Upper lip length and lower lip width were the two corolla traits exhibiting the closest phenotypic integration at the two L. latifolia populations studied. Results of experimental corolla manipulations, however, are not consistent with the interpretation that the correlated variation of these two traits is the result of correlational selection exerted by pollinators. Manipulations modified drastically the relative proportions of the upper and lower corolla lips, yet these changes had no significant effects in terms of either male or female components of pollination success. Furthermore, the direction of the significant UpperLip–LowerLip interaction effect on realized maternal fecundity was opposite to that expected if correlational selection were responsible for observed integration. Experimentally de-integrated flowers with two aberrant morphologies tended to set more seeds per fruit than control flowers with normal two-lipped corollas. The potential significance of these results and possible interpretations are discussed in the following sections.
Proportional pollen removal and number of pollen tubes are valid descriptors of per-flower male and female pollination success in L. latifolia. Measuring realized male reproductive success in hermaphroditic plants requires tracking seed paternity using genetic markers (Snow, 1989; Wilson et al., 1994; Ashman, 1998), yet pollen removal on a per flower basis is a useful comparative index to evaluate the male component of pollination success, as it provides an upper estimate of the number of pollen grains that become available for siring seeds (Young & Stanton, 1990; Wilson & Thomson, 1991; Conner et al., 1995; Wilson, 1995a; J. Herrera, 1997). Using number of pollen tubes, rather than of pollen grains on the stigma, as a measurement of the female component of pollination success was motivated by the presumption that pollen grain counts on the dried stigmas of withered flowers could be unreliable, as an unknown fraction of pollen grains could have been dislodged from the stigma as it dried up. In the study site, the number of pollen tubes in the basal portion of the style of female-stage L. latifolia flowers is closely correlated with the total number of pollen grains deposited on the stigma (C. M. Herrera, unpublished data; a similar relationship was reported by J. Herrera, 1997, for the congeneric Lavandula stoechas), hence observed variation among flowers in number of pollen tubes will closely reflect variation in pollen deposition.
Manipulations of corolla morphology performed in this study could have influenced the male and female pollination success of L. latifolia flowers through at least three different mechanisms. Firstly, experimental petal cuttings might have influenced pollinator foraging behaviour directly, regardless of the changes induced in corolla morphology. For example, petal cutting could have either enhanced or reduced the release of floral volatiles, which could in turn influence pollinator foraging. I do not have any data bearing directly on this possibility, but some indirect evidence tends to rule it out. If corolla cutting modified pollinator foraging through its effect on volatile emission, I would expect a significant decline in treatment effect from flowers with the two lips manipulated to those with only one lip cut, which was not observed (tested using a posteriori contrasts in generalized linear mixed model; results not shown).
The other two ways would be related to the induced changes in corolla morphology, and thus directly relevant to the objectives of this investigation. Manipulations of corolla morphology may modify the attractiveness of individual flowers to pollinators and, consequently, influencing the number of pollinator visits received over the entire life of the flower. All treatments reduced overall corolla size, which was expected to result in decreased pollinator attractiveness, as found in other insect-pollinated plants (e.g. Bell, 1985; Stanton & Preston, 1988; Schemske & Ågren, 1995; Conner & Rush, 1996; Ashman et al., 2000; Philip & Hansen, 2000). Furthermore, manipulations disrupted heavily the shape of flowers in front view (i.e. as perceived by an approaching pollinator) in comparison to normal flowers, which could have also affected attractiveness to visitors. I did not measure pollinator visitation rates for this study, but the lack of any significant effect of corolla manipulation on both pollen removal and deposition suggests that the number of effective pollinator visits received by individual flowers over their lifespan was unaffected by experimental modifications of the corolla. Another mechanism would involve modifications in the amount of pollen removed and/or deposited per pollinator visit, which could have occurred even if corolla manipulation had no effect on overall pollinator attraction (Campbell et al., 1991; Smith et al., 1996; Temeles & Rankin, 2000). No data are available to evaluate directly this second possibility, but if corolla manipulation actually affected mean pollinating effectiveness per visit, the effect was too weak to translate into measurable differences in per-flower pollen removal and deposition.
