Sex ratio, flowering and fruit set in dioecious Rubus chamaemorus (Rosaceae) in Labrador

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1 204 Sex ratio, flowering and fruit set in dioecious Rubus chamaemorus (Rosaceae) in Labrador Amanda L. Karst, Joseph A. Antos, and Geraldine A. Allen Abstract: Rubus chamaemorus L. is a dioecious clonal herb with highly variable fruit production. Our overall objective was to determine how fruit production (and the reproductive stages leading to fruit) for R. chamaemorus varied with habitat and population (especially sex ratio) characteristics at scales from 1 m 2 quadrats to whole populations in southeastern Labrador, Canada. During the climatically favourable year studied, reproductive variables were only weakly correlated with habitat characteristics. Strongly biased sex ratios were found at scales of 1 10 m. Male-dominated quadrats exhibited a significantly higher proportion of flowering ramets than did female-dominated quadrats. Female-biased sex ratios of flowering ramets and large distances between male and female flowers were correlated with decreased seed set. We found no differences in microhabitats of males and females. Although the biased local sex ratios could limit sexual reproduction, the high fruit set we measured clearly indicates that populations are able to produce abundant fruit during favourable years in this unpredictable environment. Key words: clone size, dioecious plants, fruit production, sex ratio, Rubus chamaemorus, seed set. Résumé : Le Rubus chamaemorus L. est une herbacée clonale dioïque ayant une productivité en fruits très variable. Les auteurs ont cherché à déterminer comment, chez le R. chamaemorus, la production des fruits (et les stades reproducteurs conduisant au fruit) varient selon les caractéristiques de l habitat et de la population (surtout la proportion des sexes), à l échelle de quadras de 1 m 2 jusqu à l ensemble de la population, dans le sud du Labrador, au Canada. Au cours d un été climatiquement favorable, les variables reproductrices ne montrent qu une faible corrélation avec les caractéristiques de l habitat. On observe des proportions sexuelles fortement biaisées aux échelles de 1 10 m. Les quadras dominés par les mâles montrent une proportion significativement plus élevée de ramètes florales comparativement aux quadras dominés par les femelles. Les proportions sexuelles, biaisées en faveur des femelles des ramètes florales, et la grande distance entre les fleurs mâles et femelles, montrent une corrélation avec une diminution de la mise à graine. On observe aucune différence de microhabitat entre les mâles et les femelles. Bien que les proportions sexuelles biaisées puissent limiter la reproduction sexuelle, la forte mise à fruit mesurée indique clairement que les populations peuvent produire une abondance de fruits dans cet environnement imprévisible. Mots-clés :dimension du clone, plantes dioïques, production de fruits, rapport des sexes, Rubus chamaemorus, mise à graine. Introduction Plants seldom mature fruits from all of the flowers they produce. Fruit production in obligately outcrossing species can be limited by various factors. Resources for reproduction may be limiting in harsh or highly variable environments, or availability of compatible pollen may be influenced by flower density, availability of pollinators, or clone size. In habitats that vary temporally, plants may produce many more flowers than can yield fruit in a typical year, and thus can take advantage of years when climatic conditions are favourable (Willson 1983; Lovett Doust and Received 19 March Published on the NRC Research Press Web site at botany.nrc.ca on 29 February A.L. Karst, 1,2 J.A. Antos, and G.A. Allen. Biology Department, P.O. Box 3020, Station CSC, University of Victoria, Victoria, BC V8W 3N5, Canada. 1 Corresponding author ( akarst@cier.ca). 2 Present address: Centre for Indigenous Environmental Resources, 245 McDermot Street, Winnipeg, MB R3B 0S6, Canada. Lovett Doust 1988). In dioecious species, large distances between males and females could adversely affect pollination and fruit set. Large clones, especially if not intermingled, can lead to substantial separation of female from male flowers. Formation of patches dominated by single clones has been implicated in reduced fruit production in a number of species (e.g., Aspinwall and Christian 1992; Eriksson and Bremer 1993). The low temperatures, short growing seasons, and low nutrient availability characteristic of boreal environments (van Groenendael and de Kroon 1990) can compromise fruit production. Bog habitats offer additional challenges for reproduction, including fluctuating water tables, low ph, and anoxic soil conditions (Mitsch and Gosselink 1993; Haslam 2003). Plants of these environments are typically slowgrowing, long-lived, and clonal (Backéus 1985; van Groenendael and de Kroon 1990; Mitsch and Gosselink 1993; Klimes et al. 1997); reproduction by seed may be infrequent or episodic. Rubus chamaemorus L. (bakeapple), a clonal dioecious plant with a boreal circumpolar distribution, is typically found in bogs (Korpelainen 1994) and is a popular and im- Botany 86: (2008) doi: /b07-127

