Stomatal development in the cycad family Zamiaceae

Abstract Background and Aims The gymnosperm order Cycadales is pivotal to our understanding of seed-plant phylogeny because of its phylogenetic placement close to the root node of extant spermatophytes and its combination of both derived and plesiomorphic character states. Although widely considered a ‘living fossil’ group, extant cycads display a high degree of morphological and anatomical variation. We investigate stomatal development in Zamiaceae to evaluate variation within the order and homologies between cycads and other seed plants. Methods Leaflets of seven species across five genera representing all major clades of Zamiaceae were examined at various stages of development using light microscopy and confocal microscopy. Key Results All genera examined have lateral subsidiary cells of perigenous origin that differ from other pavement cells in mature leaflets and could have a role in stomatal physiology. Early epidermal patterning in a ‘quartet’ arrangement occurs in Ceratozamia, Zamia and Stangeria. Distal encircling cells, which are sclerified at maturity, are present in all genera except Bowenia, which shows relatively rapid elongation and differentiation of the pavement cells during leaflet development. Conclusions Stomatal structure and development in Zamiaceae highlights some traits that are plesiomorphic in seed plants, including the presence of perigenous encircling subsidiary cells, and reveals a clear difference between the developmental trajectories of cycads and Bennettitales. Our study also shows an unexpected degree of variation among subclades in the family, potentially linked to differences in leaflet development and suggesting convergent evolution in cycads.


INTRODUCTION
The extant gymnosperms, although relatively species-poor compared with angiosperms, display a high degree of morphological disparity that reflects a long evolutionary history, extending as far back as the Devonian period of the Palaeozoic era. Extensive extinctions among all gymnosperm groups (Crisp and Cook, 2011) have removed putative morphological intermediates, making the fossil record potentially important for our understanding of the evolution of seed plants. Among the four gymnosperm lineages that include living representatives, the cycad lineage is highly significant in studies of character evolution because of its phylogenetic placement close to the root node of the spermatophyte clade, either as sister to all other extant gymnosperms in most molecular analyses (e.g. Graham and Iles, 2009;Ran et al., 2018) or as sister to all other extant seed plants in morphological analyses (e.g. Doyle, 2006;Hilton and Bateman, 2006). Cycads display many traits that are considered plesiomorphic among seed plants (Brenner et al., 2003) and are thus pivotal in helping to resolve relationships among fossil and extant groups. Among the extant cycads, in molecular phylogenies (e.g. Salas-Leiva et al., 2013) the genus Cycas L. (Cycadaceae) is placed as sister to the other nine extant genera (family Zamiaceae). Cycas is also relatively divergent morphologically from the other cycads (Table 1); for example, all cycads possess compound leaves, but Cycas leaflets display circinate vernation and a single central vein, compared with erect ptyxis and multiple veins in Zamiaceae (Stevenson, 1981). Relationships among the genera of Zamiaceae are incompletely resolved; in some analyses Dioon is sister to the rest and Bowenia is sister to two further clades, here informally termed the CSMiZ clade and the EMaL clade (Fig. 1).
Stomatal morphology has potential to help resolve the relationships among these genera and also between extant and fossil gymnosperms. Cuticles and stomata are sometimes well preserved in compression fossils, and the phylogenetic signal of stomatal traits has long been recognized (e.g. Porsch, 1905;Florin, 1931Florin, , 1933. However, descriptions based entirely on mature stomata are potentially non-homologous; for example, paracytic stomata (which possess a pair of lateral subsidiary cells) can have different developmental trajectories in different taxa. Thus, developmental studies are important to clarify homologies. One of the most influential distinctions was made by Florin, who characterized 'haplocheilic' and 'syndetocheilic' stomatal complexes in both living and fossil seed plants (Florin, 1931). This differentiation was explicitly based on development (see also Rudall and Bateman, 2019). In haplocheilic stomata, a protodermal cell functions directly as a guard-mother cell (GMC) with no prior divisions; modified subsidiary cells (where present) are derived from surrounding cells, which are termed perigene cells. In syndetocheilic stomata, a protodermal cell becomes a meristemoid that divides asymmetrically to form a GMC and one or more specialized neighbour cells, which are termed mesogene cells.
Mature stomata in cycads are characterized by a ring of cells surrounding the guard cells ( Fig. 2), typically arranged in a single layer at the poles and two to four cell layers at the lateral sides (Florin, 1931(Florin, , 1933Greguss, 1957Greguss, , 1968Bobrov, 1962;Pant and Nautiyal, 1963;Griffith et al., 2014;Magellan et al., 2018;Vovides et al., 2018;Coiro et al., 2020). In many cycad species, the stomata are sunken in deep stomatal pits (Florin, 1931;Greguss, 1957;Pant and Mehra, 1964). This threedimensional structure makes studies of development highly problematic in some cycads because they cannot readily be imaged in surface view; few photomicrographs exist of stomatal development.
In this paper, we evaluate traditional hypotheses on the origin of the subsidiary cells. We use a range of anatomical techniques to describe the development of the leaflet epidermis in six cycad species representing five genera, including all major clades of Zamiaceae. Previous detailed studies of stomatal development are restricted to Cycas and Dioon Lindl. (Florin, 1931;Pant and Mehra, 1964), and variation within Zamiaceae is hitherto unknown.

