The importance of cuticular information and systematic value of epidermal characters has been emphasized by ^{Stace (1965)} who did extensive work on of about 250 species of angiosperms. However micromorphology and anatomical studies on living cycads have been scarce and patchy in comparison to the work done on other gymnosperms i.e. Araucaria de Jussieu (^{Stockey and Taylor, 1978}), Podocarpaceae (^{Stockey, 1982}; ^{Stokey et al., 1998}, ¹⁹⁹²; ^{Mill and Schilling, 2009}) among others since the classical works of Florin cited by ^{Yoshie and Sakai (1958)}. Early work on cycad leaflets by ^{Lamb (1923)} and ^{Papadopoulos (1928)} described anatomy of selected species of nine genera and that of a hybrid cycad respectively. Work by ^{Pant and Nautiyal (1963)} described cuticle and epidermis of 59 species and ^{Tang et al. (2004)} described the leaflet anatomy of ten species from six genera. ^{Mickle et al. (2011)} studied the cuticular micromorphology with the scanning electron microscope (SEM) of five species of Cycas L. and drew taxonomic conclusions regarding the status of C. normanbyana F. Muell.

The present study is part of a leaflet anatomical study of the Neotropical cycad genus Dioon Lindl., and we report cuticular and anatomical features hitherto unexplored throughout the genus.

Transverse (TS) sections were taken from the mid portion of leaflets that were in turn taken from the mid section of healthy mature leaves from the 14 known species of Dioon held in the Mexican National Cycad Collection held at the Clavijero Botanic Garden, Xalapa. For observation with clear field microscopy sectioning was done using standard microtechnique (^{Johansen, 1940}; ^{Purvis et al., 1966}) on fresh or fixed material histochemical staining done with phloroglucinol HCl for lignin and Sudan III and IV for suberin and cutin. Permanent slides were prepared by double staining with safranin and fast green FCF and mounted in Histoclad^®. For SEM observations of outer cuticular surfaces, cuticular peels were prepared using a modified technique with Jeffery´s solution according to ^{Stace (1965)} to obtain clean surfaces as well as untreated material in order to preserve wax deposits, both dried in a desiccator and sputter coated with gold-palladium at 1.5 kv at 5 mA for eight minutes with a Jeol Fine Coat JFC 1100 sputter coater. Observations were made with a Jeol JSM-5600LV scanning electron microscope.

Transverse sections (TS) of the Dioon leaflets under clear field microscopy revealed thick adaxial cuticles ranging over nine µm in thickness depending on species, lignified epidermis and hypodermis with girder sclerenchyma varying in layers according to species (Figure 1A). Thick walled non-lignified hyaline fibers with virtually no lumen are interspersed between the hypodermis and the single row of palisade mesophyll as well as in the spongy mesophyll (intervascular) and surrounding the vascular bundles (perivascular) were observed in mature leaflets of all species. These have been referred to as bast fibers (^{Lamb, 1923}); and bast cells (^{Papadopoulos, 1928}). These are now known as tension wood fibers or gelatinous fibers (G-fibers) and have been reported for cycads by ^{Tomlinson et al. (2014)} associated in the secondary phloem in roots that undergo contraction, in Gnetum; and in angiosperms by ^{Fisher and Blanco (2014)}. G-fibers are not restricted to wood or angiosperms and may have originated during an early period of spermatophyte evolution (^{Tomlinson et al., 2014}).

Figure 1 Dioon leaflet transverse sections. A) Leaflet stained with phloroglucinol-HCl to show lignified hypodermis and girder sclerenchyma (red), note hyaline G-fibers with lignified wall (arrow). Bar: 100μm. B) Double stained with safranin and fast green-FCF, note G-fibers cellulose secondary wall (S3) green, lignified outer wall (S1 and S2) red (arrow). Bar: 20μm. C) Young leaflet showing hyaline fibers with lesser thickening of secondary wall (arrows). Bar: 40μm. D) Stomatal apparatus of D. purpusii stained with Sudan III and IV showing thickened periclinal wall of raised encircling cell, thick cuticle (arrow) and cuticular papillae protruding into the stomatal chamber. Bar: 20μm. E) Abaxial epidermal surface of D. edule under epifluorescence microscopy showing thickened cuticle surrounding pores forming Florin rings (arrow). Bar: 20μm. 

In mature leaflets of Dioon these fibers appear gelatinous-like, the first layers of the secondary wall (S1 and S2 layers) react for lignin with phloroglucinol-HCl forming a lignified sheath (Figure 1A). The rest of the secondary wall (S3 layer) does not and stains with fast green. The thick cellulose S3 layer often breaks away during sectioning that is a characteristic of G-fibers (V. Angyalossy pers. com.) being rich in cellulose and stains green when double stained with safranin and fast green (Figure 1B). The hyaline fibers of young leaflets (three to four weeks after emergence) do not show these characteristics differing by having a much less thick cellulose secondary wall with a large lumen (Figure 1C).

G-fibers or gelatinous fibers are commonly found in tension wood that forms in response to gravitational stresses. They may be clustered together forming compact tension wood or as single fibers or in small groups forming diffuse tension wood (^{Mauseth, 1988}). G-fibres are poor in lignin but rich in cellulose (^{Coté et al., 1969}; ^{Mauseth, 1988}) and have a complex chemical structure (^{Bowling and Vaughn, 2008}).

