Calyx Size in Relationship to Seed Number and Weight

Twenty-eight Phacelia stebbinsii Constance & Heckard, 28 P. marcescens Eastw. ex J.F.Macbr. and six P. quickii J.T.Howell plants from four, four, and two populations, respectively were field-collected in 1980 and 1981 with each individual plant's calyces and associated seeds divided into lots based on number of seeds produced per fruit. The calyces and seeds of each seed number lot were weighed as a group to obtain the average calyx weight and average total seed weight per fruit to assess if average calyx size was related to the number of seeds in a fruit and total seed weight per fruit.

The correlation between calyx size and total seed weight per associated capsule was further tested by individually weighing 25 field-collected calyces and their associated seeds, henceforth referred to as a fruiting structure, from one population of each of ten taxa in Phacelia subsect. Humiles. These fruiting structures were on plants previously collected to provide seed for hybridization and cytogenetic studies. These particular species' populations were used because my collections of them contained a sufficient number of fruiting structures. The fruiting structures were randomly drawn from an envelope containing hundreds of fruiting structures derived from a minimum of six different individuals per taxon. The plants sampled were field-collected from locations listed in Appendix 1. The individual plants used in the study all had high levels of seed set and largely unopened fruit.

Allometry

The study site for this experiment was located in Placer County 100 meters south of Highway 20, 0.4 km east of the Nevada County line. A 107 cm long by 104 cm wide by 54 cm high insect exclusion box constructed from 3/4” by 3/4” wooden molding and mosquito netting was placed over part of a dense uniform-sized Phacelia marcescens population as it initiated flowering. The box's function was to reduce fruit set to test if the total seed weight-calyx-weight correlation was allometric (i.e., positively correlated for developmental reasons). If calyx size and total seed weight per fruit are allometrically related, then under conditions of low fruit set, the positive correlation between them should be as strong as under conditions of high fruit set. If the relationship between them could be decoupled by a dramatic reduction in the percentage of flowers developing fruits acting as photosynthetic sinks, that would indicate the relationship was not allometric. Phacelia marcescens was chosen for this experiment because its capsules retain their seed at maturity which made fruit and seed collection easy, and its self-compatible flowers, which are not markedly autogamous (Lee, 2022), ensured pollinator exclusion would significantly reduce but not eliminate seed set.

After fruit set, 56 plants were collected, 28 covered by the insect exclusion box to reduce pollination and fruit set, and 28 control plants from outside the box. Control plants were taken from the box's northern edge so they would have experienced shading similar to within-box plants. Each harvested plant was placed in a separate envelope. Mean fruit set in the control plants was 75% (SD = 8%). Mean fruit set in the treatment group (12%, SD = 8%) was too low to make within-plant correlations so only one intact fruiting structure was sampled per plant from both groups. The fruiting structure chosen for each plant was the first one with seed in an intact capsule to shake out of each envelope. Calyces were dried at 70°C for 72 hours and weighed to the nearest 0.1 mg.

Defoliation

Phacelia orogenes Brand seeds collected along the Eagle Lake Trail in Mineral King, Tulare Co. and P. quickii seeds collected 0.2 miles southwest of Glacier Point, Yosemite National Park were grown in a growth chamber with all leaves removed as they formed. Plants were made “leafless,” by removing all leaf buds as they appeared. At no time did they have leaf tissue that reached beyond 1 mm in length, leaving them dependent upon cotyledon, stem, and floral photosynthesis. Additional plants which had no leaf tissue removed were grown for Phacelia orogenes to provide an experimental control. The growth chamber environmental regime was 16 hours light (450–550 µE/m^–2/s^–1) at 17°C, followed by eight hours of dark at 11°C. Phacelia orogenes and P. quickii were chosen for this experiment because previous work demonstrated that under long-day conditions they could flower subsequent to the first or second pair of leaves forming. Flowers were hand pollinated using a forceps to pluck stamens with dehiscing anthers and to then touch those anthers to another plant's punctiform stigma.

