Int. J. Plant Sci. 168(4):435–441. 2007. Ó 2007 by The University of Chicago. All rights reserved. 1058-5893/2007/16804-0006$15.00
SIZE-DEPENDENT REPRODUCTION AND GENDER MODIFICATION IN THE HERMAPHRODITIC PERENNIAL PLANT PAEONIA OFFICINALIS Emilie Andrieu,1 Max Debussche, and John D. Thompson Unite´ Mixte de Recherche 5175 Centre d’Ecologie Fonctionnelle et Evolutive, Centre National de la Recherche Scientifique, 1919 Route de Mende, F-34293 Montpellier, Cedex 5, France
In this study, we quantify how reproduction and allocation to male and female functions vary in relation to plant size during two consecutive but contrasting reproductive seasons in the hermaphroditic perennial herb Paeonia officinalis. Small flowering individuals in 2003 did not flower in 2004; thus, flowering probability is related to size, with an emphasis on survival. In terms of absolute numbers of stamens and ovules, large plants allocated more resources to reproduction than did small plants by the production of additional flowering stems. In contrast, in terms of relative allocation, large plants allocated a similar proportion of resources to reproduction as did small plants. Significant size-dependent gender modification occurred only in the second year of the experiment, when larger plants were more female than small plants. Hand-pollinations performed to minimize external effects on seed set and a severe drought in 2003 may have contributed to sex allocation costs in small plants. Our results suggest that differential expression of between-year reproductive costs in relation to plant size may lead to size-dependent reproduction and be a key feature driving size-dependent gender modification in perennial plants. Keywords: costs of reproduction, drought stress, flowering, gender, herbaceous perennial, resource allocation.
Introduction
male (Bickel and Freeman 1983; Ackerly and Jasienski 1990) function with size. Such gender bias may be particularly frequent in resource-poor environments (Delph and Wolfe 2005; Litrico et al. 2005) or after environmental stress (McArthur 1977; McArthur and Freeman 1982). In perennial plant species, size-dependent reproduction and gender variation may be key elements of an adaptive response to temporal environmental variation, in which a polycarpic plant maximizes its lifetime fitness. In perennials, natural selection should favor a genetically determined strategy that adjusts both flowering probability and total and relative allocation to male and female functions in relation to size and local conditions (Klinkhamer et al. 1997; Zhang and Jiang 2002). However, a lack of joint studies of flowering probability and resource allocation in relation to size in different reproductive seasons limits our understanding of the potential significance of an integrated response concerning size-dependent reproduction and gender modification in perennial plants. The purpose of this article is to quantify the effect of plant size on reproduction and sex allocation in the perennial hermaphrodite Paeonia officinalis L. (Paeoniaceae). We studied the same individuals over two consecutive years in a single population. The two years experienced very different climatic regimes. During the first year, flowering and fruiting occurred during an exceptionally hot and dry period; hence, the probability of reproduction and allocation patterns in 2004 may have been influenced by prior drought stress. We examined the following questions: (1) Is flowering probability related to plant size? (2) Do patterns of absolute and relative sex allocation depend on plant size? (3) Does the pattern of sizedependent sex allocation vary among years?
A major goal of evolutionary ecology is to explain how organisms maximize fitness relative to other individuals in a population. In plants, fitness estimation requires quantification of relative investment in reproduction, in particular the relative allocation to male and female functions, which in perennial species may vary among years in relation to pollen and resource availability, plant size, and age and costs of prior reproduction (Charnov 1982). Plant size is a key element in the expression of reproductive trait variation in herbaceous perennial plant populations. First, increasing size may increase the probability of flowering in a given year because of the existence of a threshold size for reproduction (Gross 1981; Hanzawa and Kalisz 1993; Worley and Harder 1996). This threshold size has been predicted to decline in habitats with high mortality and low size-dependent growth rates, and, indeed, in the few studies done to date, the threshold increases in favorable conditions (Wesselingh et al. 1997; Me´ndez and Karlsson 2004). Second, reproductive biomass and seed production may be size dependent (Samson and Werk 1986; Me´ndez and Obeso 1993), and several studies report an increase in absolute allocation to sexual reproduction in relation to plant size (Ohlson 1988; Worley and Harder 1996; Wright and Barrett 1999). Third, the gender of plants, i.e., their relative allocation to male and female functions, can be size dependent with an increase in allocation to either female (Wright and Barrett 1999; Me´ndez and Traveset 2003; Litrico et al. 2005) or 1
Author for correspondence; e-mail
[email protected].
