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Studies on the evolution of land plants have focused on key features of development, such as plant architecture, the histology of the shoot apical meristem (SAM), or the morphology, origin and formation of leaves, and have identified significant developmental dichotomies between land plant radiations: extant vascular plants, lycophytes and euphyllophytes (ferns and seed plants) grow and produce leaves by the activity of the sporophytic shoot apex, which in lycophytes and ferns depends on the function of apical initials, whereas the SAM of seed plants (angiosperms and gymnosperms) has a multicellular, layered histology and maintains a small stem cell population (Sussex, 1989). Stem cell homeostasis in the SAM of the model angiosperm Arabidopsis thaliana is controlled by a feedback loop between WUSCHEL (WUS) and CLAVATA (CLV) signalling (Brand et al., 2000; Schoof et al., 2000). WUS is transcribed in the organizing centre (OC) of the SAM and encodes a mobile homeodomain (HD) transcription factor (Mayer et al., 1998; Yadav et al., 2011), which promotes stem cell identity in a small population of cells above the OC. The stem cell number is limited by repression of WUS transcription, which involves heterodimeric transmembrane receptor kinases that respond to the CLAVATA3 (CLV3) ligand secreted by the stem cells (Trotochaud et al., 1999; Muller et al., 2008).
The multicellular and layered SAM of seed plants apparently coevolved with changes in the leaf developmental programme (Sussex, 1989; Harrison et al., 2005; Sanders et al., 2011). The sporophytic apex and leaves of most ferns grow by the activity of apical initials (Fig. 1), and stereotypic cell divisions create a repetitive array of several to many leaflets before the initial is ultimately consumed for a single leaflet or lobe (Sanders et al., 2011). In contrast to this acropetal mode of leaf maturation, leaves of angiosperms originate from a group of founder cells at the periphery of the multicellular SAM, and after an initial phase of synchronous cell proliferation, the leaf matures basipetally from the apical tip towards its base, where cells remain competent to proliferate and to manifest positional cues with regard to different leaf axes (Tsukaya, 2002). Although both fern and eudicot leaves are referred to as megaphylls, their morphogenesis differs profoundly in that cells proliferate either at the distal tip or at the leaf base, respectively, a difference that relates to the activity of apical or marginal initials in ferns or to the concerted function of marginal and plate meristems at the leaf base in eudicots (Fig. 1).
Figure 1. Shoot apical meristem (SAM) histology and megaphyll growth in ferns and eudicots/Gnetales. The shoot apex of ferns grows by the activity of single apical initials (ai; upper left), whereas seed plant sporophyte development is based on multicellular layered meristems (upper right) with apical/basal (tunica/corpus) and radial zonation (central (CZ) and peripheral zone (PZ)). The CZ comprises the stem cell niche, whereas cells of the peripheral zone are recruited for leaf primordia (lp) development. Most fern leaves grow by the activity of leaf apical initials (lai; lower left), reminiscent of activity of the apical initial in the shoot apex, resulting in an acropetal gradient of leaf development, with growth ceasing first at the leaf base. By contrast, the diffuse growth of eudicots and Gnetum leaves is based on the activity of marginal and plate meristems at the leaf base (lower right) and results in a basipetal mode of leaf maturation, with growth ceasing first at the leaf tip. The arrows mark the direction of leaf growth; light grey stripes indicate growth zones, apical initials/stem cells are stained dark grey.
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Traditional paleontological studies on leaf evolution have addressed the increase in the complexity of leaves from basal to higher land plants: microphylls or small leaves of lycophytes with a single vascular strand originated from tissue outgrowth (enation theory; Kenrick & Crane, 1997) whereas megaphylls according to the telome theory (Gifford & Foster, 1989) arose from planation, overtopping and webbing of lateral branches. However, these studies are usually based on interpretations of frond architecture and cannot elucidate the developmental potential of foliar meristems (Boyce & Knoll, 2002), which in Arabidopsis are responsible for leaf blade inception and expansion, respectively (Donnelly et al., 1999). By contrast, modern comparative evolutionary developmental biology (evo-devo) studies have addressed meristem evolution from a functional perspective rather than with regard to positional homology deduced from fossil records (Boyce & Knoll, 2002).
Leaf diversity based on modifications in meristem activity has been analysed in eudicots, and conserved or adapted regulatory nodes between simple and compound leaves have been identified (Blein et al., 2008; Piazza et al., 2010). Another approach has aimed to identify conserved mechanisms in micro- or megaphyllous leaf development (Bharathan et al., 2002; Harrison et al., 2005; Floyd & Bowman, 2010). For example, KNOTTED1-like homeobox (KNOX) genes encode a class of plant HD transcription factors that are expressed mutually exclusively to ASYMMETRIC LEAVES1, ROUGH SHEATH2, PHANTASTICA (ARP) genes in seed plant apices (Schneeberger et al., 1998; Waites et al., 1998; Byrne et al., 2000). Despite their presence in all vascular plant genomes, KNOX and ARP genes have been recruited differentially in lower and higher plant radiations. Those KNOX members expressed in the SAM of the extant lycophyte Selaginella kraussiana are not orthologous to those expressed in the SAM of euphyllophytes and are coexpressed with ARP orthologues in leaf primordia of lycophytes or ferns (Harrison et al., 2005). These data support differential recruitment of ancestral genes (i.e. homoplasy), which possibly relate to the independent origins of megaphylls within euphyllophytes, altered developmental programmes, but also inconsistencies in the histological classification of micro/megaphyllous leaves (Tomescu, 2009).
