What do these genetic insights into reproductive development imply for seed plant evolution, specifically the nature of integuments, ovules and associated structures? Ovules across seed plants are probably homologous, given that analyses of morphological data from both living and extinct taxa have supported their monophyly (e.g. Crane, 1985; Nixon et al., 1994; Rothwell & Serbet, 1994; Doyle, 1998; Hilton & Bateman, 2006; Doyle, 2008). However, it remains unclear how integuments, which are variable in number across seed plants, are related to one another and, similarly, what is the correspondence among ovule-bearing structures of different clades. At higher levels of organization, questions remain as to how transitions from unisexual to bisexual, as well as branched to unbranched axes, were achieved.
Integuments enclose the nucellus and form the micropylar tube through which pollen travels towards the egg cell. Presumed ovule precursors of the earliest seed plants lacked integuments that fully enclosed the nucellus, and have therefore been called pre-ovules (Stewart & Rothwell, 1993). The nucellus of pre-ovules was subtended by fused or partially fused appendages, which have been viewed by many as being derived by condensation and reduction of a group of branches or dichotomously branching telomes (e.g. Andrews, 1963; Smith, 1964; Rothwell & Scheckler, 1988; Stewart & Rothwell, 1993), or a group of megasporangia (Kenrick & Crane, 1997). Under this view, the integuments were thought to have originated by subsequent fusion of these appendages.
The genetic evidence that ovules have characteristics of meristems (Gross-Hardt et al., 2002) suggests an alternative hypothesis regarding the nature and origin of integuments. Specifically, integuments, as well as the sterile appendage of pre-ovules, could be lateral organs initiated by nucellar meristems, and are of de novo origin. The nucellar meristem appears to result from the co-option of portions of the CZ genetic module into the megasporangium developmental program. Further, given that the overexpression of WUS results in additional integuments (Gross-Hardt et al., 2002; Sieber et al., 2004), the dynamics of WUS expression in the ovule could explain both the origin of the inner integument and the variable number of integuments observed across seed plants. This variation includes a wide diversity in integument number, ranging from the second integument of angiosperms to the supernumerary integuments of taxa nested within otherwise unitegmic clades, such as Taxaceae and gnetophytes (Coulter & Chamberlain, 1917; Takaso, 1985; Takaso & Bouman, 1986; Yang, 2004), as well as the extinct Bennettitales and Erdtmanithecales (Friis et al., 2011), to third integuments in ancestrally bitegmic clades, such as Annonacaeae (Endress, 2011). The question of whether WUS homolog expression in the nucellus is conserved across angiosperms and in other clades of seed plants is critical to testing this concept of ovules and their integuments (Table 2). Notably, the WUS-like gene from Gnetum is expressed in the apex of the developing ovule primordium, indicating that this role may in fact be conserved; limited data from other gymnosperms suggest that they possess WUS-like genes, but their expression patterns are yet to be determined in detail (Nardmann et al., 2009).
Table 2. Major outstanding questions for comparative investigations of reproductive development
|1. Are WUS homologs expressed in ovules across the seed plants?|
|2. How are YABBY genes expressed in gymnosperm integuments and megasporophylls, and what do these patterns tell us about the evolution of the discrete CRC and INO functions in angiosperms?|
|3. How conserved are KNOX gene expression patterns in the tissue giving rise to ovules across the seed plants? What about other components of the peripheral zone (PZ) module, such as CUC genes and auxin trafficking?|
|4. Are teratological bisexual gymnosperms associated with the differential expression of B-class gene homologs?|
|5. How conserved are genetic pathways controlling determinacy vs indeterminacy (e.g. LFY, AG, TFL1-like genes) across seed plants?|
Patterns of ovule ontogeny from Gingko, gnetophytes, conifers and angiosperms are completely consistent with this view of integuments and their origin. In all gymnosperms and angiosperms that have been examined, the ovule primordium clearly initiates before the integuments, which subsequently arise from the flanks of the nucellus (Coulter & Chamberlain, 1917; Takaso, 1985; Takaso & Bouman, 1986; Takaso & Tomlinson, 1989a,b, 1990, 1991; Tomlinson, 1992; Tomlinson et al., 1993; Yang, 2004; Douglas et al., 2007; Rydin et al., 2010; note that comparable developmental data from cycads are lacking). In this way, the initiation of the nucellus and integuments is very like the initiation of apical meristems and lateral organ primordia (Steeves & Sussex, 1989).