Terminology of cycad stomata
In his description of the stomata of Dioon edule, Florin (1931) identified two different cell types in the stomatal apparatus: Nebenzellen (neighbour cells, directly flanking the guard cells) and Kranzzellen (crown cells, at the poles of the guard cells, as well as the three layers of cells overlying the Nebenzellen). Harris (1932) translated Florin's terminology into English, interpreting the Nebenzellen and the polar Kranzellen as subsidiary cells and the lateral Kranzzellen as encircling cells. Greguss (1968) instead translated Nebenzellen as neighbour cells and Kranzzellen as accessory cells. However, Harris' terminology has been more widely used in subsequent studies of cycad stomata (Pant and Nautiyal, 1963;Pant and Mehra, 1964;Barone Lumaga et al., 2015;Vovides et al., 2018). To avoid confusion about the use of subsidiary cells, we follow the nomenclature from Coiro et al. (2020), which distinguished between subsidiary cells and polar cells (Fig. 2).

Material examined
The material examined is listed in Table 1. (Table 1) were germinated on a perlite substrate and transferred to pumice after the emergence of the first prophyll before sampling the first developing leaflets (Fig. 3). Leaflet structure differs between species examined (Table 2; Fig. 3). In all species, leaflets were sampled at different developmental stages and immediately fixed in formalin-acetic acid-alcohol. They were subsequently transferred to 70 % ethanol and stored at 4 °C. Material for sectioning was embedded in Kulzer's Technovit 7100 (2-hydroethyl methacrylate) as described by Igersheim and Cichocki (1996). This method involves dehydration of the samples in an ethanol series and stepwise infiltration with the following ratios of 100 % ethanol : Technovit solution: 50 : 50, 25 : 75, 0 : 100. The embedded specimens were sectioned on a Microm HM 355 rotary microtome using a conventional microtome knife D. Sections of mostly 2 µm were stained with ruthenium red and toluidine blue and mounted on microscope slides in Histomount. Non-embedded sections of leaflets were mounted in 80 % glycerol or Hoyer's solution, prepared according to Coiro and Truernit (2017). Sections of mature leaflets of D. edule and Bowenia spectabilis were subject to pseudo-Schiff propidium iodide (PS-PI) staining, a technique that allows confocal imaging of cell walls, as described in Coiro and Truernit (2017), and then mounted in Hoyer's solution.

Seeds of four species
For examination of mature structure, cuticles of mature leaflets of Bowenia serrulata, Cycas circinalis and Cycas thouarsii were isolated after maceration in a mixture of H 2 O 2 and 80 % ethanol. Light and fluorescence micrographs were obtained using a Zeiss Axioscope using a brightfield filter. PS-PI-stained samples of B. spectabilis were observed using a Leica TCS SP8 microscope. Excitation was obtained using either 405-nm excitation and a DAPI emission filter or 488-nm excitation and a PI emission filter.

Bowenia
In B. spectabilis (Fig. 4), mature stomata are all similarly orientated parallel to the leaflet axis and the guard cells are not sunken in a stomatal pit (i.e. they are flush with the surface: Fig. 4A, B). Each stoma has two to five subsidiary cells (

Macrozamia
In Macrozamia communis (Fig. 6), protodermal cells are isodiametric or slightly axially elongated, with some dichotomies and anastomoses leading to a more irregular arrangement (Fig. 6B); epidermal cells become more axially elongated during later development. GMCs are oval or square and share   3A).

