Stomatal and vein band widths for distinguishing between macromorphologically similar cycad species

Cycad forensics: leaflet micromorphology as a taxonomic tool for South African cycads

Woodenberg W., Govender J., Murugan N., Ramdhani S., Sershen (2019)

Wynston WoodenbergJoelene GovenderNelisha MuruganSyd RamdhaniSershen,


Plant Syst Evol 305: 445–457 –


Cycads, a primitive group of gymnosperms, are currently facing extinction in many parts of the world. In South Africa, this is largely attributed to the illegal poaching of many threatened species. In the illegal trade of cycads, many highly threatened species are often deliberately misnamed as a more common species.

Due to macromorphological similarity between many Encephalartos species, as well as taxonomic uncertainties that exist, species identification is also problematic. This study compared the utility of selected leaflet micromorphological characters as a taxonomic tool to independently identify eight South African cycad species.

The characters, which included trichome type (if present), stomatal density and dimensions, stomatal band width and vein band width, were compared within four pairs of macromorphologically similar species. Quantitative and qualitative data on the characters were collected using stereomicroscopy, scanning electron microscopy (SEM) and variable pressure SEM.

Results indicated that the majority of these characters varied significantly (p < 0.05) between paired species. Importantly, the presence of trichomes on mature leaflets of four species appears to be previously unreported. Trichome type, stomatal width and band width, and vein band width were identified as diagnostic characters that may be used to distinguish between species.

The results validate the use of leaflet micromorphological characters, particularly stomatal and vein band widths (given the ease with which they can be measured), for distinguishing between macromorphologically similar cycad species.

Ptyxis, phenology, and trichomes in the Cycadales

Observations on ptyxis, phenology, and trichomes in the Cycadales and their systematic implications

Stevenson D.W. (1981)

Dennis W. Stevenson,

American Journal of Botany 68: 1104–1114 –

The cycad species analysed showed no significant stomatal density, stomatal index or pore-length response to changes in [CO2] or [O2].

Cycads show no stomatal-density and index response to elevated carbon dioxide and subambient oxygen

Haworth M., Fitzgerald A., McElwain J. C. (2011)

Matthew Haworth, Annmarie Fitzgerald, Jennifer C Mcelwain,


Australian Journal of Botany 59: 629–638 – DOI: 10.1071/BT11009


The stomatal density (SD) and index (SI) of fossil plants are widely used in reconstructing palaeo-atmospheric CO2 concentration (palaeo-[CO2]). These stomatal reconstructions depend on the inverse relationship between atmospheric CO2 concentration ([CO2]) and SD and/or SI. Atmospheric oxygen concentration ([O2]) has also varied throughout earth history, influencing photosynthesis via the atmospheric CO2 : O2 ratio, and possibly affecting both SD and SI.

Cycads formed a major component of Mesozoic floras, and may serve as suitable proxies of palaeo-[CO2]. However, little is known regarding SD and SI responses of modern cycads to [CO2] and [O2]. SD, SI and pore length were measured in six cycad species (Cycas revoluta, Dioon merolae, Lepidozamia hopei, Lepidozamia peroffskyana, Macrozamia miquelii and Zamia integrifolia) grown under elevated [CO2] (1500 ppm) and subambient [O2] (13.0%) in combination and separately, and compared with SD, SI and pore length under control atmospheric conditions of 380 ppm [CO2] and 20.9% [O2].

The cycad species analysed showed no significant SD, SI or pore-length response to changes in [CO2] or [O2].

Eobowenia vs Bowenia

Eobowenia gen. nov. from the early Cretaceous of Patagonia: indication for an early divergence of Bowenia?

Coiro M., Pott C. (2017)

Mario CoiroChristian Pott,

BMC Evolutionary Biology 17: 97 –



Even if they are considered the quintessential “living fossils”, the fossil record of the extant genera of the Cycadales is quite poor, and only extends as far back as the Cenozoic. This lack of data represents a huge hindrance for the reconstruction of the recent history of this important group. Among extant genera, Bowenia (or cuticles resembling those of extant Bowenia) has been recorded in sediments from the Late Cretaceous and the Eocene of Australia, but its phylogenetic placement and the inference from molecular dating still imply a long ghost lineage for this genus.


We re-examine the fossil foliage Almargemia incrassata from the Lower Cretaceous Anfiteatro de Ticó Formation in Patagonia, Argentina, in the light of a comparative cuticular analysis of extant Zamiaceae. We identify important differences with the other member of the genus, viz. A. dentata, and bring to light some interesting characters shared exclusively between A. incrassata and extant Bowenia. We interpret our results to necessitate the erection of the new genus Eobowenia to accommodate the fossil leaf earlier assigned as Almargemia incrassata. We then perfom phylogenetic analyses, including the first combined morphological and molecular analysis of the Cycadales, that indicate that the newly erected genus could be related to extant Bowenia.


Eobowenia incrassata could represent an important clue for the understanding of evolution and biogeography of the extant genus Bowenia, as the presence of Eobowenia in Patagonia is yet another piece of the biogeographic puzzle that links southern South America with Australasia.

Leaflet anatomy has a substantial amount of phylogenetic signal in the Zamiaceae

Evolutionary signal of leaflet anatomy in the Zamiaceae

Coiro M., Jelmini N., Neuenschwander H., Calonje M. A., Vovides A. P., Mickle J. E., Barone Lumaga M. R. (2020)

Mario Coiro, Nicola Jelmini, Hanna Neuenschwander, Calonje Michael A., Vovides Andrew P., MickleJ. E., Maria Rosaria Barone Lumaga,


International Journal of Plant Sciences 181(7): 697-715 – DOI: 10.1086/709372


Premise of the Research:

The morphology of leaves is shaped by both historical and current selection acting on constrained developmental systems. For this reason, the phylogenetic signal of these characters is usually overlooked.