For calyx size comparisons in the leaf removal experiment, calyces from four Phacelia orogenes plants, two with leaves removed and two with leaves left intact, were removed and weighed at anthesis. This was repeated with four other plants after seed set and just prior to senescence, at which time the sampled calyces were at their maximum size. Calyces were dried and seeds weighed as described above. Calyx dry weight was used as a surrogate for photosynthetic capacity (Herrera 2005) throughout this paper because measuring length was less precise than weight, and sepal shape and thickness were potentially confounding factors.

Carbon Allocation

The carbon invested in calyces versus leaves was examined using 22 additional Phacelia marcescens plants from the population used in the allometry experiment. They were field-collected when their fruiting structures had largely matured, but before seed dispersal had started to occur. They were dried as described above and each plant's total calyx dry weight was compared to its total leaf dry weight.

Calyx Size in Relationship to Seed Number and Weight

In the first study of the relationship between calyx size and seed production, total seed weight per capsule increased as seed number increased in the fruits of Phacelia stebbinsii, P. marcescens, and P. quickii. Calyx size typically increased as total seed weight increased (Appendix 2).

In the second study, there was a positive correlation between calyx dry weight and total seed weight per capsule for all ten taxa examined (Fig. 1). Phacelia humilis Torr. & A.Gray var. humilis was the sole case where the relationship was not statistically significant. These positive correlations indicate a resource-based relationship between calyx size and fruit size.

Fig. 1.

Spearman rank correlation coefficients (r_s), P values, regression lines and equations for calyx dry weight (10^–4 G) versus total seed weight per fruit (10^–4 G) for ten different taxa in Phacelia subsect. Humiles. A. Phacelia austromontana r_s = 0.475; p < 0.01; y = 0.84x + 4.70; B. Phacelia congdonii r_s = 0.864; p < 0.001; y = 1.26x + 3.95; C. Phacelia divaricata r_s = 0.632; p < 0.001; y = 1.13x + 11.12; D. Phacelia humilis r_s = 0.077; p > 0.10; y = 0.28x + 8.25; E. Phacelia humilis var. dudleyi r_s = 0.399; p < 0.025; y = 1.82x + 10.13; F. Phacelia purpusii r_s = 0.550; p < 0.005; y = 0.79x + 12.88; G. Phacelia quickii r_s = 0 .877; p < 0.001; y = 1.01x + 1.77; H. Phacelia stebbinsii r_s = 0.518; p < 0.005; y = 0.47x + 4.39; I. Phacelia vallicola r_s = 0.475; p < 0.01; y = 1.76x + 13.65; J. Phacelia verna r_s = 0.652; p < 0.001; y = 1.75x + 0.10. N = 25 for each sample. Collection locations in Appendix 1.

Allometry

A possible alternative explanation for the relationship between calyx size and total seed weight per capsule could be allometry, where the size of one organ is developmentally tied to the size of another.

In order to rule out allometry as a possible explanation, pollinators were excluded from a group of Phacelia marcescens plants to reduce seed set. In the control group where pollinators were not excluded, mean fruit set was 75% (SD = 8%) and calyx dry weight was strongly positively correlated with total seed weight per fruit [r_s = 0.77; P (2-tailed) < 0.001; y = 1.4126x + 1.003]. In the treatment group where pollinators were excluded, mean fruit set was reduced to 12% (SD = 8%) and the correlation between calyx dry weight and total seed weight per capsule fell to r_s = 0.21 [P (2-tailed) = 0.28; y = 0.5146x + 5.499] (Fig. 2).

Fig. 2.

Calyx dry weight (10^–4 G) versus total seed weight per fruit (10^–4 G) for low and high fruit set Phacelia marcescens plants: (A) low fruit set (inside box): r_s = 0.21; P = 0.28; y = 0.515x + 5.499; (B) high fruit set (outside box): r_s = 0.77; p < 0.001; y = 1.413x + 1.003. N = 28 for each sample.

