Plant Syst. Evol. 241: 139–151 (2003) DOI 10.1007/s00606-003-0046-6 Variation in structural gender in the hermaphrodite Helleborus foetidus (Ranunculaceae): within- and among-population patterns J. Guitia´n1, M. Medrano1,2, C. M. Herrera2, and A. M. Sa´nchez-Lafuente2 1Departamento de Biologı´a Vegetal, Universidad de Santiago de Compostela, Santiago de Compostela, Spain 2Estacio´n Biolo´gica de Don˜ ana, Sevilla, Spain Received May 22, 2002; accepted February 19, 2003 Published online: November 4, 2003 Ó Springer-Verlag 2003 Abstract. In hermaphrodite plants, variations in Key words: Helleborus foetidus, structural gender, structural gender (defined as the ratio between male sex allocation, trade-offs, pollen:ovule ratio. and female gametes) may occur at different levels (among flowers, plants, and populations). In this study, we investigated variation in four traits Introduction influencing structural gender (number of carpels, Since Darwin’s publication of The Different ovules per carpel, stamens, and pollen grains per Forms of Flowers on Plants of the Same Species stamen) within and among six distant populations (1877), there has been a continuing effort to of the hermaphrodite perennial herb Helleborus foetidus (Ranunculaceae) in the Iberian Peninsula. understand the process of plant sexual repro- Our results show that the four traits investigated duction and, more specifically, gender special- varied significantly at all levels considered. Traits ization. A critical step in the understanding of influencing the female sexual component (number gender specialization was the recognition that of carpels and ovules per carpel) showed greater in flowering plants, hermaphroditism does not variation at the lowermost levels (within flower and necessarily mean equi-sexuality, i.e. a balanced plant) than traits influencing the male component, gender with roughly similar contributions by which in turn varied more markedly among pop- male and female functions to reproduction ulations. Number of carpels per flower and number (Horovitz 1978). Following Horovitz’s (1978) of pollen grains per anther were the most important and Lloyd’s (1980a, b; 1984) initial insight, traits affecting between-plant variation in structural numerous studies have subsequently examined gender. There was no evidence of significant plant- patterns of variation in gender and sex expres- level trade-offs or correlations between the various male and female traits, which covaried differently sion in plants with hermaphroditic flowers, across populations. The observed between-popula- both from empirical and theoretical viewpoints tion variation in structural gender of Helleborus (e. g. Charlesworth and Charlesworth 1981; foetidus can be explained as a consequence of Charnov 1982; Queller 1983; Lloyd and Bawa differences in self-pollination levels related to a 1984; Broyles and Wyatt 1990, 1995; Mazer flower’s ‘‘mating environment’’. and Delesalle 1998). 140 J. Guitia´n et al.: Gender variation in Helleborus foetidus Although most investigations have recog- 1977, Mazer and Hultga˚ rd 1993, Mazer and nized two different quantitative measures Delesalle 1998, Cruden 2000, Wyatt et al. of plant gender, namely structural 2000, Ju¨ rgens et al. 2002). ( ¼ phenotypic) and functional gender (Lloyd Patterns of intraspecific variation in struc- 1980a, Lloyd and Bawa 1984), the most tural gender are interesting in their own right frequently emphasized measure has tended to for at least three reasons. First, structural be functional gender, i.e., the relationship gender is a critical factor in determining between the proportion of seeds produced functional gender. All else being equal, indi- and the proportion of seeds sired by a given vidual variation in structural gender will be individual. Reliable assessment of the func- mirrored by differences in functional gender. tional gender of a given individual requires Second, intraspecific patterns of variation in knowledge not only of the number of pollen the number of male and female gametes at the grains and seeds produced by that individual, individual flower level may provide insight into but also of sex ratio, selfing levels and levels of the relative importance of male and female inbreeding depression in the population (Lloyd functions as evolutionary driving forces for 1980a, b). Functional gender is effectively an floral phenotypes. For example, the compara- estimate of the relative proportions of an tive extent of individual variability in male- individual’s fitness that are realized via male and female-related traits may inform us about and female functions, a question that would which of the two sexual functions has played a appear to be critical for the evolution of many greater selective role in shaping floral struc- reproductive