In healthy, mature and functioning leaflets of Dioon they appear to be single fibers dispersed within the spongy mesophyll and closely clustered around the vascular bundles in varying numbers. Most cycad leaves and leaflets tend to be very coriaceous, heavy but flexible and long lived, especially in Dioon where the leaflets are strongly reinforced by a lignified hypodermis and girder sclerenchyma that adds weight to the leaf. These cycads grow in exposed environments such as rocky outcrops on steep hills or on canyon walls that are exposed to wind and direct sunlight. Thus they are exposed to mechanical stress and water loss through transpiration. In D. edule leaf flushes occur every other year (^{Chamberlain, 1919}; ^{Vovides, 1990}) and the life span of any given leaf in the field may be up to three years or more (pers. obs.). It is likely that once the long lived leaves of Dioon have reached maturity, usually less than three to six months after emergence, aspects such as flexibility, resistance to mechanical stress and the water absorbing ability of the G-fibers would thus offer some advantage to the plants occupying these harsh environments. Future research should follow the development and quantification of these fibres from early leaf emergence through to senescence.

All Dioon spp. are hypostomatic and the adult stomatal apparatus consists of three layers of encircling cells above the subsidiary cells forming an epistomatal chamber that varies in depth according to species. In TS through the stomatal apparatus a prominent thickening of the periclinal wall and cuticle of the outer or distal encircling cells occurs, forming a raised ring around the epistomatal pore. This can be seen when stained with Sudan III and IV (Figure 1D) and is reminiscent of Florin rings, for want of a better term. Florin rings are associated with the subsidiary cells observed in both living and fossil gymnosperms such as Pinus strobus by ^{Deckert et al. (2001)}, Papuacedrus (^{Wilf et al., 2009}) and in Falcatifolium (Podocarpaceae) by ^{Stockey et al. (1992)}. In Dioon these structures can also seen under epifluorescence microscopy of the abaxial cuticular surface using 400-440 nm exciter filters (Figure 1E) comparable to the Florin rings reported by ^{Wilf et al. (2009)} on fossil Papuacedrus and ^{Paull and Hill (2010)} on fossil Cupressaceae. ^{Dilcher (1969)} describes Florin rings as “conspicuous thickened rings of cutin overlying the accessory cells and surrounding the stomata.”

In the stomata of Dioon the situation differs in that there are three layers of encircling cells above the subsidiary (accessory) cells, that together form a deep stomatal chamber. The outermost (distal) encircling cells have a thickened upper periclinal wall as well as a thickened cuticle and can be seen in the transverse section of the Sudan stained cuticle in figure 1D (arrow) also under fluorescence microscopy in figure 1E (arrow) and illustration (Figure 2). Another difference is that stomatal plugs, as reported for Podocarpaceae by ^{Stockey et al. (1992)}, were rarely seen in Dioon but present occasionally.

Figure 2 Illustration showing section through stomatal apparatus of Dioon purpusii showing thickened cuticle (black), G = guard cell, S = subsidiary cell, E = encircling cells. Bar: 20 μm. 

The strongly overarching papillate subsidiary cells reported for Cycas revoluta by ^{Pant and Mehra (1964)} are unlike the structures in Dioon though their ontogeny may be similar; figure 1G in ^{Pant and Mehra (1964)} shows an overarching papilla of a subsidiary cell in a young stoma, and another subsidiary cell dividing to give rise to one encircling cell, and their figure 3F an adult stoma overarched by thickened papillae of subsidiary cells. In Dioon it is the outermost of the three layers of encircling cells with thickened periclinal wall and cuticle that forms the Florin ring-like ring structure.

SEM observations of the cuticle surface of the stomatal band of mature leaflets for all species show a variably undulating surface marking the outline of the epidermal cells. The epistomatal apertures appear raised by the encircling cells forming variably marked structures with regular, irregular or slight relief and outline according to species. We observe two types; (1) the Dioon edule Lindl. type (Figure 3A) that is a continuous raised ring that slightly overarches the stomatal pore resulting in a volcanic shape that corresponds to the Florin ring type C of ^{Yoshie and Sakai (1985)}, wax plugs sometimes referred to as Florin plugs have also been observed but are not common (Figures 3A insert and 3B); (2) the D. purpusii Rose type (Figure 3C) some presenting wax plugs (Figure 3C insert) that also corresponds to the type C ring but contrasts with the D. edule type as irregularly undulating with protruding papillae that vary in number and position, at times partially obscuring the stomatal pore (Figure 3D). The stomatal papillae vary in number, position and depth in the stomatal chamber within and between species that present the D. purpusii type structure.

Figure 3 SEM of outer surface of abaxial cuticles of Dioon. A) Dioon edule type Florin rings, insert showing wax plug. B) Detail of D. edule ring with wax plug. C) D. purpusii type rings, insert shows aper ture with wax plug. D) D. purpusii detail of pores showing protruding cuticular papillae partially occluding pores. Bar: 100 μm in A and C, 10 μm in B, 20 μm in D.