Manuscript received June 2006; revised manuscript received November 2006.
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Material and Methods Study Species Paeonia officinalis L. is an herbaceous perennial plant that occurs in the hills and low mountains of central and southern Europe (Tutin et al. 1993) and is protected in France. This polycarpic geophyte has thick tuberized roots, and individuals may live several decades. Herbaceous stems appear in early spring, bear one to 11 pinnatilobate leaves, and die back in late summer. In the study population, reproductive individuals produced one to four flowering stems, each 30–70 cm in height, the majority (79.7%, n ¼ 182) with a single flowering stem. Each flowering stem bears a single large hermaphroditic flower. In the study population, plants flowered from mid-April to mid-May, and individual flowers lasted ca. 6 d. Flowers have five to 10 pink petals and 80–300 stamens that form a crown around the one to four carpels. They do not produce nectar and attract generalist pollinators (mainly large Hymenoptera, Apoideae) that collect pollen. In July, each dehiscent follicle exposes up to 20 black 7–9-mm-long ovoid seeds. Individuals can produce a small number of seeds on selfing, albeit significantly less than on outcrossing, and a 3-yr comparison of fertility on natural- and supplementarypollinated plants produced no evidence of pollen limitation in the study site (Andrieu et al. 2007).
Study Site The study was carried out in April–July of 2003 and 2004 in a population on the Montagne de la Se´ranne (lat. 43°499N, long. 03°339E; 500–650 m above sea level), 35 km northwest of Montpellier (southern France). The site is located on limestone on west-facing slopes, with stony soils and gravel screes. The climate is classified as perhumid Mediterranean with cold winters (Daget 1977). The main vegetation is shrubs (Buxus sempervirens L., Amelanchier ovalis Medik., Viburnum lantana L., Prunus mahaleb L.) and deciduous oak (Quercus humilis Mill.) with clearings and a sparse herbaceous cover. In 2003, the study region experienced a warmer spring than usual, with mean daily temperature maxima for April and May 1°–2°C above average and an exceptionally hot summer, with mean daily temperature maxima for June, July, and August 3°–6.5°C above the average of historical records (n ¼ 27 yr; Le Caylar meteorological station, at a similar altitude, 20 km from the study site). In addition, the May–August period was much drier than usual, with a probability of recurrence of once every 8 yr (n ¼ 58 yr; St. Maurice–de–Navacelles meteorological station, 4 km from the study site). In 2004, spring and summer temperatures were close to average, except in June, which was 3°C warmer than average.