2 Karst et al. 205 portant wild food source for human populations in the circumpolar regions of the northern hemisphere. Reproductive patterns and fruit production in Scandinavian populations have been much studied (Resvoll 1929; Østgård 1964; Lohi 1974; Mäkinen and Oikarinen 1974; Ågren et al. 1986; Ågren 1987; 1988a; 1988b; 1989; Korpelainen 1994), but North American populations have received limited attention (Savory 1981; Dumas and Maillette 1987; Pelletier et al. 2001). Allocation to sexual reproduction in R. chamaemorus is low compared with allocation to vegetative structures such as underground rhizomes (Mäkinen and Oikarinen 1974), which represent up to 94% of total plant biomass (Dumas and Maillette 1987). Fruit production is strongly correlated with shelter (e.g., surrounding shrubs or trees), likely because frost and heavy rain can damage the flowers and developing fruit (Lohi 1974; Mäkinen and Oikarinen 1974; Ågren 1988a; Yudina 1993). Seedlings are rare in natural populations because of highly variable fruit production, low seed availability owing to fruit consumption, and lack of suitable microsites for germination in the thick vegetation of bogs (Resvoll 1929). Seedlings are slow growing; seeds sown in bogs cleared of vegetation required 4 years in cultivation and 7 years in the wild to attain sexual maturity (Østgård 1964). Genets (genetic individuals or clones) can extend for several metres and have many ramets (individual aboveground shoots) (Korpelainen et al. 1999). To examine variation in fruit production of R. chamaemorus in Eastern North America, we determined flowering and fruiting of individual ramets, and evaluated the components of fruit production. We also examined the sex ratios of flowering ramets at spatial scales from small plots to populations. We address the following questions. (i) How do the components of fruit production (ramet density, ratio of flowering to all ramets, fruit set, and seed set) vary with habitat? (ii) Do sex ratio biases occur, and if so, at what spatial scale? (iii) Is fruit production related to sex ratio or to distance between male and female flowers? We predicted that (i) fruit production would be correlated with vegetation shelter ; (ii) significant sex ratio biases would occur at small spatial scales as a result of clonal growth; and (iii) fruit production would be reduced in locations with a strong female-biased sex ratio and with increasing distance between male and female flowers. Materials and methods Flowers and fruits of Rubus chamaemorus Each ramet produces only a single flower; thus in R. chamaemorus the number of flowers equals the number of sexually reproductive ramets. Male flowers have numerous stamens and the female flowers have multiple pistils. The petals are larger on male than on female flowers, and male flowers produce nectar whereas female flowers do not (Mäkinen and Oikarinen 1974; Ågren 1987). The aggregate fruits are orange to red. Field sampling The study was conducted around St. Michael s Bay, in southeastern Labrador (52845 N, W). This region is characterized by extensive black spruce (Picea mariana (P. Mill.) B.S.P.) forests and peatlands (Glaser and Foster 1983). Twenty-one sites were selected, of which 18 were located on islands and 3 on the mainland. These sites were identified by a long-time local resident as areas where community members have harvested R. chamaemorus. All sites were located within 12 km of St. Michael s Bay, and ranged in elevation from 8 to 30 m a.s.l. The size of the sampling area ranged from 47 m 19 m to 377 m 336 m. Five of the sites were selected for intensive study (hereinafter, Intensive sites). At each of the Intensive sites, five transects were located, each having 10 contiguous 1 m 2 quadrats (50 quadrats per site). For the remaining 16 sites (hereinafter, the Extensive sites), four transects were located, each comprised five 1 m 2 quadrats, spaced 1 m apart (20 quadrats per site). At all sites, transects were evenly spread out to represent the range of variation in the area (e.g., moisture, shelter, species composition), with a minimum of 10 R. chamaemorus ramets