Stomatal proxy reconstructions of Mesozoic pCO2

Figure 2. Cycad and fern cuticle micromorphology
A. Lepidozamia perroffskyana (L. hopei morphology is identical). B. Zamia furfuracaea. C. Dicksonia antarctica. D. Cyathea cooperi. E. Stenochlaena palustris. F. Todea barbara. All scalebars = 100 µm.

Searching for a nearest living equivalent for Bennettitales: a promising extinct plant group for stomatal proxy reconstructions of Mesozoic pCO2

Steinthorsdottir M., Elliott‐Kingston C., Coiro M., McElwain J., (2021)

Margret Steinthorsdottira, Caroline Elliott-Kingston, Mario Coiro, Jennifer C. McElwain,


Environmental Science – DOI:10.1080/11035897.2021.1895304 – Corpus ID: 237735982 –


To understand Earth´s climate variability and improve predictions of future climate change, studying past climates is an important avenue to explore. A previously published record of pCO2, across the Triassic–Jurassic boundary (TJB, ~201 Ma) of East Greenland, showed that Bennettitales (Anamozamites and Pterophyllum) responded in parallel to the empirically proven pCO2-responders Ginkgoales, reducing their stomatal densities by half across the TJB, indicating a transient doubling of pCO2. The abundance of fossil Bennettitales in Mesozoic strata and natural history museum collections worldwide offers enormous potential for further stomatal proxy pCO2 reconstructions, but a suitable nearest living equivalent (NLE) should ideally first be identified for this extinct plant group. Using specimens from herbarium collections, three species of cycads, historically considered the best NLE, were tested for pCO2 response, as well as two species of tree ferns, grown in experimental growth chambers. None responded to changes in pCO2, and were consequently rejected as NLEs. Finally, two species of ferns were selected from the literature, and produced very similar pCO2 compared to Ginkgoales. However, these understory ferns are not appropriate NLEs for Bennettitales due to differences in habitat and a distant evolutionary relationship. Future work should test additional plant groups, in particular seed plants such as basal angiosperms and Gnetales, for suitability as NLE for Bennettitales in pCO2 reconstructions, for example through biogeochemical fingerprinting using infrared microspectroscopy. Until an appropriate NLE is identified, Bennettitales pCO2 can be reconstructed based on cross-calibration of stomatal densities with those of co-occurring pCO2 responders, such as Ginkgoales.


Reconstructing pCO2 during past climate change episodes is an important tool to understand the workings of the Earth system and better predict the path and consequences of future anthropogenic climate change. The stomatal densities of Bennettitales responded in parallel to those of proven pCO2-responder Ginkgoales to a transient doubling of pCO2 across the Triassic–Jurassic boundary of East Greenland. The potential of Bennettitales fossil leaves in palaeo-pCO2 reconstruction is of considerable interest, given the abundance of these fossils in Mesozoic strata and in museum collections worldwide. To calibrate pCO2 using stomatal densities in the stomatal proxy method, an NLE must ideally be selected for the fossil plants. An appropriate NLE should be a pCO2 responder with comparable morphological and/or ecological characters to the fossil plants investigated. Here, three species of cycads, the group historically considered morphologically and ecologically closest to Bennettitales, were first tested as potential NLEs, using herbarium material spanning the recent anthropogenic rise in pCO2, but were found to be unresponsive or weakly positively correlated to pCO2, and were rejected. Two species of tree ferns were tested next, of ancient groups somewhat morphologically similar to Bennettitales, grown in experimental chambers under ambient and elevated pCO2, but were also unresponsive, and consequently rejected. Reconstructed pCO2 using published SI data of two additional fern species as Bennettitales NLEs produced similar results to Ginkgoales pCO2 in a previous study, but are not considered appropriate NLEs either, due to distant evolutionary relationship and ecology. Future work may explore the potential of Gnetales and/or basal angiosperms as Bennettitales NLEs. Bennettitales fossil leaves may still be utilized for high-resolution stomatal proxy pCO2 reconstructions, by cross-calibration with other co-occuring, coeval pCO2 responders with known NLEs.

Stomata of Bennettitales

Fig. 2. Drawings of stomata of Bennettitales showing guard cells with characteristic thickenings (shaded dark grey), a pair of lateral subsidiary cells (light grey), and epidermal cells with lobed (sinuous) anticlinal walls. (A-C) reproduced from Thomas & Bancroft (1913); (D) reproduced from Florin (1931). (A) Otozamites feistmanteli; (B) Dictyozamites haveli; (C) Taeniopteris major; (D) Dictyozamites johnstrupii.

Leaf surface development and the plant fossil record: stomatal patterning in Bennettitales

Rudall P. J., Bateman R.M. (2019)

Paula J. Rudall, Richard M. Bateman,


Biological Reviews of the Cambridge Philosophical Society 94: 1179–1194 –

Fig. 3. Photomicrographs of abaxial leaf surfaces of Bennettitales. (A-C) Dictyozamites johnstrupii; (D) Pseudocycas roemeri; (E, F) Otozamites bornholmiensis; (G) Zamites schmidtii; (H) Nilssoniopteris tenuinervis; (I) Otozamites pterophylloides. Scale bars: A = 100 μm; B-I = 20 μm.