It seems safe to conclude that, under the conditions of this study, variations in the morphology of the corolla were inconsequential for both male and female pollination success of individual L. latifolia flowers. One possible explanation for this finding could be that, during the study period, pollinators were so abundant in relation to the number of flowers available that they foraged indiscriminately over all floral phenotypes and included in their foraging bouts flowers with unpreferred corolla morphologies to obtain sufficient nectar or pollen returns. This would lead to pollinator visits becoming so frequent as to ‘saturate’ the pollen removal and deposition functions irrespective of floral phenotype (Wilson, 1995b). Some indirect evidence partly supports this view. For the vast majority of flowers, the number of pollen tubes was well in excess of the number of ovules, thus suggesting an effective saturation of the female function. For the male function, results are more difficult to interpret in relation to the saturation hypothesis. On one side, removal of pollen grains was far from complete, as only 53% of pollen grains were removed on average. This proportion is considerably lower than the 80–90% pollen removal reported for bee-pollinated flowers in general (Mitchell & Waser, 1992; Wilson, 1995a, b), and also lower than the 82% reported by J. Herrera (1987) for the congeneric, bee-pollinated Lavandula stoechas. These observations would contraindicate male function saturation of L. latifolia flowers in this study. But, on the other side, one would expect that mean number of undispersed pollen grains per flower will approach different asymptotic values depending on the specific composition of pollinators and their thoroughness at removing pollen. Casual observations on experimental plants during this study indicated that, in accordance with previous studies in the same locality (e.g. Herrera, 1987, 1988, 2000), flowers were visited by a diverse insect assemblage composed not only of pollen-gathering female bees, but also of nectar-feeding butterflies and male solitary bees that only passively remove pollen. The modest pollen removal figures found in this study might still reflect a situation of male saturation for the particular specific configuration of pollinators prevailing during the study period.
Seed production per L. latifolia flower is generally insensitive to artificial increases in the quantity of pollen reaching the stigmas, but it does respond positively to experimental increases in resource availability and the quality (cross vs. self) of the pollen (Herrera, 1987, 1990, 1991, 2000). Results of the present study also suggest that failure of flowers to produce some seed was very rarely due to insufficient pollination. Flowers that eventually failed to set seed (N=147) had a mean (±1 SD) of 12.6 ± 7.3 pollen tubes in the style, and only 12% of them had pollen tubes/ovules ratios <1.
The only statistically significant effect of corolla manipulation found in this study was the interaction of UpperLip and LowerLip on seed set. Flowers that had either the upper lip alone or the lower lip alone manipulated tended to set more seeds than ‘normal’ unmanipulated flowers. This result cannot be related to differences between floral morphologies in pollen deposition, as there was no significant effect of UpperLip, LowerLip or their interaction on number of pollen tubes. Differences among floral morphologies in the quality of the pollen received may account for the observed results. In L. latifolia, even slight differences in pollinator composition may result in seed set differences, presumably because of variations in the proportion of selfed vs. outcrossed pollinations performed by different species (Herrera, 2000). The different pollinators of a given plant species may respond differentially to corolla variations (Johnson et al., 1995; Thompson, 2001). Consequently, the seed set advantage of floral ‘morphs’ with either the upper or the lower lips manipulated found in this study may be due to these flowers being visited proportionally more often by species predominantly performing cross pollinations, like butterflies. Testing this hypothesis will require direct observations of pollinator foraging responses to floral manipulations. Preliminary data, however, suggest that this is a plausible possibility, as butterflies tend to select preferentially L. latifolia shrubs characterized by relatively longer upper lips and smaller lower lips (C. M. Herrera, unpublished data).