per quadrat. At the Extensive sites, the total numbers of ramets and flowers of R. chamaemorus in each quadrat were counted between 23 June and 14 July 2004 (the first blossom was observed on 8 June 2004). At Intensive sites, the numbers of ramets, male flowers, female flowers, and fruits were counted in each quadrat. One female flower was randomly selected within each quadrat and the distance to the nearest male flower was measured, to a maximum of 900 cm. Five nearly ripe fruits from each quadrat were randomly selected, weighed, and the numbers of developed and undeveloped ovules counted. Several habitat variables were measured in each quadrat at all sites. Peat depth was measured, to a maximum of 1 m, near a R. chamaemorus plant. Three estimates of shelter (defined as any vegetation over 15 cm in height, including herbs) were made: average shelter height; shelter density (percentage of a quadrat with plants > 15 cm tall); and proportion of the edge of the quadrat from which a line extending 45 degrees from quadrat edge would contact vegetation. The percentage cover of vascular plant species and moss was recorded (nomenclature follows Meades et al. (2000); mosses were identified to genus). For Intensive sites, a composite substrate sample for each quadrat (from four samples representing the four quarters of the quadrat) was obtained at a 10 cm depth. Samples were weighed while wet, then frozen and later dried at 70 8C for 48 h, after which dry mass and ph (measured in a mixture of equal volumes of soil and distilled water) were determined. Data analysis Fruit set was calculated in each quadrat as number of fruits / total number of female flowers, and seed set as: number of developed ovules / total number of of ovules on the five unripe fruit collected. The ramet sex ratio was expressed as the proportion of female flowers (number of female flowers / total number of male + female flowers) in each quadrat. The proportions of female, male, and nonflowering ramets were calculated, based on the total number of ramets (the gender of nonflowering ramets could not be determined). The average mass per druplet was calculated as: average berry mass / average number of fertilized carpels. For the Intensive sites, cover per ramet was calculated as: total ramet cover / total ramet density within a quadrat.

3 206 Botany Vol. 86, 2008 Table 1. Summary statistics for variables in Intensive sites (n = 50 per site); values are mean ± SE. Variable Site 1 Site 2 Site 3 Site 4 Site 5 Peat depth (cm) 61.1± ± ± ± ±2.70 Soil moisture 7.81± ± ± ± ±0.22 ph 3.92± ± ± ± ±0.02 Ramet density (no./m 2 ) 132.3± ± ± ± ±8.48 Cover per ramet (% total cover/no. of ramets per m 2 ) 0.261± ± ± ± ±0.011 Nonflowering ramets/total ramets 0.716± ± ± ± ±0.016 Proportion of ramets flowering 0.285± ± ± ± ±0.016 Fruit set (no. of fruits/no. of female flowers) 0.779± ± ± ± ±0.033 Seed set (no. offertilized ovules/total ovules) 0.699± ± ± ± ±0.028 Mass of berry (g) 0.49± ± ± ± ±0.045 Averaged druplet mass (g) 0.041± ± ± ± ±0.004 Total ovules per flower 12.3± ± ± ± ±0.366 Female ramets/total ramets 0.122± ± ± ± ±0.013 Male ramets/total ramets 0.162± ± ± ± ±0.023 Distance to nearest male flower (cm) ± ± ± ± ±2.83 Sex ratio (number of female flowers/ number of female flowers + number of male flowers) 0.463± ± ± ± ±0.044 Analyses of relationships to environmental variables were conducted at two levels, among sites (using all 21 sites; i.e., Extensive study) and within sites (using the five Intensive sites individually; i.e., Intensive study). For the among site analysis, sample size at the Intensive sites was adjusted to that of the Extensive sites by randomly omitting one transect at each site and using only odd numbered quadrats. Peat depth measurements were converted to a scale of 1 6 (1, 0 20 cm; 2, cm; 3, cm; 4, cm; 5, cm; 6, >100 cm). The three shelter measurements (density, average height, and proportion at 45 degrees ) were converted to scales of 0 2 (0, 0; 1, 1% 26% density, or, 31 cm height, or, 36% at 45 degrees; 2, >26%, or, 31 cm, or, 36%), and then added together to form a Shelter Index (0 6). Soil moisture was calculated as: wet mass dry mass / dry mass. We used nonmetric multidimensional scaling (PC-ORD version 4.25, McCune and Mefford 1999) to summarize species cover data, using cover values converted to a modified Braun Blanquet scale and using the Sorensen distance measure. Analysis of the Extensive site data was performed at the site level (using quadrat means); each Intensive site was analyzed separately at the quadrat level. Species present in two or fewer quadrats were omitted from the analysis. Ordination axis scores were used