In Arabidopsis, the expression patterns of leaf polarity genes in the integuments (Fig. 2d) also support the interpretation of integuments as lateral organs, as does the presence of ANT transcripts in both leaves and integuments (Elliott et al., 1996). A common feature of leaves and integuments is the expression of HDZIPIII and KANADI genes in the respective adaxial and abaxial surfaces of inner and outer integuments (Fig. 2d; Kelley & Gasser, 2009; Kelley et al., 2009). It is important to note, however, that, unlike leaves, neither Arabidopsis integument utilizes the adaxial identity locus AS1 and the inner integument lacks YABBY expression. With regard to the AS1 gene, it may be that the lack of PZ identity in the ovule negates a requirement for AS1 to down-regulate the KNOX genes. In the case of the differences between the inner and outer integument, these could reflect a fundamental difference in their derivation, perhaps with the inner being derived from branches (e.g. Kelley & Gasser, 2009), as predicted by the telomic theory of origin (see pre-ovule discussion earlier in this section). However, it is equally possible that the developmental programs of the outer and inner integuments have diverged because of their different morphology, or simply as a result of developmental system drift (True & Haag, 2001). An added complication is that the YABBY lineage itself is seed plant specific (Floyd & Bowman, 2007), and it is unknown how the timing of its appearance relates to the origination of either integument. Further data from angiosperms and gymnosperms are clearly needed to distinguish among the alternatives (Table 2). The polarity genes are members of gene families with complex evolutionary histories (Floyd & Bowman, 2007; Yamada et al., 2011) and, although the expression of the YAB locus INO appears to be conserved across angiosperms (Yamada et al., 2003), it remains to be determined whether expression patterns of other polarity genes are similarly conserved in flowering plants. Likewise, few data exist about the distribution and expression of polarity genes outside of angiosperms, although ANT has been detected in gymnosperm integuments (Shigyo & Ito, 2004; Yamada et al., 2008).
Developmental geneticists often use interchangeably the phrases ‘female identity’ and ‘carpel identity’, but, clearly, female identity is determined by the presence and development of a megasporangium, a structure that long predates the origin of seed plants, let alone carpels. Therefore, it may be more productive to hypothesize the following: female identity in seed plants is determined by the elaboration of a meristematic tissue, the placenta, which initiates one or more ovules; the expression of this basic female identity program leads to the modification of the formerly sterile surrounding tissues, and this pattern of modification has evolved along different trajectories in various clades of seed plants, leading to diverse reproductive architectures. The questions then become: what genetic pathways lead to the elaboration of the placenta and initiation of the ovule, and are they shared across seed plants? As with questions about integuments, a full characterization of candidate gene families in terms of evolution and expression patterns is needed to identify common determinants of ovule identity (Table 2). Regardless of the outcome, the results will set the stage for subsequent experiments to investigate how the basic female identity program interacts with other meristem and organ identity genes to produce the architectures found in different clades of seed plants.
There is limited evidence that allows us to consider the genetic basis for ovule identity. As already discussed, this so-called ‘D’ function is closely associated with homologs of the AG subfamily, but it is important to remember that the often ovule-associated STK lineage is derived from an angiosperm-specific duplication event. Gymnosperms possess AG family members that predate this duplication, but these have experienced their own independent duplication events (J. Winther & E. M. Kramer, unpublished). Based on the Arabidopsis model (Pinyopich et al., 2003), we would expect members of the AG lineage s.l. to determine ovule identity in other seed plants. Consistent with this, data available from conifers suggest that multiple AG-like genes are broadly expressed in both male and female cones, with expression becoming more localized to different tissues as development proceeds (Rutledge et al., 1998; Jager et al., 2003; Zhang et al., 2004; Englund et al., 2011; Groth et al., 2011). This would seem to indicate that, in gymnosperms, AG-like genes act in the entire reproductive axis, but further sampling and better detail in expression patterns will be important for an accurate interpretation of these findings, especially in the light of previously unrecognized complexity that has been detected within the conifer AG-like gene lineages (J. Winther & E. M. Kramer, unpublished). In addition to testing all the AG-like paralogs, it would be equally important to investigate other components of the ovule identity and development pathway (reviewed in Skinner et al., 2004; Kelley et al., 2009) to gain an understanding of their potential functions across seed plants.
3. Ovule-bearing structures
In living seed plants, ovules are variously borne on the inner walls of carpels (angiosperms), on leafy or reduced megasporophylls (cycads), on axillary stalks subtended by leaves (Ginkgo), at the termini of condensed axillary shoots (gnetophytes) or on the surface of a cone scale that represents a condensed axillary shoot (conifers).