Megaphyllous leaves of gymno- and angiosperms most likely have a common origin (Tomescu, 2009) and characters shared by multiple taxa, and their most recent common ancestors in cladistics are synapomorphies or symplesiopmorphies, if shared with an earlier common ancestor. Based on the fossil record, complex laminated leaves evolved in the Paleozoic in at least four vascular plant lineages: progymnosperms (extinct), sphenopsids, ferns and seed plants. In each lineage, leaf complexity followed the same sequence of morphologies: from dichotomizing single-veined leaves, via multiveined leaves with divergent venation, to convergent and lastly reticulate venation patterns (Boyce & Knoll, 2002). The sequence of increasingly complex leaf architectures suggests that the last common ancestor (LCA) of all four clades had already evolved developmental mechanisms that served as the basis for parallel evolution. The venation pattern of a leaf reflects the mode by which it grows (i.e. marginal vein endings indicate marginal meristem activity), whereas a reticulate venation pattern with veins of multiple orders and free-ending internal veinlets characteristic of extant angiosperm species, at least of eudicots, also requires nonmarginal diffuse intercalary leaf growth (Boyce & Knoll, 2002) or plate meristem activity (Fig. 1). The long independent trajectory of angiosperms and gymnosperms provides a valuable resource with regard to leaf meristem evolution, because the major gymnosperm orders, Cycadales, Ginkgoales, Gnetales, Pinales and Cupressales (Christenhusz et al., 2011), display different modes of leaf growth. The genus Gnetum comprises c. 30–35 species of the order Gnetales and is the only nonangiosperm seed plant genus with a reticulate venation pattern (Fig. 1), whereas exclusively marginal vein endings are found in Cycads, Ginkgo and some conifers (Boyce, 2005).
Apart from the KNOX gene family mentioned earlier, members of the WUS-related homeobox or WOX gene family are particularly suitable for studies on meristem evolution. Beside its founding member WUS, several other family members are associated with specific stem cell niches: WOX5 in the quiescent centre of the root meristem (RM) in Arabidopsis, maize and rice (Kamiya et al., 2003; Nardmann et al., 2007; Sarkar et al., 2007); WOX4 in the vascular cambium of Arabidopsis, tomato, rice and maize (Ji et al., 2010b; Suer et al., 2011; Ohmori et al., 2013); WOX3 or PRESSED FLOWER (PRS) in lateral domains and margins of new organ primordia in Arabidopsis and maize (Matsumoto & Okada, 2001; Nardmann et al., 2004); and WOX1 in blade expansion of Petunia hybrida (MAEWEST; Vandenbussche et al., 2009), Medicago truncatula (STENOFOLIA) and Nicotiana sylvestris (LAM1; Tadege et al., 2011) or together with PRS/WOX3 in Arabidopsis (Nakata et al., 2012). In addition, WOX2 mediates apical cell fate after the first asymmetric division of the zygote during Arabidopsis embryogenesis (Breuninger et al., 2008) and this transcription pattern is conserved in the maize embryo (Nardmann et al., 2007). Some genes are functionally interchangeable when expressed under the appropriate promoter, that is, WUS and WOX5 can replace each other in the SAM or RM (Sarkar et al., 2007) and WUS can substitute for WOX3 in leaf margins (Shimizu et al., 2009). All these genes group in the WUS clade (Deveaux et al., 2008; Nardmann & Werr, 2012) and members of this clade are absent in the genomes of lower plant species (i.e. the moss Physcomitrella patens or the lycophyte Selaginella moellendorffii), but have recently been identified in two leptosporangiate ferns, Ceratopteris richardii and Cyathea australis (Nardmann & Werr, 2012). The cell type-specific expression of WOX1/WOX3 and WOX4 in leaf meristems and the vasculature system of eudicots, respectively, prompted us to search for orthologues in gymnosperms, as their expression patterns might provide insights into the evolution of different modes of euphyllophyte leaf development and venation patterns.
Here, we show that the genomes of Ginkgo biloba, Gnetum gnemon and Pinus sylvestris consistently contain WOX3 and WOX4 orthologues. The inclusion of the full-length gymnosperm WOX sequences and those of the basal angiosperm Amborella trichopoda refines phylogenetic reconstructions that robustly distingiush the WUS clade from two other sub-branches in the WOX gene family. This phylogeny, together with cellular expression studies, leads to four major conclusions: genes in the WUS clade were amplified before the separation of gymnosperms and angiosperms; the genome of the LCA of seed plants contained at least WOX2, WOX3 and WOX4 orthologues, in addition to a previously described WUS/WOX5 pro-orthologue (Nardmann et al., 2009); WOX2, WOX3 and WOX4 were subfunctionalized to the embryo proper, marginal meristems and the vascular system, respectively, at the base of seed plants and comprise discrete symplesiomorphic characters; and the shift of the concerted function of WOX3/WOX1 and WOX4 to nonmarginal leaf domains relates to the evolution of plate meristems, which enabled the new, basipetal mode of leaf development with reticulate venation pattern characteristic of eudicots and Gnetales.