in subsequent analyses to represent environmental variation as indicated by variation in species composition. We used Spearman s rank correlations to determine relationships among R. chamaemorus variables (abundance and reproductive stages) and environmental factors at both among and within site levels (SYSTAT 1998). An r s of 0.400, which corresponds to P = 0.01 for a sample size of 40, was used as a guideline for significance to prevent overinterpretation of correlations. For analysis of Extensive site data, quadrats were averaged within each transect (to give four means per site). Each Intensive site was analysed separately, at the quadrat level. To determine whether sex ratios differed significantly from a 1:1 ratio at each of the Intensive sites, we performed a 2 test on the site means. To examine sex ratio variation within the Intensive sites we compared the sex ratio distribution at two scales (1 m 2 quadrats; n = 250) and transects (10 m long; n = 25) with that expected for a binary distribution of male and female ramets. We also determined the Pearson correlation of the sex ratio for quadrats separated by different distances (1 9 m) along transects to examine how sex ratio biases change with distance. ANOVA was used to analyse relationships between reproductive variables and sex ratio. For comparisons, quadrats were separated into male dominated (<50% females) and female dominated (>50% females). Results Habitat characteristics A total of 39 species occurred in quadrats with R. chamaemorus in the Extensive site data set. The most common species in the quadrats were Empetrum nigrum L., Rhododendron (Ledum) groenlandicum (Oeder) Kron & Judd, Sphagnum spp., Cladonia spp., Vaccinium angustifolium Ait., Vaccinium oxycoccus L., and Drosera rotundifolia L. (Appendix A, Table A1). There were only a few significant relationships between R. chamaemorus variables and environmental variables. All five Intensive sites had low ph, high soil moisture, and peat depth averaging between 45 and 70 cm among the sites studied (Table 1). Among the 21 Extensive sites, neither of the two R. chamaemorus variables (ramet density and proportion of ramets flowering) was significantly correlated with peat depth, shelter index, or either ordination axis (data not presented). For Intensive sites, cover per ramet, ramet density, flower to ramet ratio, seed set, and average druplet mass all showed at least one significant correlation with an ordination axis (Table 2), indicating relationships to species composition. Reproduction and sex ratios Average ramet density was 157/m 2 for Extensive sites and 133/m 2 for Intensive sites. Site 2 had the highest mean ramet density, which was most similar to the average density for the Extensive sites. Sites 2 and 5 had both a low cover per ramet and a low flower to ramet ratio compared with the other sites, but both sites had the greatest mean druplet mass. Fruit set was high at all of the Intensive sites, averaging 0.75, as was seed set, which averaged 0.66 (Table 1).

4 Karst et al. 207 Table 2. Spearman s rank correlations of both environmental and reproductive variables with ordination axes (from nonmetric multidimensional scaling of species cover data) for each of the five Intensive Rubus chamaemorus sites (based on 50 quadrats per site). Site 1 Site 2 Site 3 Site 4 Site 5 Ordination axis 1 Cover per ramet Ramet density Seed set Average druplet mass Ordination axis 2 Cover per ramet Ramet density Proportion of ramets flowering Seed set Average mass druplet Note: Values in bold are considered significant (see text). Total ovules per flower averaged 10, being highest at Site 1 and lowest at Site 2. The proportion of nonflowering ramets was high in all sites, ranging from 0.63 to 0.81 (Table 1). Site 4, the only site with a higher proportion of female than male floral ramets, had the lowest means for fruit set and seed set. The mean distance between flowering male and female ramets was greatest at Site 1 and least at Site 5. The sex ratio was significantly male biased at two sites (Site 2, 2 = 8.74; Site 5, 2 = 11.9; P < 0.05) and female biased at one site (Site 4, 2 = 6.99; P < 0.05); two sites had means not significantly different from a 1:1 ratio (Sites 1 and 3) (Table 1). Sex ratios of individual quadrats were much more biased (both male and female) than expected for a random distribution of male and female ramets (Fig. 1a). The same pattern occurred at the scale of 10 m long transects (Fig. 1b). In over half of the transects, >80% of the flowering ramets were of one sex, and in 15 of 25 transects all 10 quadrats had a sex ratio bias in the same direction; both of these are situations