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Abstract Divergence at reproductive traits can generate barriers among populations, and may result from several mechanisms, including drift, local selection and co-adaptation between the sexes. Intersexual co-adaptation can arise through sexually antagonistic co-evolution, a timely hypothesis addressed in animals but, to our knowledge, not yet in flowering plants. We investigated whether male and female population of origin affected pollen competition success, offspring fitness and sex ratio in crosses within/between six genetically differentiated populations of the white campion, Silene latifolia. Each female was crossed with pollen from one focus male from the same population, and pollen from two focus males from two distinct populations, both as single-donor and two-donor crosses against a fixed tester male with a 2-h interpollination interval (n = 288 crosses). We analysed paternity with microsatellite DNA. Male populations of origin significantly differed for siring success and in vitro pollen germination rates. In vitro pollen germination rate was heritable. Siring success also depended on sex ratio in the female family of origin, but only in between-population crosses. In some female populations, two-donor crosses produced less female-biased sex ratios compared with single-donor crosses, yet in other female populations the reverse was true. Offspring sex ratio varied with donor number, depending on the female population. Within/between population crosses did not differ significantly in seed set or offspring fitness, nor were siring success and offspring fitness significantly correlated. Altogether this suggests reproductive divergence for traits affecting pollen competition in S. latifolia.
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Spatial and temporal variations in biotic and abiotic factors may influence trait evolution and lead to local adaptation (Kawecki & Ebert, 2004). Divergence among allopatric populations can lead to speciation, whereby divergence at reproductive traits is particularly important in generating barriers among extant populations (Parker & Partridge, 1998; Howard & Berlocher, 1998; Kirkpatrick & Ravigne, 2002; Knight & Turner, 2004; Salzburger et al., 2006). Furthermore, several processes including antagonistic co-evolution between the sexes can lead to reproductive divergence and local co-adaptation of males and females in traits affecting post-mating success (Holland & Rice, 1998; Andres & Arnqvist, 2001; Knowles & Markow, 2001; Rowe et al., 2003). To our knowledge, this timely idea has yet to be investigated for male and female traits affecting post-pollination success in plants (Arnqvist & Rowe, 2005).
In plants, competition among different pollen donors may frequently occur, even when some flowers are pollen limited (e.g. Bernasconi et al., 2006), due to multiple pollinator visitation, pollen carryover and floral structures (syncarpy, Armbruster et al., 2002) that impose a common race of pollen tubes towards the ovules. The fertilization success of each competing pollen donor may depend on both the male and female traits expressed in the sporophyte or in gametophyte (pollen tube and embryo sac). Indeed, fertilization success may depend on male traits affecting pollen competitive ability (Snow & Spira, 1991; Snow et al., 2000; Hedhly et al., 2005) and female traits influencing pollen germination on the stigma, pollen tube growth through the style, fertilization, seed and fruit abortion (Marshall, 1988, 1991; Snow & Spira, 1991; Baker & Shore, 1995; Cruzan & Barrett, 1996; Krauss, 2000; Lankinen et al., 2006). Male and female optima over these traits and over the outcome of fertilization may differ, analogous to sexual conflict in animals (Chapman et al., 1995; Rice, 1996; Rice & Holland, 1997). Whereas each male (pollen donor) will increase its fitness by maximizing its share of paternity, females (pollen recipients) may benefit by increasing diversity of pollen and sires to avoid inbreeding, genetic incompatibility and selfish genes (Willson & Burley, 1983; Delph & Havens, 1998; Baker & Shore, 1995; Bernasconi et al., 2004). Indeed, pollination with large and diverse pollen loads can increase female fitness through higher offspring number and quality (Schlichting et al., 1987, 1990; Winsor et al., 1987; Karron & Marshall, 1990; Snow, 1990; Mitchell, 1997; Niesenbaum, 1999; Paschke et al., 2002; Bernasconi et al., 2003; Bernasconi, 2003; Armbruster & Rogers, 2004; Herrero, 2003; Vergnerie, 2006; Young & Young, 1992).