The experimental design used in this study was based on the premise that the two lips of the corolla played different roles in the pollination of L. latifolia flowers. While the conspicuous, vertical upper lip presumably acts enhancing insect attraction, the lower lip serves as a landing platform and foothold to pollinating insects (Herrera, 2000, Plate 1), thus probably affecting their foraging efficiency and flower handling times. The attractive role of the upper lip of Labiatae flowers was long ago demonstrated experimentally by Clements & Long (1923) for the insect-pollinated Monarda fistulosa. The influence of the lower lip of Labiatae flowers on pollinator handling time and pollen deposition and removal has been documented, for example, by Temeles & Rankin (2000) for the hummingbird-pollinated Monarda didyma. These observations thus lend biological sense to the experimental dissection of the effects of upper and lower lips attempted in this study, and provide biological and statistical justifications for questions about their possible interactions.
Results of this study are clearly contrary to the interpretation that correlated variation of upper and lower lips in L. latifolia corollas is the result of correlational selection exerted by pollinators. Artificial enhancement of morphological variation and the phenotypic de-integration of the two lips was inconsequential in terms of pollination success. Even more importantly, two aberrant floral morphologies characterized by strong de-integration relative to the normal phenotype had a distinct realized maternal fecundity advantage over normal, two-lipped corollas. These results suggest that pleiotropic and/or developmental effects are possibly more important that pollinator selection in maintaining the phenotypic integration of the upper and lower corolla lips in L. latifolia. This interpretation is not at odds with what is known on the genetic basis of corolla morphology and relative size of corolla parts in other zygomorphic flowers. In Antirrhinum majus and Linaria vulgaris, single regulatory genes (cycloidea and Lcyc, respectively) are responsible for the zygomorphism characteristic of these species, which results from the arrangement of the five corolla lobes into two unequal, bilaterally symmetric parts (Coen & Nugent, 1994; Luo et al., 1995; Cubas et al., 1999). Mutations affecting these genes produce drastic transformations from zygomorphic corollas with two unequal lips to radially symmetrical ones with five similarly sized lobes. This suggests that, in these species, the relative dimensions of corolla parts (lips in the case of wild-type zygomorphs, lobes in the case of mutant actinomorphs) are most likely subject to strong pleiotropic effects.
Two aspects must be kept in mind when interpreting the results of this study. Firstly, the ability to detect selection on floral traits may be strongly dependent on variations in the abundance of pollinators, as emphasized by Wilson (1995b). I have censused L. latifolia pollinators at the study locality every year in the period 1982–1987 (Herrera, 1988), in 1991 (Herrera, 2000), and again during 1996–1998 (C. M. Herrera, unpublished data), thus spanning a 17-year period. These long-term data reveal that abundance and composition of the pollinators of L. latifolia fluctuate from year to year at the study locality, hence the likelihood of detecting selection may likewise vary. Some results shown here may therefore be contingent on pollinators being very abundant during the study year, as noted earlier. Were this the case, however, it must be emphasized that there are reasons to suspect that similar results would be obtained in other years as well, and are probably not unusual for the L. latifolia population studied. Although I did not conduct pollinator censuses during this study, my subjective impression in the field was that pollinator abundance in 2000 was not above the average levels for the period 1982–1998. A second aspect to be considered is that results of this investigation might be dependent on aspects of the experimental design used. From an evolutionary viewpoint, it is differences between individual plants in reproductive success, not between individual flowers, that matters. Results of this study, based on between-flower comparisons, need to be corroborated in a between-plant context before rigorous evolutionary inferences can be done. In short, it is not possible to know if the irrelevance of corolla morphology for the pollination success of L. latifolia flowers found in this study would persist in years with fewer pollinators, or in populations where pollinators are consistently scarcer than in the Aguaderillos site (e.g. populations on arid slopes, located well away from permanent streams; Herrera, 1988), and/or if whole plants, rather than single flowers, were the experimental units chosen. Future studies will consider these two possibilities.