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Flowering plants (angiosperms) are today the most diverse and ecologically important group of land plants world-wide. They achieved global dominance over gymnosperms during the Cretaceous and early Tertiary, and have formed the foundation of nearly all terrestrial ecosystems ever since. In considering features responsible for angiosperm success, carpel closure (and concomitant germination of pollen on stigmas rather than ovule micropyles) and carpel fusion (syncarpy) are commonly invoked as among the most important key innovations (Stebbins, 1974; Mulcahy, 1979; Endress, 2001). These features, in combination with animal pollination, are thought to be partly responsible for angiosperms gradually displacing gymnosperms as the dominant photosynthetic organisms on land (Regal, 1977; Mulcahy, 1979; Carr & Carr, 1961; Stebbins, 1974; Endress, 1982, 2001; Armbruster et al., 2002; but see Bond, 1989; Berendse & Scheffer, 2009 for additional hypotheses).
Adaptive explanations for the frequency of transitions to syncarpy and their contribution to angiosperm success have focused on enhanced physical protection of the ovules and economy of ovary construction (Stebbins, 1974), greater floral precision in pollination (Armbruster et al., 2002, 2009), improved dispersal capacity (Stebbins, 1974; Endress, 1982), intensified pollen competition (Endress, 1982; Williams et al., 1993; Armbruster et al., 2002), and fertilization of a larger proportion of the ovules (‘pollen-tube dispersion’; Carr & Carr, 1961; Endress, 1982; Armbruster et al., 2002). Recent research has suggested that improvements in the number and quality of offspring through pollen-tube dispersion and intensified pollen competition are probably the most important factors (Endress, 1982; Armbruster et al., 2002). However, all analyses of the evolutionary trends in, and adaptive significance of, syncarpy are based on the assumption that movement of pollen tubes between separate carpels is impossible when carpels are physically isolated (apocarpy), except when there is obvious adhesion of stigmas or secretions between them (‘extra-gynoecial compita’; Endress, 1982; Endress et al., 1983). New research calls this assumption into question and may force us to change our ideas about the evolution of carpel fusion in angiosperms.
One question to arise from the adaptive analysis of syncarpy is that, if syncarpy is so advantageous, why do so many angiosperms (c. 20%; Soltis et al., 2005) lack syncarpy and why have there been multiple reversals to apocarpy (Endress, 2001, 2011; Armbruster et al., 2002; Endress & Doyle, 2009; Rudall et al., 2011)? Further, if a major advantage of angiosperms over gymnosperms accrued through syncarpy (Mulcahy, 1979; Endress, 1982), why did apocarpous angiosperms diversify, possibly at the expense of the gymnosperm? We suggest that the advantages of increased fertilization and pollen competition normally associated with syncarpy may accrue also to many apocarpous plants by virtue of having cryptic routes of pollen-tube communication between carpels (extra-gynoecial pollen-tube growth (EGPG)). More specifically, infra-stylar EGPG has been largely overlooked and presents adaptive advantages not seen in the more widely observed supra-stylar EGPG.
Previous studies have shown unusual pollen-tube growth in a few aquatic plants, for example, pollen tubes growing into receptacle, pedicel, or even stem tissues (Philbrick, 1984; Wang et al., 2002, 2006; Huang, 2003). Other studies have shown unusual pollen-tube growth patterns in cleistogamous flowers, where pollen germinates in undehisced anthers and tubes grow down the filaments and receptacle to the carpels (Anderson, 1980; Márquez-Guzmán et al., 1993). One study of a basal angiosperm showed pollen-tube communication (EGPG) between free carpels via the ‘apical residuum’ or receptacle (Williams et al., 1993). Finally, many additional angiosperms, especially in the basal paleodicots, have EGPG, where tubes grow between carpels before, or without, transiting the style (see Endress & Igersheim, 2000). We call this supra-stylar and extra-stylar EGPG, respectively. Such unexpected pollen-tube behavior has been largely regarded as exceptional, solving special fertilization problems (but see Endress & Igersheim, 1997, 1999, 2000). However, if pollen tubes commonly surmount physical barriers to pass between free carpels, we would have to re-evaluate not only major evolutionary trends in angiosperms (see Endress & Doyle, 2009), but also the adaptive significance of the numerous independent origins (and reversals) of syncarpy and the role of syncarpy as a key innovation in the diversification and success of angiosperms.
Here we examined nine apocarpous species in six families to assess whether apocarpy generally precludes pollen-tube growth to unfertilized ovules in other carpels or whether, instead, EGPG is common and widespread. We were particularly interested in whether pollen tubes passed between carpels (EGPG) before or after travelling down the styles. We therefore investigated in detail the routes of pollen-tube growth and examined carpel anatomy in these species to explore why pollen tubes can grow between carpels in some species but not in others. Our survey reveals that infra-stylar EGPG is widespread, with about half of the apocarpous species surveyed showing this pattern of inter-carpellary fertilization. This observation, in combination with previous published work, forces us to re-evaluate the commonly cited advantages of morphological syncarpy and may provide an explanation for the frequency of reversals from syncarpy to apocarpy in flowering plants.