What genetic pathways might interact with those that determine female reproductive identity to shape this architecture? And exactly how do variations in the pathways and their interactions result in the variety of reproductive architecture observed in seed plants?
To address these questions, let us first return to our characterization of the carpel as a complex leaf that uses the PZ genetic module in a female reproductive context, which we could simply call ‘PZ + C’. This is an intriguing model, but considerable additional work is required in angiosperms to determine whether it is broadly applicable. Keeping that significant caveat in mind, it is still interesting to examine how the PZ + C model might help to explain the diversity in ovule-bearing structures. First, if we consider the PZ module alone, we know that it can be expressed in two completely different contexts: in terminal or axillary meristems, it helps drive the production of entire phytomers, whereas, in leaves, it plays a narrower role in promoting leaflet/lobe initiation. What if the PZ + C module is similarly labile? The laminar megasporophylls of angiosperms evolved from within a diverse assemblage of seed plants that were themselves apparently derived from lineages that produced terminally borne pre-ovules (Friis et al., 2011). What if the PZ + C module first arose in the context of a meristem rather than a lateral organ? This hypothesis would hold that, when PZ + C is expressed in a meristematic context, it can produce an ovule-bearing stalk, either axillary or terminal, but when co-opted into a lateral organ, would produce a laminar structure bearing ovules, similar to that seen in angiosperm carpels or cycad megasporophylls. Although this idea is, admittedly, highly speculative, it suggests specific lines of investigation into the nature of ovule production in extant gymnosperms, as well as potential explanations for the genetic basis of diversity seen in fossil seed plants.
The first area of needed research concerns the nature of female reproductive identity. Although we typically think of ‘C’ function as primarily related to AG homologs, which have already been discussed, carpel identity is also promoted by the YABBY paralog CRC. Orthologs of this gene are expressed in all angiosperm carpels examined to date, including those of members of the ANITA grade (Yamada et al., 2004, 2011; Fourquin et al., 2005; Ishikawa et al., 2009). Functional tests are more limited, but are still consistent with a model that the role of CRC in carpel identity is broadly conserved, although, in certain lineages, it may perform additional developmental functions (Yamaguchi et al., 2004; Lee et al., 2005; Orashakova et al., 2009). Current data suggest that the CRC lineage is angiosperm specific, without obvious gymnosperm precursors (Yamada et al., 2011), and so it is critical to obtain a more detailed picture of the YABBY lineage in gymnosperms in order to understand the potentially novel origin of their role in carpels.
A useful starting place for a discussion of the role of the PZ + C module in the diversification of seed plant female structures is with a description of these structures. A megasporophyll, leafy or reduced, is the fundamental ovule-bearing structure in both angiosperms and cycads. As with the carpel, the leafy megasporophylls of Cycas are candidate analogs of complex leaves expressing the PZ/lateral primordium pathway along the margins. In more distal positions along the megasporophyll, leaflets are produced, whereas, in more proximal positions, ovules arise. By contrast, a modified axillary shoot is the fundamental ovule-bearing structure shared by Ginkgophytes, conifers (living and extinct) and gnetophytes. In Ginkgo biloba, the ultimate product of modification is a stalk bearing a pair of ovules, with each stalk borne in the axil of a leaf. In conifers, ovule-bearing stalks of the axillary shoot were fused with sterile subtending scales into a cone-scale, which, in turn, was more or less fused with the bract that originally subtended the axillary shoot, leading to a branch-scale complex. The branch-scale complex is the basic unit of the ovulate conifer cone, and they are variously aggregated to produce the diversity of cones in modern conifers. In gnetophytes, axillary shoots, with terminal ovules subtended by sterile scales, are condensed and aggregated into cones of varying degrees of laxness, that is, more or less elongated and condensed.
