Stomata in cycads

Leaf micromorphology as a possible tool in cycads systematics

by Barone Lumaga M. R., Coiro M., Erdei B., Mickle J. (2012)


In Conference Botany 2012, Columbus, Ohio, USA, July 7–11, Abstract ID: 306 –



The genus Ceratozamia (Cycadales; Zamiaceae) was classically divided into two groups based on gross leaf morphology, but recent molecular phylogenetic analyses has identified three clades. On a larger scale, Ceratozamia appears closely related to Stangeria and to the neotropical genera Microcycas and Zamia. Whole leaf and isolated cuticle specimens from eight Ceratozamia species (C. euryphyllidia, C. hildae, C. kuesteriana, C. latifolia, C. matudae, C. mexicana, C. miqueliana, C. norstogii), Stangeria eriopus, Microcycas calocoma, and Zamia amblyphyllidia were examined using SEM for features of inner and external surfaces.

Samples were collected from the middle region of leaflets of mature leaves of greenhouse-grown plants. For external surfaces, samples were air dried or fixed in FAA (10:5:50) and critical-point dried. For the inner cuticle surface, isolated cuticles were obtained using 20% CrO3.

Characteristics in common to these species include hypostomy with the exception of S. eriopus showing stomata also on the adaxial side (near the midrib), occasional presence of hair scars, adaxial epidermal cells longitudinally elongated and arranged in rows, and smooth adaxial exterior cuticle (with the exception of S. eriopus showing irregular ridges).

Stomatal complexes are not contiguous and are oriented parallel to the leaflet axis (with the exception of S. eriopus showing randomly oriented stomata), and are of the diperigenous to tetraperiginous type in Ceratozamia species, M. calocoma and Z. amblyphyllidia, with S. eriopus showing stomata of polyperigenous type.

Lightly granulate epicuticular wax borders stomatal pits in M. calocoma and Z. paucijuga, and it is granulate in C. matudae and C. robusta.

Epicuticular wax occuring as granules to ridges borders the pits in C. euryphyllidia, C. miqueliana, C. norstogii and as reticulate ridges in C. hildae, C. kuesteriana, C. latifolia, C. mexicana.

The distribution of different kinds of epicuticular waxes in Ceratozamia species closely reflect the phylogenetic relationships that has emerged from molecular data. The presence of granulated wax in M. calocoma and Z. amblyphyllidia suggests that this character is ancestral in Ceratozamia.

The closeness of S. eriopus to the other taxa is not supported by cuticular micromorphology. The close correspondence between molecular and micromorphogical data in Ceratozamia confirms that micromorphology can provide useful data for rapidly and efficiently assessing systematics in other cycad taxa.

Stomata in Ceratozamia kuesteriana(Zamiaceae, Cycadophyta)

Morphological aspects of stomata, cuticle and chloroplasts in Ceratozamia kuesteriana Regel (Zamiaceae)

by Barone Lumaga M. R., Moretti A., De Luca P. (1999)


in Plant Biosystems 133 (1): 47-53 – –


Light and scanning electron microscopy were utilised to study stoma and cuticle morphology whereas transmission electron microscopy was used to observe plastid ultrastructure in Ceratozamia kuesteriana Regel (Zamiaceae). 

Results show that in C. kuesteriana a diperigenous‐type stoma (or a derivation of a diperigenous type) occurs and that protein crystalloids and prolamellar bodies are simultaneously present in the chloroplast.

Epidermal morphology of Dioon (Zamiaceae) with comments on climate and environment

Fig 1. Dimensions taken from inner cuticle stomatal flange and polar cell flanges of Dioon: A, length and width of stomatal flange; B, length of polar cell flanges. 


Epidermal morphology and leaflet anatomy of Dioon (Zamiaceae) with comments on climate and environment

by Vovides A. P., Clugston J. A. R., Gutiérrez-Ortega J. S., Perez Farrera M. A., Sanchez Tinoco M. Y., Galicia S. (2017)

Andrew P. Vovides, Instituto de Ecología, AC (INECOL)
James A.R. Clugston,
José Said Gutiérrez-Ortega, Chiba University

Miguel Angel Perez Farrera, Universidad de Ciencias y Artes de Chiapas, (UNICACH), Tuxtla Gutiérrez, Chiapas

M. Ydelia Sánchez Tinoco, Universidad Veracruzana, Xalapa de Enríquez, Mexico

Sonia Galicia,

in Flora – Morphology Distribution Functional Ecology of Plants 239: 20–44 – DOI 10.1016/j.flora.2017.11.002 –

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Fig. 3. Epidermal peels of Dioon leaflets: A, C, E, G, I, showing abaxial surface stomatal band with interband (insert); B, D, F, H, J, adaxial surface (bright field microscopy): A, B Dioon angustifolium; C, D D. argenteum; E, F D. califanoi; G, H D. caputoi; I, J D. edule. Note papillae in epistomatal chambers in C, E and G. All bars = 50 μm.

Anatomical descriptions can be effective for solving systematic issues, but these studies are relatively scarce for cycads.
Therefore, we present here a leaflet and cuticle anatomical study on the genus Dioon, to provide a set of epidermal traits that clarify species delimitation and relationships between species and their habitats.
We used standard micro-technique for leaflet sectioning, and cuticular peel preparation for light microscopy. Also, we used the chromium trioxide method for scanning electron microscope observations on cuticles. Measurements were taken on 10 randomly chosen replicates of each cell or tissue type, for each of the leaflets sampled per taxon.