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Our study of extra-stylar pollen-tube growth (EGPG) leads us to distinguish among three types of EGPG: supra-stylar, extra-stylar, and infra-stylar. Supra-stylar EGPG depends largely on appressed stigmas and/or secretion of fluids through which pollen tubes can grow to reach stigmas and styles of carpels other than those on which the pollen originally landed. Extra-stylar EGPG is found in species in which pollen tubes do not penetrate the stigmas, and is seen in only a few specialized situations (e.g. cleistogamous flowers). In contrast, infra-stylar EGPG involves pollen tubes growing down the stigma and style on which they landed and exiting the ovary to reach unfertilized ovules in other carpels. Our investigation of nine species showed that infra-stylar EGPG occurs in species with carpels that have openings through which pollen tubes can exit, but appears to be absent in species with carpels that are completely sealed. This suggests that infra-stylar EGPG is influenced by carpel morphology.
We found EGPG to be surprisingly common, even in species whose flowers did not have obvious extra-gynoecial compita (see Endress & Igersheim, 1997, 1999, 2000). For example, in the apocarpous monocot genus Sagittaria, we found that ‘extra’ pollen tubes grew out of the ovary through a basal opening of the incompletely sealed carpel, and across the surface of the receptacle, to other carpels, thus fertilizing virgin ovules. In this species, the opening provides a passage for pollen tubes to exit the ovary, allowing them to grow freely across the receptacle to other carpels, and form a network of pollen tubes interconnecting otherwise separate carpels.
The distinction among supra-stylar, extra-stylar, and infra-stylar EGPG has not been previously emphasized (although see Armbruster et al., 2002; Endress, 2011). It is important not only because of the markedly different morphologies and developmental patterns involved, but because of the different adaptive consequences of the three types of EGPG. Supra-stylar and extra-stylar EGPG can improve the seed set of flowers under pollen limitation, as can infra-stylar EGPG. However, only infra-stylar EGPG can consistently increase the intensity of pollen competition and reduce rates of fertilization by genetically inferior male gametophytes (pollen). (Certain types of extra-stylar EGPG can potentially also lead to pollen competition, e.g. cleistogamous Malpighiaceae). The logic of this is the same as expressed for partially syncarpous pistils (Armbruster et al., 2002): pollen tubes that are committed to a single carpel above the style compete only with pollen tubes in their own style. By contrast, pollen tubes that can cross between carpels below the style effectively compete with all pollen tubes in all styles. Because infra-stylar EGPG is more difficult to recognize than morphological mechanisms promoting pollen competition (e.g. morphological compita; Endress, 1982), its frequency and importance have probably been seriously underestimated.
The discovery that infra-stylar, extra-gynoecial pollen-tube growth is phylogenetically widespread (Fig. 3) and perhaps common in apocarpous angiosperms leads us to reassess several aspects of angiosperm evolutionary history. First, redistribution of pollen tubes among carpels and effective pollen competition are not restricted to plants with fused ovaries (syncarpy), as noted by Endress and collaborators (e.g. Endress, 1982, 2011; Endress & Doyle, 2009). This means that the repeated shifts from apocarpy to syncarpy may not always be driven by selection for the increased quantity and quality of offspring via pollen-tube redistribution, given that one or both of these capacities may already occur in the apocarpous ancestor. This increases the balance of evidence for alternative hypotheses that have been proposed but hitherto thought less important, at least recently, including reduced reproductive investment in ovary walls per seed and/or increased protection of developing ovules for a given investment (Stebbins, 1974), improved seed-dispersal mechanisms (Stebbins, 1974; Endress, 1982), and/or increased floral precision and adaptive accuracy in pollination (Armbruster et al., 2002, 2009). Also, the shifts from syncarpy to apocarpy seen repeatedly in angiosperms may not incur the costs of reduced offspring quantity and quality. This may help explain the occurrence of these otherwise surprising transitions (Soltis et al., 2005; Endress & Doyle, 2009; Endress, 2011; Rudall et al., 2011). For example, if pollen tubes in other apocarpous monocots behave as we observed in Sagittaria and Ranalisma rostratum, there would be little or no selective cost in terms of pollen redistribution (seed set) and pollen competition (seed quality) in the four or more transitions from syncarpy to apocarpy in monocots (Fig. 3; Endress & Doyle, 2009 (Fig. 10b); Rudall et al., 2011).