If the calyx size and seed weight per capsule positive correlation was due to allometry, then low fruit set should not affect it. If the correlation represents a functional source-sink relationship, when fruit set is low then calyx-produced photosynthate from unfertilized flowers could provide an alternate carbon source for fertilized flowers' seeds, thereby lessening the effect an individual calyx might have on its associated capsule's yield (expressed as total weight of seed produced). This experiment's results support a source-sink hypothesis because the relationship between calyx size and fruit weight was uncoupled under low fruit-set conditions when photosynthate from unfertilized flowers calyces presumably would be more available to developing fruits throughout the plant. This interpretation is further supported by the slope in the regression lines. The slope for the regression line in the low fruit set group is just 36% of the size of the slope in the high fruit set group, indicating that calyx size in low fruit set conditions has a smaller effect on total seed weight of the associated fruit than it does in high fruit set conditions.

Defoliation

The P. orogenes plants grown without leaves thrived in the growth chamber, exceeding in size many field-collected specimens (Fig. 3). In fruit, sepals from leafless P. orogenes plants became significantly larger than the control group's sepals even though they were smaller at anthesis (Table 1). Both sets of plants produced viable seed. Although measurements were not made, in leafless P. quickii plants growth was dramatically reduced and not all plants survived to produce seed, but those that flowered did expand their sepals to well beyond their normal sizes (Fig. 4). The increase in size of both species' calyces when leaves were removed further demonstrates the plasticity of Phacelia sepals and that the degree of accrescence can increase when the plant is stressed by defoliation and would benefit from activation of an alternate source of photosynthate.

Fig. 3.

Growth chamber grown Phacelia orogenes. The plant was completely defoliated at all times post-germination. All of the foliaceous tissue in the photo are sepals which were abnormally large for the species.

Table 1.

Kruskal-Wallis Test Comparing Calyx Dry Weight At Anthesis and after Accrescence for Phacelia orogenes Plants Grown With and Without Leaves. At anthesis the plants with leaves had significantly heavier calyces (H = 10.49; P < 0.025) while after accrescence, the calyces of the plants grown without leaves were significantly heavier (H = 36.98; P < 0.001).

Fig. 4.

Growth chamber grown Phacelia quickii. The plant was completely defoliated at all times post-germination. Note the abnormally large sepals developed subsequent to defoliation.

Carbon Allocation

One measure of the importance of calyces as photosynthetic structures in Phacelia is the amount of carbon invested in calyces versus leaves. In P. marcescens, which has smaller calyces than most species in Phacelia subsect. Humiles, average total dry weight of calyces from 22 field-collected P. marcescens plants was comparable to the dry weight of each plant's leaves over a wide range of plant sizes (Table 2). In 13 of the 22 individuals examined, total dry calyx weight per plant actually exceeded total leaf dry weight per plant and overall averaged 106.43% (SD = 35.22%, n = 22) of the leaves' total dry weight. While there was considerable variation in the calyx dry weight to leaf dry weight ratio amongst individual plants (Table 2), the overall relationship between the two was surprisingly linear over a wide range of plant sizes (Fig. 5).

Table 2.

Total Leaf Dry Weight (10^–4 g) and Total Calyx Dry Weight (10^–4 g) For Field-collected Phacelia Marcescens Plants.

Fig. 5.

Total calyx dry weight (10^–4 g) versus total leaf dry weight per plant (10^–4 g) at fruit maturation for 22 field-collected Phacelia marcescens plants.

Morphology

Many species in Phacelia subsect. Humiles have calyces with sepals which are markedly unequal in size. In every instance of markedly unequal sized sepals, the smallest one is adaxial and located between the capsule and peduncle with the larger ones located abaxially. The smallest sepal is thus in the most shaded position and the largest are in positions presumably receiving the most illumination. In these otherwise actinomorphic flowers, unequal-sized sepals may represent a modification for enhancing calyx photosynthesis.