We investigated genetic variation among native populations of the white campion, Silene latifolia for male reproductive success under conditions of pollen competition. We addressed whether male and/or female population of origin influence paternity in within- and between-population crosses. Effects of male genotype (population of origin) are expected if populations differ, e.g. due to locally variable selection on traits affecting siring success (for instance, through variation in the intensity of pollen competition) or drift (e.g. at the Y chromosome). Effects of male × female genotype are expected under local co-adaptation between the sexes, either as a result of sexual conflict (Rowe et al., 2003; Arnqvist & Rowe, 2005), or if co-adaptation occurs for ‘female preference’ (Andres & Arnqvist, 2001). Recent studies indicate that sexual conflict does not necessarily result in males from foreign populations always obtaining highest paternity success, but that different scenarios are possible (reviewed in Arnqvist & Rowe, 2005). Although between-population crosses often reveal significant interactions between the sexes (e.g. Andres & Arnqvist, 2001), they cannot on their own conclusively demonstrate sexually antagonistic coevolution. Instead, the functional analysis of single traits and their consequences for male, female and offspring fitness must be taken into account. Whereas antagonistic effects should balance within populations, they might become apparent in between-population crosses (Brandvain & Haig, 2005). We therefore also investigated whether paternity success of focus males correlated with female or offspring fitness, to examine whether variation in pollen competitive ability is associated with fitness costs (a specific prediction of sexual conflict, see Pizzari & Snook, 2003) or benefits to females. The white campion is a suitable study organism because it has separate sexes and does not reproduce clonally, and thus depends entirely on sexual reproduction for its fitness. Moreover, multiple within-fruit paternity is common in natural populations (S. Teixeira & G. Bernasconi, unpublished manuscript). Frequent pollen competition generates selection for male traits increasing pollen competitive ability, and strengthens the potential for conflict (Brandvain & Haig, 2005).
Specifically, we used a design involving within- and between-population crosses in two blocks of three populations each (Fig. 1). These populations are geographically separated (Table 1) and a microsatellite DNA analysis revealed significant genetic divergence (Jolivet & Bernasconi, 2007). Furthermore, European S. latifolia populations show strong genetic structure for variation at the Y chromosome (Ironside & Filatov, 2005). We reared an F1 generation under standardized conditions to reduce the effect of maternal environmental conditions and thus to better isolate genetic differences among populations. With these F1 plants, we conducted two-donor crosses on the same female plant with pollen from within the same population of origin of the female, and pollen from two males from two distinct populations, using a fixed tester male as a competitor. Additionally, we conducted control crosses with pollen from each male as a single donor. This control allows testing the effect of providing pollen competition on seed set, offspring sex ratio and offspring fitness, and to correlate performance as single donor (e.g. seed set) with performance in competition (e.g. paternity). A total of 288 crosses were conducted. We assessed paternity in the two-donor crosses using molecular markers, examined potential correlates of siring success (pollen germination in vitro, the number of pollen grains per anther, sex ratios of the paternal and maternal family of origin, seed set in single-donor crosses) and measured female and offspring fitness, including sons’in vitro pollen germination rates. This design addresses the following questions: (i) is there genetic variation among male and female populations (and families) of origin for traits affecting siring success? Can we identify such traits through a correlation analysis of pollen, male and female traits with siring success? (ii) Is there an interaction between male and female genotype (population of origin) in determining paternity, as might be expected under local co-adaptation? Is the direction of such an interaction indicating that pollen from the ‘home’ population is more or less successful than ‘away’ pollen? (iii) Do male and female populations of origin affect offspring fitness, and does this point at inbreeding or outbreeding depression? (iv) Is in vitro pollen germination heritable? and (v) Do the number of donors (single- vs. two-donor crosses) and among-male variation in male siring success influence female fitness, offspring sex ratio or offspring fitness?