Another interesting facet of infra-stylar EGPG is that the average pollen-tube length is increased dramatically for those pollen grains participating in between-carpel fertilizations. This means that the potential intensity and effectiveness of pollen competition are increased (hence potentially increasing mean offspring fitness) relative to both intra-gynoecial and supra-stylar EGPG (Mulcahy, 1983; Armbruster et al., 1995). This feature may be especially important in species such as Schisandra sphenanthera and Sagittaria spp. that have relatively short styles (Fig. 1).
Mechanism of tropism in extra-gynoecial pollen-tube growth
For successful fertilization, pollen must germinate on the stigma, grow tubes, usually through the style, and find and penetrate the ovule micropyle. These processes require growing pollen tubes to undergo numerous changes in growth orientation. The cues in the pistil that guide pollen-tube orientation are believed to be mechanical and chemotropic, but their nature is not well understood (Hülskamp et al., 1995; Johnson & Preuss, 2002; Holdaway-Clarke & Hepler, 2003; Chae & Lord, 2011). The guidance of the pollen tube has been assumed to depend on the architecture and chemical properties of the female reproductive tissues and/or ovules to provide a signal for the target-directed growth of the pollen tube. We observed pollen tubes growing freely but not randomly among carpels and apparently attracted by unfertilized ovules. This observation demonstrates that, if there is a signal to direct pollen tube growth, the signal is probably released only by ‘virgin’ ovules (Okuda & Higashiyama, 2010).
Significance of extra-gynoecial pollen-tube routes to pollen-tube reallocation
The repeated evolution of syncarpy is one of the dominant features of angiosperm macroevolution. A minimum of 17 independent evolutionary transitions from apocarpy to syncarpy have occurred; about three-quarters of these transitions allowed pollen tubes to cross between carpels and fertilize ovules that would otherwise be left unfertilized (Armbruster et al., 2002). This will generally occur if there is a joint pollen-tube transmission tissue shared by the carpels (the ‘compitum’; Carr & Carr, 1961), allowing pollen tubes to cross between carpels (Carr & Carr, 1961; Walker, 1978; Endress, 1982; Williams et al., 1993; van der Schoot et al., 1995). This condition is thought to be the rule in flowers with fully syncarpous ovaries that are unilocular or incompletely multilocular, but occurs also in many flowers with multilocular ovaries and post-genital (after initial formation) fusion of styles or stigmas, forming a compitum (Carr & Carr, 1961; Endress, 1982; Endress et al., 1983).
However, some apocarpous flowers possess extra-gynoecial compita (allowing EGPG). In such cases, pollen tubes can travel on or through a functional (extra-gynoecial) compitum to cross between separate carpels, usually through secretions joining appressed or adjacent ovaries or stigmas (Walker, 1978; Endress, 1982; Endress et al., 1983; Renner et al., 1997). The present study shows the importance of distinguishing between supra- and infra-stylar crossings between carpels, whether by intra-gynoecial compita, obvious extra-gynoecial compita, or other forms of extra-gynoecial pollen-tube growth, such as through the receptacle. Whereas both supra- and infra-stylar crossings of pollen tubes between carpels potentially enhance seed set under pollen limitation, only the latter can enhance offspring quality though intensified pollen competition.
In several apocarpous species, we found long, narrow stylar canals with obvious openings at each end. Pollen tubes could travel along this track and reallocate between carpels by exiting the base of the canal. This allows apocarpous flowers to function more like syncarpous flowers in terms of the redistribution of pollen tubes between carpels and pollen competition. Our detailed study of S. potamogetifolia revealed that inter-carpellary pollen-tube growth can be very extensive (Fig. 1) and may therefore play a major role in increasing both the quantity and the quality of seeds produced by flowers of apocarpous species with infra-stylar EPGP.
Phylogeny of extra-gynoecial pollen-tube growth
Endress and colleagues (Table 1; Endress & Doyle, 2009) have shown that extra-gynoecial pollen-tube growth (EGPG; via extra-gynoecial compita) is widely distributed among basal angiosperms and is probably the basal state (Table 1; Endress & Doyle, 2009). The work presented here adds support to this conclusion. Most basal angiosperms with EGPG have supra-stylar EGPG, but some Austrobaileyales have infra-stylar EGPG (Fig. 3). Repeated evolution of both forms of EGPG in primitively or secondarily apocarpous lineages (Fig. 3) supports the hypothesis that selection for increased offspring quantity and/or quality has promoted this transition.
Experimental studies are needed to assess whether pollen competition is more intense and offspring quality is improved in plants with infra-stylar extra-gynoecial pollen-tube growth. We anticipate that such experiments will reveal that many apocarpous angiosperms indeed benefit from greater seed set and more intense pollen competition through infra-stylar EGPG, much like in plants with fused carpels and intra-gynoecial compita. Experimental confirmation of these benefits would help explain both ‘anomalous’ reversals to apocarpy and the early success and radiations of apocarpous angiosperms and their role in replacing gymnosperms as the dominant higher plant life form.