with nil probability assuming a random distribution of male relative to female ramets. In the most extreme case, a single transect had 557 male ramets but only one female ramet, suggesting that one or a few male clones strongly dominated in an area 10 m in length. The sex ratio was highly correlated between adjacent 1m 2 quadrats (r = 0.885). When sex ratios were correlated between quadrats at different distances, the relationship decreased gradually with distance such that the correlation was still high (r = 0.592) at 9 m (Fig. 2), which is consistent with frequent occurrence of large clones (the x-intercept of the linear regression was 26 m). The sex ratio of flowering ramets within quadrats showed a few significant relationships to habitat variables at some sites, but relationships differed among sites (Table 3), providing no support for consistent habitat differences between males and females. Reproductive variables showed pronounced correlations to the sex ratio of flowering ramets. The proportion of ramets flowering was significantly and inversely correlated with sex ratio at two sites (Table 3) and for all quadrats in the Intensive sites combined (r s = 0.450). Seed set showed a similar pattern of correlations: significantly negative at three sites (Table 3) and for all quadrats combined (r s = 0.518) (Fig. 3a). Average druplet mass was inconsistently related to sex ratio at individual sites (Table 3), but showed a significant negative relationship for all quadrats combined (r s = 0.471) (Fig. 3b). When quadrats were separated into female-biased and male-biased categories, the groups were significantly different (ANOVA) for all three variables. Female-dominated quadrats had a lower proportion of ramets flowering (0.230 vs ; F = 12.26, P < 0.001), seed set (0.567 vs ; F = 41.58, P < ), and average druplet mass (0.038 g vs g; F = 18.06, P < ). Distance to the nearest male flower showed inconsistent correlations with reproductive variables among sites (Table 3). However, seed set showed a significant negative correlation when all quadrats were combined (r s = 0.472) (Fig. 4). Discussion Environmental factors and species composition Although previous studies of R. chamaemorus have found strong correlations between fruit production and shelter (Lohi 1974; Mäkinen and Oikarinen 1974; Yudina 1993), we found no such relationship. This may be a function of the array of habitats surveyed in this study area, which represents only a portion of the habitats occupied by this species. It may also reflect favourable climatic conditions. No frost or heavy rain occurred during the flowering and fruiting period in our study; thus shelter would not have benefited fruit production. Information from local communities indicated that production of fruit by R. chamaemorus was high all along the coast of Labrador during the summer of We found no relationship of reproductive stage to either ph or moisture, perhaps because the range of values among and within the sites sampled was too low to detect differences. The low ph values ( ) were consistent with values reported in previous studies (Lohi 1974; Thiem 2002). Species assemblages associated with R. chamaemorus in our study area were similar to those of other R. chamaemorus habitats in Newfoundland and Labrador (Savory 1981; Glaser and Foster 1983). In general, species composition was fairly uniform across all sites. The most consistent relationship observed between ordination axes and other variables was with the Shelter Index, suggesting that species are separated along a gradient from sheltered to open areas. Fruit set and sex ratios Over-initiation of flowers in R. chamaemorus populations (i.e., the production of more flowers in a season than can develop into fruits) may be an adaptation to highly variable climatic conditions, allowing plants to take advantage of favourable years (Lovett Doust and Lovett Doust 1988; Ågren 1988a). Because of the lack of extreme weather conditions during the period of R. chamaemorus fruit development in our study, we expected that fruit set would be high compared with other studies if over-initiation of flowers was re-

