The mating system of flowering plant populations evolves through selection on genetically based phenotypic variation in floral traits. The physical separation of anthers and stigmas within flowers (herkogamy) is expected to be an important target of selection to limit self‐fertilization. We investigated the pattern of phenotypic and genetic variation in herkogamy and its effect of self‐fertilization in a broad sample of natural populations of Aquilegia canadensis, a species that is highly selfing despite strong inbreeding depression. Within natural populations, plants exhibit substantial phenotypic variation in herkogamy caused primarily by variation in pistil length rather than stamen length. Compared to other floral traits, herkogamy is much more variable and a greater proportion of variation is distributed among rather than within individuals. We tested for a genetic component of this marked phenotypic variation by growing naturally pollinated seed families from five populations in a common greenhouse environment. For three populations, we detected a significant variation in herkogamy among families, and a positive regression between parental herkogamy measured in the field and progeny herkogamy in the greenhouse, suggesting that there is often genetic variation in herkogamy within natural populations. We estimated levels of self‐fertilization for groups of flowers that differed in herkogamy and show that, as expected, herkogamy was associated with reduced selfing in 13 of 19 populations. In six of these populations, we performed floral emasculations to show that this decrease in selfing is due to decreased autogamy (within‐flower selfing), the mode of selfing that herkogamy should most directly influence. Taken together, these results suggest that increased herkogamy should be selected to reduce the production of low‐quality selfed seed. The combination of high selfing and substantial genetic variation for herkogamy in A. canadensis is enigmatic, and reconciling this observation will require a more integrated analysis of how herkogamy influences not only self‐fertilization, but also patterns of outcross pollen import and export.
We studied 10 populations of A. canadensis located in eastern Ontario, Canada in the northern portion of the species' geographical range, and eight populations in the southern Appalachian region at the center of the species' range in Virginia and North Carolina, U.S. (Appendix 1). These populations spanned the range of population size, plant density, and habitats typical of this species, from large, dense patches of up to 2000 individuals on exposed rocky outcrops to small, scattered clusters of a few dozen individuals under a forest canopy.
To examine the phenotypic variation in floral morphology, a single observer measured (to 0.1 mm) herkogamy as the minimum distance between stigmas and anthers midway through anthesis, maximum pistil length from the receptacle to the stigma, and the length of one nectar spur from the nectar gland to the tip of the lamella on one flower per plant. Stamens could not be measured without destroying flowers (see below). On a subset of 71 plants, we measured each flower twice and assessed measurement error as the intraclass correlation coefficient (rI ). All floral measurements were highly repeatable for this (herkogamy rI = 0.989; pistil length rI = 0.978; spur length rI = 0.991; all P < 0.0001) and all other analyses reported below. We used spur length as a general index of floral size, because it correlates positively with the size of other floral organs (e.g., sepals and pistils, Herlihy and Eckert 2005). We sampled ∼65 and ∼80 plants per northern population in 1999 and 2000, respectively, and ∼50 plants per central population in 2000. We collected fruits produced by all measured flowers for genetic analysis (see below).
To contrast the amount of phenotypic variation between different floral traits, we used a modification of Levene's median ratio test (Schultz 1985) in which individual values (i) for a floral measurement are expressed as absolute relative deviations from the population median
We compared the population mean deviations (MdRi) of herkogamy to those for pistil length and spur length using paired t ‐tests. We compared individual MdRi values between floral traits within each population using nonparametric Wilcoxon tests (because sample variances in individual MdRi values were not homogeneous).