoides (Brassicaceae). Oecologia 91: 129– 133. ———. 1993. Sex and the single mustard: population density and pollinator behavior effects on seed-set. Ecology 74: 2145–2160. ———. 1997a. Population biology and rarity: on the complexity of density dependence in insect-plant interactions. In W. E. Kunin and K. J. Gaston [eds.], The biology of rarity, 150–169. Chapman and Hall, New York, NY. ———. 1997b. Population size and density effects in pollination: pollinator foraging and plant reproductive success in experimental arrays of Brassica kaber. Journal of Ecology 85: 225–234. KVA˚LSETH, T. O. 1985. Cautionary note about R2. American Statistician 39: 279–285.
June 1999]
BOSCH
AND
WASER—POPULATION
DENSITY, POLLINATION, AND REPRODUCTIVE SUCCESS
KWAK, M. M. 1987. Pollination and pollen flow disturbed by honeybees in bumblebee-pollinated Rhinanthus populations? In J. van Andel, J. P. Bakker, and R. W. Snaydon [eds.], Disturbance in grasslands, 273–283. Dr. W. Junk, Dordrecht. ———. 1994. Populatie structuur en bestuiving: effecten van ruimtelijke rangschikking bij de Zwartblauwe rapunzel. Landschap 11: 15–24. ———, C. VAN DEN BRAND, P. KREMER, AND E. BOERRICHTER. 1991. Visitation, flight distances and seed set in populations of the rare species Phyteuma nigrum (Campanulaceae). Acta Horticulturae 288: 303–307. LYONS, E. E., AND T. W. MULLY. 1992. Density effects on flowering phenology and mating potential in Nicotiana alata. Oecologia 91: 93–100. MITCHELL, R. J. 1997. Effects on pollination intensity on Lesquerella fendleri seed set: variation among plants. Oecologia 109: 382–388. NEWMAN, J. A., J. BERGELSON, AND A. GRAFEN. 1997. Blocking factors and hypothesis tests in ecology: is your statistics text wrong? Ecology 78: 1312–1320. PLEASANTS, J. M., AND M. ZIMMERMAN. 1990. The effect of inflorescence size on pollinator visitation of Delphinium nelsonii and Aconitum columbianum. Collectanea Botanica 19: 21–39. PRICE, M. V., AND N. M. WASER. 1979. Pollen dispersal and optimal outcrossing in Delphinium nelsoni. Nature 277: 294–297. PYKE, G. H. 1978. Optimal foraging in bumblebees and coevolution with their plants. Oecologia 36: 281–293. ———. 1984. Optimal foraging theory: a critical review. Annual Review of Ecology and Systematics 15: 523–575. ROLL, J., R. J. MITCHELL, R. J. CABIN, AND D. L. MARSHALL. 1997. Reproductive success increases with local density of conspecifics in a desert mustard (Lesquerella fendleri). Conservation Biology 11: 738–746. SAS. 1990. SAS/STAT user’s guide, version 6, 4th ed. SAS Institute, Cary, NC. SCHAAL, B. A. 1978. Density dependent foraging on Liatris pychnostachya. Evolution 32: 452–454. SCHMITT, J. 1983. Flowering plant density and pollinator visitation in Senecio. Oecologia 60: 97–102. SIH, A., AND M.-S. BALTUS. 1987. Patch size, pollinator behavior, and pollinator limitation in catnip. Ecology 68: 1679–1690. SMITH, J. W. 1998. Effects of species rarity and plant density on the pollination of eight montane herbs. Master’s thesis, University of California, Riverside, CA. SOKAL, R. R., AND F. J. ROHLF. 1995. Biometry, third edition. W. H. Freeman, New York, NY. THOMSON, J. D. 1981. Spatial and temporal components of resource
879
assessment by flower-feeding insects. Journal of Animal Ecology 50: 49–59. ———. 1988. Effects of variation in inflorescence size and floral rewards on the visitation rates of traplining pollinators of Aralia hispida. Evolutionary Ecology 2: 65–76. VAN TREUREN, R., R. BIJLSMA, H. J. OUBORG, AND W. VAN DELDEN. 1993. The effects of population size and plant density on outcrossing rates in locally endangered Salvia pratensis. Evolution 47: 1094–1104. ———, ———, ———, AND M. M. KWAK. 1994. Relationships between plant density, outcrossing rates and seed set in natural and experimental populations of Scabiosa columbaria. Journal of Evolutionary Biology 7: 287–302. VITOUSEK, P. M. 1994. Beyond global warming: ecology and global change. Ecology 75: 1861–1876. WASER, N. M. 1978. Competition for hummingbird pollination and sequential flowering in two Colorado wildflowers. Ecology 59: 934–944. ———. 1982. A comparison of distances flown by different visitors to flowers of the same species. Oecologia 55: 251–257. ———. 1987. Spatial genetic heterogeneity in a population of the montane perennial plant Delphinium nelsonii. Heredity 58: 249– 256. ———, AND M. V. PRICE. 1981. Pollinator choice and stabilizing selection for flower color in Delphinium nelsonii. Evolution 35: 376– 390. ———, AND ———. 1990. Pollination efficiency and effectiveness of bumblebees and hummingbirds visiting Delphinium nelsonii. Collectanea Botanica 19: 9–20. ———, AND ———. 1991. Outcrossing distance effects in Delphinium nelsonii: pollen loads, pollen tubes and seed set. Ecology 72: 171– 179. ———, AND ———. 1994. Crossing distance effects in Delphinium nelsonii: outbreeding and inbreeding depression in progeny fitness. Evolution 48: 842–852. ———, AND ———. 1998. What plant ecologists can learn from zoology. Perspectives in Plant Ecology, Evolution and Systematics 1: 130–150. ———, L. CHITTKA, M. V. PRICE, N. WILLIAMS, AND J. OLLERTON. 1996. Generalization in pollination systems, and why it matters. Ecology 77: 279–296. WATKINS, L, AND D. A. LEVIN. 1990. Outcrossing rates as related to plant density in Phlox drummondii. Heredity 65: 81–89. WATT, W. B., P. C. HOCH, AND S. G. MILLS. 1974. Nectar resource use by Colias butterflies. Oecologia 14: 353–374. WEBER, W. A., AND R. C. WITTMANN. 1996. Colorado flora: western slope. Revised edition. University Press of Colorado, Niwot, CO.