Ceratozamia
Leaf ptyxis reflexed; leaflets of each side overlap each other, with the adaxial side of the proximal pinna facing the adaxial side of the distal pinna.

Cycas
Circinate vernation and a single central vein Dioon Leaf ptyxis erect; leaflets of each side overlap each other, with the adaxial side of the proximal pinna facing the adaxial side of the distal pinna (Fig. 3B).

Macrozamia
Leaves simply pinnate, with erect ptyxis of the whole leaves; leaflets of each side overlap each other, with the adaxial side of the proximal pinna facing the adaxial side of the distal pinna.

Stangeria
Leaves simply pinnate, each leaflet possessing a clear midrib. Leaf ptyxis strongly inflexed. The primordia of the leaflet midrib are conduplicate, and the lamina of each leaflet is also conduplicate.

Zamia
Leaves simply pinnate, with plicate leaflets in Z. roezlii. Leaf ptyxis reflexed; leaflets overlap each other. the same lineage as one of the polar cells, which is therefore mesogenous (Fig. 6A). Other subsidiary cells originate from neighbour cells (Fig. 6D-H). Two neighbour cells can divide further to give rise to three to five encircling cells; further divisions in the lateral neighbour cells result in two rings of proximal subsidiary and distal encircling cells. During development, the neighbour cells also elongate distally, resulting in a stomatal pit (Fig. 5H).

Stangeria
In Stangeria eriopus (Fig. 7), protodermal cells are arranged in irregular squares or rectangles, lacking a clear direction of division (Fig. 7C). GMC differentiation commences early in leaflet differentiation and continues throughout leaf development, resulting in stomatal complexes at different developmental stages in close proximity to each other ( Fig.  7D-F). Division and differentiation of new GMCs continues until late in leaflet differentiation, resulting in intercostal axial rows of stomata. Early-formed stomata are mostly axially orientated, but later-developing stomata are often randomly orientated (Fig. 7D-F). Subsidiary cells are formed from neighbour cells. They undergo divisions parallel to the margin of the guard cells, resulting in subsidiary cells (Fig.  7A). Pavement epidermal cells often have slightly sinuous walls in surface view. Subsidiary cells differ from pavement cells in their shape; some also maintain a nucleus and cytoplasm and have a slightly thicker cuticle (Fig. 7A, B). Crystals are often present in the epidermal cells, often in the polar cell and encircling cells (Fig. 7A). Mature guard cells are flush with the surface; they have a thickened cell wall both dorsally and ventrally; and they contact the subsidiary cells and some mesophyll cells on their dorsal wall (Fig. 7B).

Zamia
In Zamia (Fig. 8), early development was observed in Z. integrifolia (Fig. 8A, B) and later development in Z. roezlii (Fig. 8C-G). Protodermal cells are angular and isodiametric (Fig. 8A), soon becoming axially elongated (Fig. 8B). GMCs are angular in surface view (Fig. 8B), although they can appear more oval later in development. Subsidiary cells develop from cells flanking the GMC. Later in development, stomata are orientated parallel to the leaflet axis ( Fig. 8C-E). Neighbour cells undergo divisions, resulting in subsidiary cells. Subsidiary cells also elongate distally to form a stomatal pit (Fig. 7G).

DISCUSSION
Our investigation of stomatal and epidermal development and stomatal anatomy in six of the nine genera of Zamiaceae has highlighted traits shared with other gymnosperm groups as well as potentially synapomorphic traits of the cycads such as the presence of encircling cells and sunken stomata. It has revealed a clear difference between the developmental trajectories of cycads and Bennettitales. The anatomy and consistent presence of subsidiary cells opens the possibility of investigating the functional role of the stomatal morphology of cycads. Moreover, we show an unexpected degree of variation between subclades in the family, potentially connected to differences in whole leaflet development, and validate hypothesis of convergent evolution of the stomatal morphology in the Stangeriaceae, while strengthening the relationship between Stangeria and the Ceratozamia-Zamia-Microcyas clade.