features, such as floral display tural traits (Stanton et al. 1986). Third, a and inflorescence design (Cruzan et al. 1988, conspicuous trend exists in angiosperm evolu- Fishbein and Venable 1996, Fritz and Nilsson tion, from basal primitive lineages with an 1996). Relatively few studies, however, have indeterminate number of male and female attempted a detailed examination of patterns gamete-bearing structures, towards more ad- of variation in structural gender in hermaph- vanced lineages with extraordinarily reduced roditic species. By structural gender we mean variability in the number of such structures per the relative number of male and female flower (Stebbins 1974). The study of patterns gametes produced by an individual plant. of intraspecific variation in structural gender in Clearly, measuring structural gender in natural some of these primitive lineages may provide populations, and assessing its patterns of insight into some of the factors potentially variation, are considerably easier than involved in these macroevolutionary patterns measuring functional gender. Why, then, the characterizing angiosperm floral evolution. relative neglect of structural gender as an This paper presents a descriptive analysis objective of plant reproduction studies? One of patterns of variation in structural gender possible reason is that number of stamens, within and among six distant populations of carpels and ovules generally exhibit little or no the hermaphroditic perennial herb Helleborus intraspecific variability, and these floral traits foetidus (Ranunculaceae) in the Iberian Penin- are commonly used to separate families and sula. This species is particularly well suited to genera (Cronquist 1981), so little opportunities this type of inquiry, as it exhibits broad seems to be left for variability to occur in intraspecific variation in all the four floral structural gender in most plants. Perhaps for traits that may contribute to variation in this reason, most studies that have examined structural gender, namely number of carpels the relationship between the number of pollen per flower, number of ovules per carpel, grains and the number of ovules (pollen:ovule number of stamens per flower, and number ratio) have focused on interspecific variation, of pollen grains per anther. Our objectives are and have largely ignored the possibility of (1) to document patterns of within and significant intraspecific variation (Cruden between population variation in structural J. Guitia´n et al.: Gender variation in Helleborus foetidus 141 gender of individual plants; (2) to assess the study these plants were pooled into a single data relative magnitudes of individual variability in set, because preliminary analyses showed almost no the male- and female-related components of variation between populations in a given region in structural gender, and determine the relative any of the four traits analyzed. Five flowers were importance of variation in each of these four collected from 26–67 plants in each population, and components as determinants of individual var- preserved in 2.5–2.5–95% formaldehyde-acetic acid-ethyl alcohol (FAA). The number of stamens iation in structural gender; and (3) to identify and carpels, and the number of ovules per possible trade-offs between same-sex compo- carpel, were determined for all collected flowers. nents (e.g. number of stamens vs. number of To account for possible differences in the number pollen grains per anther, number of carpels vs. of carpels and stamens between early- and late- number of ovules per carpel) and between opening flowers, we attempted to sample flowers in different-sex components (total number of each locality as closely as possible to the local ovules vs. total number of pollen grains). flowering peak. A subset of the flowers collected in 1999 (from CAU, MAG, MON, GRA and PIR; Fig. 1) was Material and methods used for pollen counting. Pollen was counted using a particle counter (Coulter CounterÒ Z2, Beckman Study system. Helleborus foetidus L. is a self- Coulter Inc.) equipped with a 100 lm aperture tube compatible, perennial herb distributed over much and a particle-size channelizer accessory. From of western, central and southwestern Europe (Wer- each flower we removed a subsample of completely ner and Ebel 1994). In the Iberian Peninsula, it closed anthers and separately counted the number typically occurs in clearings, forest edges, and the of pollen grains in each (N ¼ 1380 anthers, 1–5 understory of deciduous and mixed forests. Flow- anthers per flower; N ¼ 314 flowers, 1–5 flowers per ering mainly takes place from January to March. plant; N ¼ 76 plants, 10–25 plants per population). Plants consist of one to several ramets that develop Each anther was placed in a clean 1.5 ml polypro- a terminal inflorescence