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Fig. 4. Epidermal peels of Dioon leaflets: A, C, E, G, I, showing abaxial surface stomatal band with interband (insert); B, D, F, H, J, adaxial surface (bright field microscopy): A, B Dioon holmgrenii; C, D D. mejiae; E, F D. merolae; G, H D. purpusii; I, J D. rzedowskii. Note papillae in epistomatal chambers in G. All bars = 50 μm.

Micromorphological variation among species was calculated for each trait. Finally, we reconstructed the ancestral states of the observed epidermal fibre-like cell and pore shapes, by tracing the characters on the species phylogenetic tree of Dioon. We were able to describe the leaflet anatomy, cuticles, and epidermal features for 14 Dioon species.
The quantitative analysis was useful to reveal five geographically structured species groups. Character tracing on the phylogenetic tree of Dioon has amplified our current understanding on species relationships with respect to habitats.

Fig. 11. T.S. through leaflet of Dioon stevensonii abaxial epidermis note isodiametric to narrow elongate lignified epidermal cells with thick external periclinal walls (arrows). The slight pink colouring in the cuticle is artefactual caused by the Safranin stain and does not indicate the presence of lignin, previous tests with phloroglucinol HCl did not show reaction for lignin in the cuticle. Stain Safranin O and Fast Green FCF. Bar = 50 μm.

The presence/absence data suggest that the evolutionary acquisition-deletion of structural shapes is phylogenetically independent, thus climate seems to play a very important role in the variation of cuticular and stomatal traits.
Many epidermal traits, especially adaxial cuticle thickness and epistomatal pore width and depth, might be adaptations resulting from a long-term influence of climate, since they appear to have correlation with climatic conditions in relation to their biogeography.
We conclude that the variation of all traits are mostly sustained and intrinsic to the species, and are of promising taxonomic value. The combination of the epidermal traits with other characters has potential for taxonomic resolution at species level.

The stomata of a hybrid cycad combine distinctly the peculiarity found in the contour of the guard cells in both parents.



A morphological comparison of leaflets of a hybrid cycad and the two parents

by Papadopoulos S. (1928)

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Sophia Papadopoulos,


in Botanical Gazette 85(1): 30–45 –


A morphological study of the leaflets of a hybrid cycad obtained from a cross between Ceratozamia mexicana and a new species, Zamia monticola, shows that some of the structures distinctly resemble either one parent or the other, and that other structures show characteristics of both parents.
The stomata illustrate the latter point clearly, as they combine distinctly the peculiarity found in the contour of the guard cells in both parents.


Stomata in Cycas



Scanning electron microscopy studies of cuticle micromorphology in Cycas L. (Cycadaceae).

by Mickle J. M., Barone Lumaga M. R., Moretti A., De Luca P. (2011)


in Plant Biosystems 145: 191–201 – –


A scanning electron microscopy (SEM) study of Cycas cuticle characteristics was undertaken in order to expand our knowledge of microscopic characters not observable under light microscopy and to clarify unresolved affinitites among some species within the genus.

Whole leaf and isolated cuticle specimens from the middle region of leaflets of greenhouse-grown plants of Cycas revolutaCycas rumphiiCycas circinalisCycas media, and Cycas normanbyana were examined using SEM for interior and exterior features.

Characteristics in common include hypostomy, hair bases on abaxial and adaxial surfaces, adaxial cells randomly arranged, adaxial exterior cuticle smooth, and stomata sunken to various degrees but stomatal pit always formed by two layers of epidermal cells.

Stomatal complex is of the polyperigenous type.

Stomata randomly dispersed and oriented, and except C. revoluta, are not contiguous.

Stomata deeply sunken in C. revoluta, intermediate in C. rumphii and C. normanbyana, and less sunken in C. circinalis and C. media.

Aperture between guard cells extends the entire stomatal length in C. rumphii and C. normanbyana, ∼80% in C. circinalis and C. media, and ∼50% in C. revoluta.

Cuticular features of C. revoluta show the greatest difference from the other species in complex relief of exterior cuticle and interior cuticular structure of subsidiary cells; C. media and C. circinalis show close similarity to each other and their stomatal complex dimensions fall within the same unique cluster using principal component analysis under normalized variables.

C. normanbyana and C. rumphii show the most similarity to each other in cuticular micromorphology. Stomatal complex dimensions of these two species fall into a second cluster that also includes C. revoluta. These data contrast with current taxonomy placing C. normanbyanasynonymous to C. media.

Stomata in Cycadaceae

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Cycas (Plat e I. Figs., 1, la , 2, 2a) – Microcycas (Plat e I. Figs. 3, 3a) – Stangeria (Figs, 4, 4a) – Bowetiia (Figs. 5, 5a) – Dioon (Figs. 6, 6a)


Determination of Cycas genera as suggested by leaf epidermis structure

by Greguss P. (n.d.)

Director of Institute for Botany of the University, Szeged, Hungary

* Detail of the author’s work : Xylotomy and leaf epidermis of recent Cycadaceae, in preparation now  –

We hope to offer some help to botanists dealing with recent Cycadaceae but also to paleontologists, when attempting to determine the individual genera by the structure of leaf epidermis on the basis of the following key of determination and two photo-plates. The magnification of the leaves of characteristic representatives of the individual genera (both from surface an d backside) is uniformly 300X .

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Encephalartos (Plate II., Figs. 7, 7a) – Macrozamia, (Lepidozamia) (Figs. 8, 8a, 10, 10a, 11, 11a) – Ceratozamia (Plat e II., Figs. 9, 9a ) – Zamia (Plat e II. Figs. 12, 12a)