Figure 1. Design for intra- and interpopulation crosses in Silene latifolia. Crosses were conducted on F1 plants. Parents (P) were field collected as seeds (from 15 females per population), raised (20 seeds per fruit) in the greenhouse and crossed within populations between families (shown only for one population) to obtain an F1 generation free of maternal environmental effects. The six populations were assigned to two different blocks (three populations per block). In each block, F1 females were crossed with one male from a different family but the same population, and two males from the other two different populations (shown as different colours). Each replicate thus consisted of three females (from three different populations) and three males (from three different populations); per block there were eight such replicates. Pollinations for each focus male were conducted both as single-donor pollinations and as competitive pollinations against a fixed tester male. In total, we conducted 288 crosses [(three males × three females × two levels of pollen competition) per replicate × eight replicates per block × two blocks]. Twenty seeds per fruit from competitive pollinations were raised for paternity analysis (in four replicates per block and a total of 1440 offspring genotyped). We determined seed family sex ratio (F1, F2) and offspring fitness components (F2: age at first flowering; for a subset, size at first flowering) of 20 offspring per fruit.
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Table 1. (a) Geographic origin of Silene latifolia populations sampled for intra- and interpopulation crosses to assess effect of male and female population of origin on paternity success and offspring fitness. Size = number of flowering individuals at sampling date. (b) Sample sizes for crosses performed for two blocks of three populations each. In each block and male–female combination, for fitness measures there were eight replicates (each replicate using plants from different families, total n = 288 crosses); genetic analysis of paternity in two-donor crosses was conducted for four replicates (total n = 72 crosses, 1440 offspring genotyped); offspring sex ratios were determined for the offspring analysed for paternity and within-population, single-donor crosses of the same focus males (total n = 96 crosses). Tester competitors for two-donor crosses all originated from an independent population (AL).
| CT||Cottendart||CH||46°58′30′′N 6°50′50′′E||1000||Block 1|
| HO||Millingerwaard||NL||51°52′45′′N 6°00′55′′E||2000||Block 1|
| PA||Gagny||F||48°53′11′′N 2°32′36′′E||400||Block 1|
| SC||Sesto Calende||I||45°44′08′′N 8°37′00′′E||> 100||Block 2|
| VN||Village-Neuf||F||47°36′25′′N 7°33′31′′E||80||Block 2|
| GR||Saint-Martin d'Uriage||F||45°09′51′′N 5°51′34′′E||60||Block 2|
| AL||Göttingen||D||51°33′20′′N 9°58′01′′E||24||Tester males (competitors)|
|Focus male origin||Single-donor crosses||Two-donor crosses|
|Female origin||Competitor origin||Female origin|
| CT||8||8||8|| || || ||AL||8||8||8|| || || |
| HO||8||8||8|| || || ||AL||8||8||8|| || || |
| PA||8||8||8|| || || ||AL||8||8||8|| || || |
| SC|| || || ||8||8||8||AL|| || || ||8||8||8|
| VN|| || || ||8||8||8||AL|| || || ||8||8||8|
| GR|| || || ||8||8||8||AL|| || || ||8||8||8|
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We thank M. Kölliker, W. Salzburger, G. Bowman and the reviewers for comments on the manuscript and B. Schmid for statistical advice. R. Candeias, B. Gautschi (EcoGenics GmbH) and S. Teixeira helped with microsatellite DNA analysis, B. McDonald's laboratory with automated DNA extraction, U. Becker, A. Biere, J.-H. Blanc, J. Elzinga, M. Kleih, D. Lang and I. Till-Bottraud with field collection, M. Dufay with pollen counts, R. Felix-Keller (felix visual media) with drawing Fig. 1, and P. Busso, B. Künstner and T. Zwimpfer with greenhouse maintenance. We acknowledge financial support from Swiss NSF (3100A0-10331/1; PPOOA-102944/1), Fondation Mercier pour la Science, Roche Research Foundation, Faculté de Biologie et Médecine and Bureau Egalité Hommes/Femmes of Lausanne University.