Experimental Procedure In April 2003, 40 flowering plants (in a single large clearing and adjacent smaller clearings) were randomly chosen for use in the study of sex allocation and constituted a representative sample of the number of flowering stems per plant in the studied population. To estimate potential seed production (female function) unbiased by any effects of pollen limitation, all flowers of each plant were hand-pollinated in 2003 and
2004 using a mix of pollen collected from different individuals (n 4) outside the study sample. Hand-pollination was performed three times by brushing pollen onto the stigma every 2 d until flowers wilted. In geophyte species, aerial biomass is proportional to underground biomass and is thus a good surrogate for total biomass (Hanzawa and Kalisz 1993; Worley et al. 2000). Plant size was estimated from leaf surface area measurements to avoid the destruction of individuals (our study lasted two successive years and involved a protected species). We measured leaflet length and width on all leaves of each stem on each individual to estimate total leaf surface area per stem and per plant. To test whether leaf surface area per plant was correlated with leaf biomass, in 2003 we sampled (with permission) 30 individuals on which we measured the length and the width (61 mm) of each leaflet of one randomly chosen leaf. Leaves were weighed (60.1 mg) after being ovendried for 1 wk at 60°C. Leaf surface area (i.e., the sum of all values of leaflet length 3 width) was closely correlated with leaf dry weight (rSp ¼ 0:923, P < 0:001). To estimate female reproductive function, we counted the number of carpels, ovules, and seeds per flower. Male fertility was estimated by counting anther number per flower and pollen grain number per anther. We counted anther number per flower using digital photos, a method shown to be accurate by linear regression of direct anther count (d) on picture anther count (p) for 32 flowers outside the study sample (d ¼ 1:12p, r2 ¼ 0:99, P < 0:001). Before dehiscence, we removed a sample of 10 anthers per studied flower for pollen counts using a Casy Model TT cell counter with a 150-mm aperture capillary. Anthers were left in open 1.5-mL polypropylene centrifuge tubes in a dry and warm place to release pollen. Tubes were then filled with distilled water and placed for 10 min in an ultrasonic bath to further promote pollen release. Anthers were then removed, and one-fourth of each tube was transferred to a vat filled with 5 mL of CASYton (isotonic dilution liquid). Pollen grains were then counted from three 400-mL aliquots (parameters of the curve smoothing: step ¼ 2, width ¼ 8), and mean values were used to estimate the number of pollen grains per anther.
Sex Allocation Phenotypic gender was estimated using the formula of Lloyd and Bawa (1984), which calculates standardized femaleness (Gi) of an individual i in a population of n individuals as Gi ¼
di ; di þ li 3 E
ð1Þ
where di is an estimate of female allocation based on ovule or seed number and li is an estimate of male allocation (based on anther number); the equivalence factor E is the ratio of female to male allocation in the population as a whole: Pn di E ¼ Pin¼ 1 : i ¼ 1 li
ð2Þ
Estimates of gender were based on anther number in relation to ovule number (Go) or seed number (Gs). We used anther number because, given the large number of anthers
ANDRIEU ET AL.—REPRODUCTION AND GENDER IN PAEONIA OFFICINALIS per flower (up to ca. 300 per flower), it is impossible to count all the pollen grains produced by a flower. In addition, if we cut all the anthers to count pollen, this could have an effect on resource allocation to female reproduction. To quantify total pollen grain number would require multiplication of pollen grain number in a small proportion of anthers by the number of anthers in a flower; this measurement is correlated with anther number and is thus redundant (first flowering stem of each individual: rSp ¼ 0:466, P ¼ 0:003, n ¼ 38). Our examination of pollen number in 10 anthers per flower showed that pollen production per anther does not vary significantly in relation to anther number in a flower (first flowering stem of each individual: rSp ¼ 0:152, P > 0:05, n ¼ 38); hence, anther number provides practical and valid surrogate for male function in our study system. We assessed variation in standardized relative gender in relation to variation in total reproductive investment (Sarkissian et al. 2001). In both study years, Spearman rank correlations were calculated to analyze the relationship between plant size (i.e., leaf surface area) and sex allocation. All statistical analyses were performed using Statistica (StatSoft France 2005). A sequential step-down Bonferroni procedure was used to correct threshold values for significance testing with multiple comparisons. Occasional predation of floral structures caused sample sizes to differ from the number of plants in the total sample of each year.