Phacelia subsect. Humiles species with equal-sized sepals, e.g., P. curvipes Torr. ex S.Watson, P. davidsonii A.Gray, and P. douglasii (Benth.) Torr., typically have longer pedicels than species such as P. quickii, P. purpusii Brandegee, and P. mohavensis A.Gray with unequal-sized sepals. In taxa with equal-sized sepals, adaxial sepal-shading may be minimized, not by reducing that sepal's size, but by moving it away from the peduncle. In compact inflorescences, adaxial sepal-shading could still be a problem, but in Phacelia subsect. Humiles long pedicels and equal-sized sepals are often associated with less congested inflorescences, or with inflorescences, if distally congested, that are proximally laxer in fruit, as in P. orogenes. This is consistent with the observation that calyx shading should be less in distal flowers than for those located in more basal positions in the inflorescence. Another common pattern is unequal calyx lobes, short pedicels, and uncongested inflorescences in fruit, as in P. vallicola Congdon ex Brand., P. pringlei A.Gray, and P. leonis J.T.Howell. Phacelia humilis var. humilis and P. humilis var. dudleyi J.T.Howell are unusual in Phacelia subsect. Humiles as they have equal-sized sepals, short pedicels, and compact inflorescences, a poor combination of characteristics for reducing sepal shading. They were also the taxa in which the calyx size-fruit yield correlation was weakest (Fig. 1). For the most part, though, the observed inflorescence character combinations would appear to increase the light-harvesting potential of calyces, but whether or not this is functionally significant is beyond the scope of this paper.

Physiological Ecology

Photosynthesis that maximizes carbon gain and minimizes water loss is a documented attribute of some photosynthetic reproductive structures. For example, Earley et al. (2009) found that inflorescences in different genotypes of Arabidopsis thaliana contributed on average 55% of lifetime carbon gain, but only 25% to lifetime water loss. Bort et al. (1996) found that the awns of lemmas, which are modified sepals, increased water- use- efficiency (WUE) in barley. Weyhrich et al. (1995) found that awns increased WUE in wheat. However, unlike agricultural crops, the open habitats most Phacelia species occupy become very hot and dry as the growing season progresses. Therefore, if Phacelia calyces have greater WUE than their leaves, this could explain the relatively large late-season investment of carbon in calyces as compared to leaves.

Phacelia sepals are generally smaller than their leaves and morphologically, the calyx resembles a palmately dissected leaf. The temperature of small leaves and dissected larger leaves stays closer to ambient air temperatures than undivided large leaves because a thinner boundary layer enables heat to be convected away more effectively, thereby reducing the need for transpirative cooling and increasing WUE (Bannister 1976). In addition, Phacelia flowers are usually elevated relative to leaves, presumably for pollinator attraction, but this also elevates them above soil surfaces (and positions them well for light interception). As air temperature is usually highest at the soil surface and decreases upward (Geiger, 1950), Phacelia inflorescences may be experiencing a lower air temperature microenvironment than leaves. Calyces would therefore require less water per surface area than leaves for transpirative cooling, enhancing plant WUE.

The variability in and length of Phacelia pedicels and peduncles may also contribute to thinner boundary layers and/or better light interception. The plasticity of sepals, pedicels and peduncles; their relationship to seed production; the water use efficiency WUE of sepals versus leaves; the percentage of total lifetime carbon gain contributed by sepals; the effect of partial defoliation and its timing, such as might occur in nature, on sepal accrescence; the movement and distribution of calyx-produced photosynthate in the plant; and determining the prevalence and importance of the phenomenon described in this paper in other sections of the genus are all avenues for further research. In summary, the taxonomically important calyx characteristics of accrescence, size, and sepals equal or unequal in combination with pedicel length and inflorescence density merit investigation to determine the extent to which they represent adaptive syndromes to enhance calyx photosynthesis.