We investigate morphology of the leaflets of all genera of the Zamiaceae using multiple microscopical techniques to test whether leaf characters present a phylogenetic signal, and whether they are useful to define clades at a suprageneric level.

Pivotal results:

Our investigation shows that most genera are quite uniform in their leaflet anatomy, with the largest genera (Zamia, Encephalartos) presenting the highest degree of variation. Using both Bayesian and Parsimony methods on two different molecular scaffolds, we are able to show that leaflet anatomy has a strong phylogenetic signal in the Zamiaceae, and that many clades retrieved by molecular analyses present potential synapomorphies in their leaflet anatomy. Particularly, the placement of Stangeria in a clade with Zamia and Microcycas is supported by the presence of both an adaxial and abaxial girder sclerenchyma and the absence of sclerified hypodermis. The placement of Stangeria as sister to Bowenia, on the other hand, is not supported by our analysis. Instead, our results put into question the homology of the similar guard cell morphology in the two genera.


We show that leaflet anatomy has a substantial amount of phylogenetic signal in the Zamiaceae, supporting relationships that are not supported by general morphology. Therefore, anatomical investigation represent a promising avenue for plant systematists.

Stomatal structure and development in Zamiaceae

Schematic generalized drawings of stomata of Zamiaceae in middle transverse, polar transverse and longitudinal section. Cell wall in black, cuticle in grey. The guard cells (g, in dark blue), subsidiary cells (s, in red), encircling cells (e, in yellow) and polar cells (p, in light blue) are highlighted.

Stomatal development in the cycad family Zamiaceae

Coiro M., Barone Lumaga M. R., Rudall P. J. (2021)

Mario CoiroMaria Rosaria Barone LumagaPaula J Rudall,

Annals of Botany 128(5): 577–588 –

Bowenia spectabilis. (A, B). Transverse section of stomatal complex of an adult leaflet stained with pseudo-Schiff-propidium iodide observed using confocal laser scanning microscopy and imaged using (A) UV excitation, (B) propidium iodide excitation. (C) Fluorescence micrograph of isolated cuticle from adult leaflet stained with Auramine O, showing mature stomata in axial cell files. (D) Fluorescence micrograph of isodiametric protodermal cells in developing leaflet. (E–G) Development of stomatal complexes at different stages. Scale bars = 50 µm. Abbreviations: GMC, guard mother cell; GC, guard cell; LSC, lateral subsidiary cell.


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.

(A, B) Ceratozamia hildae; (C–F) Dioon edule. (A) Early development showing squared (quartet) arrangement of protodermal cells. (B) Slightly later stage, indicating protodermal cell enlarging to form a guard-mother cell (GMC). (C, D) Tangential sections of developing leaflets showing developing stomata in an intercostal stomatal band, all similarly orientated in axial cell files along the leaflet axis. Crystals are present in cells over veins in the slightly later stage in D. (E, F) Transverse sections of leaflets showing stomata; neighbour cells elongating periclinally in E and divided in F. Scale bars = 50 µm. Abbreviations: C, crystal; GMC, guard mother cell; GC, guard cell.


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 CeratozamiaZamia 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.

Macrozamia communis, developing leaflets imaged using (A) differential interference contrast, and (B, D–H) fluorescence micrography. (A, B) GMCs in axial cell files. (C–E) Stomatal development in surface view showing (C, D) GMCs and (E) guard cells. (F–H) Stomatal development in transverse section at successive stages. Scale bars = 50 µm. Abbreviations: GMC, guard mother cell; GC, guard cell.


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.

Stangeria eriopus. (A) Paradermal view of mature epidermis showing stoma with guard cells containing cytoplasm and nuclei; encircling subsidiary cells also with active nuclei. Calcium oxalate crystals present in surrounding intercostal epidermal cells. (B) Transverse section of stomatal complex showing guard cells with thickened cell walls containing lignin–pectin deposits, and encircling cells with calcium oxalate crystals. (C–F) Fluorescence and differential interference contrast images of cleared leaflets showing stomatal development. (C) Protodermal cells interspersed with both GMCs and stomata. (D) Slightly later stage, with guard cells and a GMC undergoing division. (E) Leaf clearing showing both differentiated and developing stomata in intercostal regions, most similarly axially orientated. Costal region (in different focal plane) with trichomes (hairs). (F) Later stage showing similarly orientated differentiated stomata; some smaller stomata in different orientation. Scale bars = 50 µm. Abbreviations: Cr, crystal; GMC, guard mother cell; GC, guard cell; St, stomata.
Zamia. (A, B) Zamia integrifolia, early developmental stages; (C–G) Zamia roezlii, differentiated stomata. (A) Protodermal cells. (B) Early stage with GMCs undergoing division, arranged in axially orientated cell files. (C) Surface view showing stomatal openings surrounded by encircling cells. (D) Young stoma with dividing lateral subsidiary cells. (E) Mature stoma with encircling cells and wall thickenings on guard cells. (F, G) Transverse sections showing successive stages of maturing stomata with guard cells already differentiated; in G the guard cells are sunken due to enlargement of the encircling subsidiary cells. Scale bars = 50 µm. Abbreviations: GMC, guard-cell mother cell; GC, guard cell.

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 –

Screen Shot 2018-03-30 at 20.10.16
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.

Screen Shot 2018-03-30 at 20.12.40
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)

Screen Shot 2017-12-10 at 20.47.50

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.