Results In both years of the study, carpel, ovule, seed, and anther numbers were significantly correlated with overall plant size (table 1). These correlations were not significant when analyzed at the level of individual flowering stems for the two years of the study (table 1). Likewise, a separate analysis of flowers on only the first flowering stem produced no signifi-
Table 1 Spearman Rank Correlations between Plant Size (Leaf Surface Area) and Reproductive Traits at the Plant, Flowering Stem, and Within-Flower Levels 2003 Level and trait Plant: Carpels Ovules Seeds Anthers Flowering stem: Carpels Ovules Seeds Anthers Within-flower: Ovules Seeds Pollen grains
2004
n
r
t(n2)
P
n
r
t
39 37 38 40
.58 .55 .62 .58
4.31 3.92 4.75 4.38
0:05) and anthers (T ¼ 90, Z ¼ 0:56, P > 0:05) per flower. In 2004, 21 of the 37 study plants flowered, while 16 became vegetative. Plants that were small in 2003 remained small in 2004 and mainly became vegetative, whereas large plants remained large and flowered again. Indeed, the size in 2003 of those individuals that flowered in 2004 was significantly greater than the size in 2003 of those individuals that became vegetative in 2004 (Mann-Whitney U ¼ 66, P < 0:01) (fig. 2A). In 2004, the size of those individuals that flowered was significantly greater than the size of those individuals that had become vegetative (U ¼ 72, P < 0:01) (fig. 2B). The size of individuals that flowered in 2003 and became vegetative in 2004 did not differ significantly among years (Wilcoxon signed-rank tests T ¼ 81, P > 0:05), and neither did that of individuals that flowered in both years (T ¼ 53, P > 0:05). The variances in size of reproductive plants in 2003 and reproductive plants in 2004 were not significantly different (P > 0:05) in a comparison of correlated variances test (Snedecor and Cochran 1967). In 2003, there was no significant difference at the flowering stem level in anther number (U ¼ 124, P > 0:05) or ovule number (U ¼ 140:5, P > 0:05) for plants that either became vegetative or flowered again in 2004. However, individuals that flowered in 2004 had significantly (U ¼ 87, P < 0:05) more pollen grains per anther in 2003 (120;225 6 13;692)
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Fig. 1 Relationship between plant leaf surface area and both anther number and ovule number at the whole-plant (A, B) and flowering stem (C, D) levels in 2003. Filled diamonds ¼ one flowering stem; open diamonds ¼ two flowering stems; filled squares ¼ three flowering stems; open squares ¼ four flowering stems. Results of correlation tests are in table 1.
than did individuals that became vegetative (73;202 6 13;200) and significantly (U ¼ 92, P < 0:05) more seeds per flowering stem (15:2 6 1:8) than individuals that became vegetative in 2004 (81:9 6 1:1). Although the Go in 2003 of the two groups was not significantly different (U ¼ 167, P > 0:05) (fig. 3A), the mean Gs in 2003 of plants that flowered again in 2004 (0:52 6 0:02) was significantly greater (U ¼ 102:5, P < 0:05) than that of plants that became vegetative in 2004 (0:41 6 0:03 in 2003) (fig. 3B). Interannual variations of Go and Gs were assessed only for plants that flowered in the two consecutive years, using Wilcoxon signed-rank tests. We detected a marginally significant (T ¼ 59, P ¼ 0:0496, n ¼ 21) decrease in Go between the two years of study, from 0:501 6 0:020 in 2003 to 0:482 6 0:022 in 2004 (fig. 3C), and a sig-
nificant (T ¼ 39, P ¼ 0:014, n ¼ 20) decrease in Gs (2003, Gs ¼ 0:518 6 0:023; 2004, Gs ¼ 0:467 6 0:030) (fig. 3D).
Discussion Our study provides strong evidence for size-dependent reproduction and sex allocation in a perennial plant. The patterns of temporal variation across years we detected provide an original demonstration of the links between size-related variation in flowering probability and sex allocation and reemphasize the need to conduct repeated studies of flowering and gender for the true nature of variation in sex allocation to be assessed in perennial plants.
Table 2 Results of Polynomial Regressions of Number of Ovules or Anthers against Standardized Leaf Surface Area (LSA) Models
Regression parameters
F
P
a
b
c
.591
24.54