Mature stomatal structure
Our comparative observations confirm those of earlier researchers (e.g. Florin, 1931Florin, , 1933 in demonstrating the presence of a more or less distinct ring of subsidiary cells and polar cells in most extant Zamiaceae (Fig. 2). A similar arrangement also occurs in the sister genus Cycas (Fig. 9), in which both mature stomata and stomatal development have previously been described (Pant and Nautiyal, 1963;Pant and Mehra, 1964;Griffith et al., 2014). The stomatal apparatus of Bowenia differs in lacking encircling cells in a mature stomatal complex due to the lack of divisions of the lateral neighbour cells (Coiro and Pott, 2017). Based on mature leaflet anatomy, Coiro et al. (2020) suggested that this heterochronic difference could be due to relatively rapid elongation and differentiation of the pavement cells during leaflet development in Bowenia. Our study shows that axial elongation is synchronous with GMC division in Bowenia, and that the neighbour cells undergo divisions in all species examined except B. spectabilis.
In most cycads, mature stomata are predominantly axially orientated with their apertures more or less parallel to the long axis of the leaflet (with some exceptions; see below and Table 3). This pattern resembles the condition in most other land plants (e.g. conifers) but contrasts with fossil Bennettitales, in which the apertures are transversely orientated (Rudall and Bateman, 2019). GMCs are more or less square in all species examined and no asymmetric divisions were observed, indicating the absence of amplifying divisions that would lead to random stomatal orientation. A consistent axial orientation indicates linear epidermal expansion and an absence of amplifying divisions.
Although highly speculative, the consistent presence in cycad stomata of subsidiary cells, often with persistent cytoplasm and nucleus (unlike the almost completely sclerified pavement cells), could indicate a physiological role. A physiological connection between the guard cells and subsidiary cells is well known in grasses, where the lateral subsidiary cells are involved in the mechanisms of stomatal opening via exchange of osmolytes with the guard cells (Franks and Farquhar, 2007;Raissig et al., 2017;Gray et al., 2020). Some authors have reported a relatively rapid mechanism of closure and opening and high water efficiency in the Cycadales (Haworth et al., 2011;Álvarez-Yépiz et al., 2017). On the other hand, encircling cells and polar cells are almost completely sclerified at maturity in all genera except Stangeria, in which they contain oxalate crystals.
Mature stomata are sunken in stomatal pits in all species examined except B. serrulata and S. eriopus, in which the guard cells lie at the same level as surrounding epidermal cells or are only slightly sunken. As we have demonstrated, and Florin (1931) also demonstrated in D. edule (Fig. 2), the stomatal pit results from anticlinal enlargement of the subsidiary cells and the polar cells, often followed by division of the subsidiary cells. In S. eriopus, the initial division is periclinal, while it is oblique in the other species. In D. edule, the precursor of the encircling cells divides further, resulting in a three-layered encircling chamber in all members of this genus (Barone Lumaga et al., 2015). The 'flush' guard cells of Stangeria and Bowenia result from differing ontogenetic paths (Table 3), strengthening the hypothesis of parallel evolution for this supposed synapomorphy (Coiro and Pott, 2017;Coiro et al., 2020). This convergent morphology could be linked to leaflet economics in these two genera. Both genera share a similar habit, with subterranean stems, few large leaves and growth mainly in shaded areas, although both Bowenia and Stangeria can also grow in sunny habitats. The sunken guard cells of most cycads have traditionally been associated with adaptation to aridity. In some genera, such as Dioon (Barone Lumaga et al., 2015;Vovides et al., 2018), the depth of the stomatal pits is associated with plants living in more xeric environments. However, it is unclear whether the depth of the pits is an adaptation to avoid water loss, since there is little direct evidence either from models (Roth-Nebelsick et al., 2009) or from comparative analyses of other groups (Jordan et al., 2008). Deep stomatal pits could be a way of reducing the already elevated mesophyll resistance provided by the thick cells walls of gymnosperms (Veromann-Jürgenson et al., 2017;Carriquí et al., 2020). Thicker leaves, which are potentially adaptive in arid climates, would necessitate deeper pits, in a similar fashion to the evolution of crypts in sclerophyllous taxa . Bowenia and Stangeria have among the thinnest leaves in the Zamiaceae, and thus lack stomatal pits.