The starting point for these structures is thought to have been a lax axillary shoot, similar to that of extinct Cordaitales (e.g. Florin, 1951; Clement-Westerhoff, 1988), and analyses of combined morphological and molecular data suggest that Ginkgo, conifers and gnetophytes share a common ancestor with Cordaitales (Mathews et al., 2010), as do some analyses of morphological data alone (Doyle, 2006, 2008; Hilton & Bateman, 2006). Inasmuch as ovules in Cordaitales were terminal (e.g. Florin, 1951; Stewart & Rothwell, 1993), these observations indicate that living gymnosperms may represent two basic trajectories in the evolution of reproductive architecture: one in which the placental/ovule meristem pathways have been transferred onto the megasporophyll, as may have happened in cycads and angiosperms, and one in which these pathways have been maintained in an essentially terminal position. This suggests that a synthetic understanding of the evolution of reproductive development may require at least three models: one each for angiosperms and cycads, and one for a gnetophyte, conifer or Gingko. This should begin by the characterization of the relevant gene families in gymnosperms, followed by the documentation of expression patterns of their members. Intuitively, we might predict the greatest similarity between cycads (particularly Cycas) and angiosperms, with type I KNOX and CUC genes expressed along the margins of the megasporophyll. Conversely, the cones of conifers and gnetophytes and the stalked ovules of Ginkgo represent compound structures for which the question is whether the expression of KNOX genes, and other genetic markers associated with placental development, is associated with the tissues that immediately give rise to the ovules.
Hermaphroditic axes occur in angiosperms, gnetophytes and Bennettitales, and are occasionally observed in some conifers. Nonetheless, dioecy and monoecy predominate in seed plants. The two most recent models to explain the transition from dioecy and monoecy to hermaphroditism in angiosperms are the Mostly Male (MM) and the Out of Male/Female (OOM/F) models (Frohlich & Parker, 2000; Theissen et al., 2002; Theissen & Melzer, 2007). The MM model was based on a premise of ectopic identity expression rather than complete homeosis, specifically that ovule identity was expressed on the surface of a microsporophyll, which subsequently became sterilized to enclose the ovule. Although key aspects of this model have been definitively disproven (Vazquez-Lobo et al., 2007), it represents a critical first step in the process of integrating developmental genetic data into our understanding of angiosperm evolution. The OOM/F model makes a clear case for homeosis as the driving force underlying the evolution of hermaphroditism. Quite simply, a male strobilus could become hermaphroditic if B homolog expression was eliminated from the distal sporophylls or, alternatively, a female strobilus would become hermaphroditic if B homologs were ectopically expressed in proximal sporophylls (Theissen et al., 2002). Baum & Hileman (2006) expanded on this idea to produce a more detailed model for how such a shift in gene expression might have occurred in terms of transcriptional regulation.
How can we determine whether the OOM/F model is accurate? Ideally, we would manipulate the expression of homeotic B class homologs in gymnosperms to test whether such simple transformations are possible but, unfortunately, no extant gymnosperms are currently tractable for functional genetics. In lieu of such tests, we might consider the predictions of a homeotic identity program. Most notably, we would expect the occurrence of hermaphroditic teratologies, as are observed throughout angiosperms. Indeed, this has been well documented: occasional bisexual strobili are observed throughout conifers, and also in Gnetum, most commonly represented by male cones that have distal sporophylls transformed to female identity (reviewed in Coulter & Chamberlain, 1917; Flores-Renteria et al., 2011; Rudall et al., 2011). In these cases, the proximal lateral organs have fertile microsporophyll identity, whereas the distal nodes have fertile ovule identity. Although it is yet to be decisively demonstrated, the expectation is that these transformations are the result of the differential expression of homologs of B-class homeotic genes. Other types of teratology have been described in Ginkgo, where normally unisexual short shoots produce both male and female organs, albeit on separate strobili, and, in other cases, chimeric leaves bear ectopic ovules. The former case could be explained by inconsistent expression of B gene homologs within the short shoot axillary meristem, whereas the latter could result from imprecise delimitation of leaf boundaries within the short shoot meristem (Douglas et al., 2007). This would be analogous to mutants of Arabidopsis, in which perturbation of primordium positioning can result in chimeric organs (Levin & Meyerowitz, 1995; Wilkinson & Haughn, 1995), although, in the case of Ginkgo, it would be a chimera of leaf and axillary female strobilus. Homoplastic evolution of hermaphroditism also provides evidence that components of the homeotic program may be widely conserved. Perhaps the most notable examples of this are Welwitschia and some species of Ephedra, which express a cryptic bisexuality, much like the moneocy of angiosperms (Endress, 1996). Furthermore, certain extinct lineages exhibit forms of bisexuality, most notably representatives of the Bennettitales (Friis et al., 2011). Thus, the lability inherent in such a homeotic identity program, together with the teratologies and instances of bisexuality (cryptic and obvious) in various clades whose relationships remain to be convincingly resolved, suggest that bisexuality could be homoplastic in living and extinct seed plants.