5 208 Botany Vol. 86, 2008 Fig. 1. The distribution of sex ratios [, /(, + <)] of Rubus chamaemorus for (a) 1m 2 quadrats (n = 247) and (b) 10 m long transects (n = 25). Expected distributions are based on the binomial distribution for a sex ratio of 0.381, the ratio for the 8701 flowering ramets sampled, and are conservative because for quadrats the expected distribution is based on 20 ramets per quadrat whereas the actual mean is 34.8, and for transects, 100 ramets per transect is used, whereas the mean is 348 and only one transect had less than 100 ramets (it had 97). lated to variability of weather. However, the average fruit set (74%) was comparable to values reported from northern Sweden and Finland (72.5% 75.6%; Ågren 1989), and was intermediate between values reported in two Quebec studies (57% and 94%) (Dumas and Maillette 1987; Pelletier et al. 2001). The values we found are also similar to average fruit set reported in a summary of dioecious species (0.724; Sutherland 1986). Sex ratios in dioecious species are commonly male biased (Lloyd and Webb 1977; Melampy and Howe 1977; Willson 1983; Allen and Antos 1993; but see Crawford and Balfour 1983; Niesenbaum 1992). Overall, our results tend to sup-

6 Karst et al. 209 Fig. 2. Pearson correlation coefficients of the sex ratio of R. chamaemorus between quadrats separated by 1 9 m along 25 transects, each 10 m long (10 contiguous quadrats per transect). Sex ratios were arcsin transformed before correlation coefficients were calculated. Fig. 3. (a) Seed set (r s = 0.518) and (b) average druplet mass (r s = 0.471) vs. sex ratio [, /(, + <)] of R. chamaemorus for all Intensive site quadrats (n = 207). port this generalization, but sex ratios varied greatly among our populations, suggesting multiple causal factors. Observed sex ratio biases of flowering ramets can result from various factors, including differential mortality, flowering frequency, and clonal growth (Allen and Antos 1993), any of which could contribute to the overall male-biased ramet sex ratio that we obtained (0.381 for all of the 8701 flowering ramets at the five Intensive sites). Lower mortality of male R. chamaemorus could result either from their greater resistance to frost and defoliation damage (Mäkinen and Oikarinen 1974; Ågren 1987) or their lack of fruit production costs (Ågren 1988a). As is typical of dioecious species (e.g., Allen and Antos 1988; Abe 2002), female R. chamaemorus have much higher total reproductive effort (Ågren 1987; Korpelainen 1994). Differential flowering of males and females yields an apparent bias (Lloyd and Webb 1977, Lovett Doust and Lovett Doust 1988; Allen and Antos 1993). Although the sex of nonflowering ramets could not be determined, the greater proportion of ramets that were flowering in male-dominated quadrats suggests a higher flowering propensity of males than females, which is consistent with previous observations for this species (Ågren 1988b). Clonal growth could also contribute to the overall sex-ratio bias if male clones produced more ramets than female clones. Rhizomes of male ramets begin growth earlier in a season than those of fruiting females (Ågren 1988b), but a contribution of differential rhizome growth to malebiased ramet sex ratios has not been conclusively established. However, the local sex ratio biases we observed, which are both male and female biases, likely relate to clonal growth. The significant relationships of reproductive variables with sex ratio and with distance to nearest male flower, both within sites and among all Intensive sites collectively, suggest that pollen availability may affect reproductive output in these populations. The significant relationships of sex ratio to seed set found in this study have not been observed in studies of other species (Bullock and Bawa 1981; Barrett and Thomson 1982) or R. chamaemorus populations; however, Ågren et al. (1986) detected limitations on seed set in female dominated habitats using hand-pollination experiments. The more pronounced negative relationship of sex ratio (proportion of female flowers) to seed set than to fruit set suggests that the skewed sex ratio limited efficient pollen flow. Reduction in visitation by pollinators would have more affect on the repeat visits necessary to increase the number of fertilized ovules than on the initial visits affecting fruit set. There may be too few male flowers in an area of female clones to attract pollinators, because female R. chamaemorus provide no rewards to the pollinator. This effect is also suggested by the negative relationship between distance to nearest male flower and seed set, a trend that was also found in Silene dioica (L.) Clairville (Kay et al. 1984). Female flowers in close proximity to males are more likely to attract insects by deceit and thus receive pollen (Ågren et al. 1986). Some studies have found male and female plants spatially segregated along resource gradients (Cox 1981; Willson 1983; Bierzychudek and Eckhart 1988; Lovett Doust and Lovett Doust 1988; Nicotra 1998), and a few studies indicate that female R. chamaemorus clones are more abundant in moist than in dry habitats (Dumas and Maillette 1987; Ågren 1987). The inconsistent relationship we found be-