Stomata play a critical ecological role as an interface between the plant and its environment. Although the guard-cell pair is highly conserved in land plants, the development and patterning of surrounding epidermal cells follow predictable pathways in different taxa that are increasingly well understood following recent advances in the developmental genetics of the plant epidermis in model taxa. Similarly, other aspects of leaf development and evolution are benefiting from a molecular–genetic approach. Applying this understanding to extinct taxa known only from fossils requires use of extensive comparative morphological data to infer ‘fossil fingerprints’ of developmental evolution (a ‘palaeo-evo-devo’ perspective).

Fig. 4. Abaxial leaf surfaces of Dictyozamites johnstrupii (Bennettitales). (A-C) Photomicrographs showing stomata mostly oriented transversely to veins, with some exceptions in B. (D) drawing based on C, with lateral subsidiary cells coloured pink, guard cells yellow, and epidermal pavement cells blue (darker over veins). Blue arrows denote course of nearest veins, indicated by elongated epidermal cells over veins (i.e. in costal regions). Red arrow in D indicates likely direction of leaf expansion (see Section IV).

The seed-plant order Bennettitales, which flourished through the Mesozoic but became extinct in the Late Cretaceous, displayed a consistent and highly unusual combination of epidermal traits, despite their diverse leaf morphology. Based on morphological evidence (including possession of flower-like structures), bennettites are widely inferred to be closely related to angiosperms and hence inform our understanding of early angiosperm evolution. Fossil bennettites – even purely vegetative material – can be readily identified by a combination of epidermal features, including distinctive cuticular guard-cell thickenings, lobed abaxial epidermal cells (‘puzzle cells’), transverse orientation of stomata perpendicular to the leaf axis, and a pair of lateral subsidiary cells adjacent to each guard-cell pair (termed paracytic stomata). Here, we review these traits and compare them with analogous features in living taxa, aiming to identify homologous – and hence phylogenetically informative – character states and to increase understanding of developmental mechanisms in land plants.

Fig. 5. Stomatal development in species with paracytic stomata. Photomicrographs (right-hand column) made from specimens held in the microscope slide collection of the Royal Botanic Gardens, Kew. Diagrams with cells coloured/textured as follows: light grey = meristemoid, guard-mother cell or guard cell, black = first mesogene lateral subsidiary cell (LSC), dark texture = second mesogene LSC, light stipple = perigene LSC. In A-D, both LSCs are mesogene; the stomatal meristemoid divides twice and passes through a distinct ‘triad’ stage before achieving the full complement of four cells in the stomatal complex. In E and F, both LSCs are perigene. In G, one LSC is mesogene and the other perigene. (A) Gnetum (Gnetales), drawing adapted from Kausik (1974), photomicrograph: G. gnemon. (B) Welwitschia (Gnetales), adapted from Florin (1934b), photomicrograph: W. mirabilis. (C) Equisetum (Equisetales), drawing adapted from Chatterjee (1964) and Cullen & Rudall (2016), photomicrograph: E. myriochaetum. (D) Magnolia (Magnoliaceae), drawing adapted from Payne (1970), photomicrograph: M. guatemalensis. (E) Zea (Poaceae), drawing adapted from Campbell (1881) and many subsequent authors, including Rudall et al. (2017), photomicrograph: Z. mays. (F) Claytonia (Montiaceae), drawing adapted from Payne (1970), photomicrograph: Calandrinia crassifolia (Montiaceae). (G) Amborella (Amborellaceae), drawing adapted from Rudall & Knowles (2013), photomicrograph: A. trichopoda.

We propose a range of models addressing different aspects of the bennettite epidermis. The lobed abaxial epidermal cells indicate adaxial–abaxial leaf polarity and associated differentiated mesophyll that could have optimised photosynthesis. The typical transverse orientation of the stomata probably resulted from leaf expansion similar to that of a broad-leaved monocot such as Lapageria, but radically different from that of broad-leafed eudicots such as Arabidopsis. Finally, the developmental origin of the paired lateral subsidiary cells – whether they are mesogene cells derived from the same cell lineage as the guard-mother cell, as in some eudicots, or perigene cells derived from an adjacent cell lineage, as in grasses – represents an unusually lineage-specific and well-characterised developmental trait. We identify a close similarity between the paracytic stomata of Bennettitales and the ‘living fossil’ Gnetum, strongly indicating that (as in Gnetum) the pair of lateral subsidiary cells of bennettites are both mesogene cells. Together, these features allow us to infer development in this diverse and relatively derived lineage that co-existed with the earliest recognisable angiosperms, and suggest that the use of these characters in phylogeny reconstruction requires revision.

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.