Stomatal development
Our comparative observations show that in Zamiaceae the stomatal subsidiary cells are derived from protodermal cells adjacent to the GMC, rather than from the sister cell to the GMC. In a few cases where epidermal cell elongation precedes GMC formation, we did observe that one of the polar cells is mesogenous, for example in M. communis (Fig. 6A), where the GMC is apparently the sister to the adjacent cell. Indeed, as Rudall and Bateman (2019) noted, in narrow linear leaves with axially elongated cell files, the GMC is invariably sister Table 3. Summary of stomatal patterning and development in the ten extant cycad genera

Species
Mature leaf epidermis (this paper; Florin 1931Florin , 1933Greguss 1957Greguss , 1968 Mature stomata (this paper ;Florin 1931Florin , 1933Greguss 1957Greguss , 1968 Stomatal differentiation and pre-patterning  to one of the polar neighbour cells, as also observed in conifers (Pinus: Johnson and Riding, 1981) and monocots (Rudall et al., 2017). However, in most cycad species the protodermal cells are isodiametric and remain relatively short. This perigenous pattern of development agrees with the haplocheilic definition of Florin, and matches studies of early stomatal development in Cycas, Ceratozamia and Dioon (Florin, 1931(Florin, , 1933Pant and Mehra, 1964;Barone Lumaga et al., 1999). A similar pattern of development was also reported in several other extant gymnosperms, including conifers (Florin, 1931;Johnson and Riding, 1981) and Ephedra of Gnetales (Rudall and Rice, 2019), but not in other Gnetales (Gnetum and Welwitschia: Takeda, 1913a, b;Rudall and Rice, 2019), in which the subsidiary cells are clearly derived by division of the meristemoids. In Ginkgo, which has fanshaped leaves with relatively chaotic epidermal patterning, both perigenous and mesogenous neighbour cells are observed (Rudall et al., 2012).
We also observed some differences among extant Zamiaceae in epidermal patterning and pre-patterning. The most common condition within the family is axial patterning with consistent orientation of stomata and consistent timing of the development of adjacent stomata (Table 3). For example, in D. edule and M. communis, the epidermal precursor cells are already arranged in clear axial files. In B. spectabilis (Fig. 2), axial cell files are discernible but less regular during early pre-patterning, with occasional transverse divisions; elongation of the pavement cells occurs before or synchronously with GMC division, and thus precedes guard-cell differentiation in this species. GMCs differentiate almost synchronously in the same section of the leaflet in Dioon, Bowenia and Macrozamia.
Stangeria is also unusual within Zamiaceae in that new GMCs differentiate during later development, often at different orientation to the early-formed stomata, resulting in a close proximity of stomatal complexes at different stages of development (Fig. 7E, F). The presence of both 'quartet' pre-patterning and successive development is partly correlated with leaf development. In Stangeria, growth of the leaf lamina starts from a continuous marginal meristem that develops after elongation of the leaflet midrib. In Zamia and Ceratozamia, the leaflet primordia undergo not only longitudinal expansion, but also lateral expansion that results in variation in leaf width between species (Medina-Villarreal et al., 2019).
Stangeria shares with Cycas non-synchronous stomatal differentiation and inconsistent stomatal orientation (Table  3). Previous observations on Cycas leaflet development as well as the mature shape of the epidermal cells show a 'quartet' pre-patterning (Coiro and Pott, 2017), indicating that this pattern could be either ancestral in cycads or occurred independently in Cycas and the CSMiZ clade. However, the absence of a clear outgroup for cycads makes resolution of this issue currently unfeasible. Preliminary inferences suggest that fossil cycads also show both squared and linear prepatterning, but difficulties in placement of these fossils in the cycad phylogeny (Erdei et al., 2019) make determining character polarities highly problematic. The long evolutionary time span between the origin of Cycadales and crown-group Zamiaceae and Cycadaceae limits our ability to infer character history using outgroup comparison.

CONCLUSIONS
Although the perigene origin of subsidiary cells in cycads is confirmed by our results, the division of the lateral cells into subsidiary and encircling cells suggests that the traditional separation between 'haplocheilic' and 'syndetocheilic' stomata fails to fully capture the variability in stomatal development and morphology observed between the extant gymnosperm groups, especially following translation in different languages. This potential difficulty in making accurate comparisons, together with recent new observations on stomatal development in other extant and extinct groups Rudall and Bateman, 2019;Rudall and Rice, 2019), suggests that a revision of the stomatal characters in the morphological matrices of the seed plants (Doyle 2006;Hilton and Bateman 2006) might be necessary to improve the resolution of the relationships between these groups.
The similarity between early development of the epidermis in cycads and other gymnosperms suggests that the lack of response of cycad stomatal density to CO 2 (Haworth et al., 2011) might not be linked to developmental constraints. Further physiological studies are needed, including electrophysiological investigations, stomatal mechanics, and response to desiccation or abscisic acid, to test whether the responses of cycad stomata differ radically from those of the more efficient angiosperm stomata.