7 210 Botany Vol. 86, 2008 Table 3. Spearman s rank correlations of sex ratio and distance to nearest male flower with other variables for each of the five Intensive R. chamaemorus sites. Site 1 Site 2 Site 3 Site 4 Site 5 Sex ratio Shelter index Peat-depth scale Ordination axis Ordination axis Cover per ramet Proportion of ramets flowering Fruit set Seed set Average druplet mass ln distance to nearest male Shelter index ph Proportion of ramets flowering Fruit set Seed set Average druplet mass Note: Values in bold are considered significant (see text). Fig. 4. Distance to nearest male flower (log-transformed) vs. seed set (r s = 0.472) of R. chamaemorus for all Intensive quadrats (n = 207). tween sex ratio and shelter (positive in two sites, negative in two sites) and lack of relationships between sex ratio and other environmental variables suggest that there was no spatial segregation of male and female plants along the environmental gradients measured. Although strong effects of microhabitat differences on sex-related mortality or flowering propensity could contribute to the very pronounced local sex ratio biases we observed, we found no evidence for such differential effects of habitat; clonal growth is a much more parsimonious explanation for the local sex ratio biases. Fruit production in R. chamaemorus populations is controlled by a variety of factors that are likely to vary greatly in importance among years and locations. Climatic variables, site factors, and population characteristics all interact to affect fruit production. During the study summer, climatic conditions that normally might have a major influence on fruit production were extremely mild, allowing population characteristics, such as sex ratio, to have the most prominent influence on fruit production. The large variation in local sex ratios suggests that a reliance on vegetative growth has resulted in large clone sizes in this area. The presence of large clones has led to limitations on sexual reproduction, as indicated by decreased seed set with local female-biased sex ratios and with increasing distances between male and female flowers. However, given the high fruit set measured, it appears that the strong reliance on clonal growth does not compromise the ability of R. chamaemorus to produce abundant fruit during favorable years in this unpredictable environment. Acknowledgements This research was funded by Natural Sciences and Engineering Research Council, Coasts Under Stress, Northern Scientific Training Program, and the Labrador Metis Nation. We thank John Kippenhuck for providing boat services and Lael Kippenhuck for field assistance. Thanks to Dr. Luise Hermanutz for her assistance with the project, and her lab for providing support during ph analysis. We thank Dr. Peter Scott for help with plant identification; Susan Meades for inquiries pertaining to Labrador plants; and Dr. Mark Schlessman and anonymous reviewers for comments on the manuscript. References Abe, T Flower bud abortion influences clonal growth and sexual dimporphism in the understorey dioecioius shrub Aucuba japonica (Cornaceae). Ann. Bot. (Lond.), 89: doi: /aob/mcf111. PMID: Allen, G.A., and Antos, J.A Relative reproductive effort in the dioecious shrub Oemleria cerasiformis. Oecologia, 76: Allen, G.A., and Antos, J.A Sex ratio variation in the dioecious shrub Oemleria cerasiformis. Am. Nat. 141: doi: / Aspinwall, N., and Christian, T Clonal structure, genotypic

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9 212 Botany Vol. 86, 2008 Table A1. Species found in quadrats with Rubus chamaemorus and percentage of quadrats (out of 420) in which a species was present. Species Percent of quadrats Species Percent of quadrats Empetrum nigrum 92.6 Equisetum spp 8.1 Rhododendron groenlandicum 88.3 Carex trisperma 6.4 Sphagnum spp Trichophorum cespitosum 5.0 Polytrichum spp Andromeda glaucophylla 4.5 Vaccinium angustifolium 71.7 Kalmia angustifolia 3.6 Vaccinium oxycoccus 68.3 Lycopodium annotinum 3.1 Drosera rotundifolia 67.4 Gaultheria hispidula 2.9 Cladonia spp Carex microglochin 2.6 Kalmia polifolia 58.1 Diapensia lapponica 2.1 Vaccinium vitis-idaea 52.6 Cornus Canadensis 1.7 Chamaedaphne calyculata 39.5 Eriophorum vaginatum 1.7 Maianthemum trifolium 35.7 Coptis trifolia 1.4 Carex rariflora / C. pallescens 22.9 Chamerion angustifolium 0.7 Vaccinium uliginosum 22.1 Linnaea borealis 0.7 Picea mariana 18.1 Trientalis borealis 0.7 Larix laricina 17.9 Eriophorum angustifolium 0.4 Myrica gale 13.6 Arctous alpina 0.2 Rhododendron tomentosum 10.0 Carex bigelowii 0.2 Geocaulon lividum 9.5 Sibbaldiopsis tridentate 0.2