A likely occurrence of polyploidy in Sphenobaiera huangii may result in underestimated paleo-CO2 when applying mechanistic method due to an increase in the size of the stomatal complex

An inter-comparison study of three stomatal-proxy methods for CO2 reconstruction applied to early Jurassic Ginkgoales plants

Zhou N., Wang Y., Ya L., Porter A. S., Kürschner W. M., Li L., Lu N., McElwain J. C. (2020)

Ning Zhou a-b, Yongdong Wangb, Li Ya b, Amanda S. Porter c, Wolfram M. Kürschner d, Liqin Li b, Ning Lu b, Jennifer C. McElwain c,

a Department of Geology, Northwest University, Xi’an 710069, China

b State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology, and Center for Excellence in Life and Paleoenvironment, Chinese Academy of Sciences, Nanjing 210008, China

c Department of Botany, School of Natural Sciences, Trinity College Dublin, the University of Dublin, Dublin 2, Ireland

d Department of Geosciences, University of Oslo, N-0316 Oslo, Norway


Palaeogeography, Palaeoclimatology, Palaeoecology 542: 109547 – https://doi.org/10.1016/j.palaeo.2019.109547



• A high degree of consistency in pCO2 and trends are observed in three methods.

• The performance of mechanistic method is improved.

• The genome size may result in overestimated pCO2 when applying mechanistic method.


The inverse relationship between concentrations of CO2 in the atmosphere (pCO2) and the stomatal index of vascular plant has been widely used to estimate ancient levels of atmospheric CO2. However, some atmospheric concentration of CO2 in the geological past (paleo-CO2) estimates show little congruence because they are derived using different correlative methods, or from different fossil plant species with different calibration approaches. Here we apply three methods, including (1) the empirical method of McElwain (1998), (2) the empirical method of Barclay and Wing (2016) and (3) the mechanistic method of Franks et al., (2014) to a single fossil Ginkgo species (Ginkgoites marginatus) to track and assess their consistency of pCO2 estimates for the Early Jurassic. By using an inter-comparison of three methods, a high degree of consistency in pCO2 estimates and trends has been observed in two empirical proxy methods. In addition, the mechanistic method and both the empirical methods also show generally good consistent paleo-CO2 estimates at the bed-level. To test the congruence of paleo-CO2 estimates, we also apply all three methods to one additional Ginkgoalean fossil species (Sphenobaiera huangii). All three methods show species-dependent uncertainty in paleo-CO2 estimates when applied to different Ginkgalean fossil species collected from the same fossiliferous bed. Moreover, considering the potential effect of guard cell size to the mechanistic method, the genome size of fossil and living Ginkgo taxa was analyzed based on the significant positive relationship between genome size and guard cell size. The result demonstrates that a likely occurrence of polyploidy in Sphenobaiera huangii may result in underestimated paleo-CO2 when applying mechanistic method due to an increase in the size of the stomatal complex.

Stomatal density worldwide was responding to significant changes in pCO2 across the K–Pg

Global trends of pCO2 across the Cretaceous-Paleogene boundary supported by the first Southern Hemisphere stomatal proxy based pCO2 reconstruction

Steinthorsdottir M., Vajda V., Pole M. (2016)

Margret Steinthorsdottir a, Vivi Vajda b-c, Mike Pole d,

a Department of Geological Sciences and Bolin Centre for Climate Research, Stockholm University, SE 109 61 Stockholm, Sweden

b Department of Palaeobiology, Swedish Museum of Natural History, SE 104 05 Stockholm, Sweden

c Department of Geology, Lund University, SE-223 62 Lund, Sweden

d Nanjing Institute of Geology and Palaeontology, Academia Sinica (the Chinese Academy of Sciences), 39 East Beijing Road, Nanjing 210008, PR China


Palaeogeography, Palaeoclimatology, Palaeoecology 464: 143–152 – https://doi.org/10.1016/j.palaeo.2016.04.033



• A new fossil Lauraceae leaf database from New Zealand spans the K–Pg boundary.

• Latest Cretaceous–mid Paleocene pCO2 was reconstructed using the stomatal proxy.

• On average, pCO2 decreased by ~ 45%, from ~ 570 to ~ 310 ppm, during this time.

• Results are consistent with previously published Northern Hemisphere pCO2 records.

• However, a spike of extremely high pCO2 previously reported at K–Pg was not confirmed.


Reliable reconstructions of atmospheric carbon dioxide concentrations (pCO2) are required at higher resolution than currently available to help resolve the relationship between mass extinctions and changes in palaeo-pCO2 levels. Such reconstructions are needed: 1, at a high temporal resolution for constraining the pre- and post-extinction atmospheres; and 2, at a sufficient spatial resolution to constrain potential inter-hemispheric differences. Here we estimate pCO2 based on fossil Lauraceae leaf cuticle specimens derived from three localities with strata spanning the latest Cretaceous to the mid-Paleocene, including a new Cretaceous–Paleogene boundary (K–Pg) locality, in New Zealand. We use two independent methods of stomatal density-based pCO2 reconstructions; a transfer function calibrated using herbarium material and the stomatal ratio method, producing three calibration sets. Our results based on the mean values of each of the three calibration methods indicate pCO2 ranging between ca. 460 and 650 ppm during the latest Cretaceous, falling precipitously to average values between ca. 360 and 430 ppm across the K–Pg boundary, and further to ca. 305–320 ppm in the mid-Paleocene. A ‘spike’ of extremely high pCO2 at the K–Pg could not be confirmed, but our results are, nonetheless, consistent with previously published pCO2 records from the Northern Hemisphere, and show that stomatal density worldwide was responding to significant changes in pCO2 across the K–Pg.

Stomatal densities of fossil plants can be used to reconstruction past CO2 levels

Sulphur dioxide fumigation effects on stomatal density and index of non-resistant plants: implications for the stomatal palaeo-[CO2] proxy method

Haworth M., Elliott-Kingston C., McElwain J. C. (2012)

Matthew Haworth a, Caroline Elliott-Kingston b, Angela Gallagher c, Annmarie Fitzgerald b, Jennifer C. McElwain b,

a CNR – Istituto di Biometeorologia (IBIMET), Via Giovanni Caproni 8, 50145 Firenze Italy b

School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland

c Department of Hydrology and Geo-environmental Sciences, Vrije Universiteit, De Boelelaan 1085–1087, 1081 HV Amsterdam, The Netherlands


Review of Palaeobotany and Palynology 182: 44–54 – https://doi.org/10.1016/j.revpalbo.2012.06.006



► Stomatal densities of fossil plants can be used to reconstruction past CO2 levels.

► SO2 may also affect stomatal development and therefore estimates of CO2.

► Seven plants with no resistance to SO2 were grown in controlled environments.

► SO2 resulted in an increase in the ratio of stomatal density to index.

► This ratio may be employed to differentiate between SO2 and CO2 effects on stomata.



The inverse relationship between the number of stomata on the surface of a leaf and the atmospheric concentration of carbon dioxide ([CO2]) in which it developed permits the use of fossil plants as indicators of palaeo-atmospheric [CO2] level (palaeo-[CO2]). This “stomatal method” to reconstruct palaeo-[CO2] is dependant upon stomatal initiation being determined by [CO2]. However, global perturbations to the carbon cycle and climate system throughout earth history are not only characterised by fluctuations in the level of atmospheric [CO2], but also by the release of large volumes of toxic gases such as sulphur dioxide (SO2) into the atmosphere. Recent studies have raised uncertainties into the efficacy of stomatal palaeo-[CO2] proxies during episodes of SO2 fumigation. This study aims to assess the effect of exposure to 0.2 ppm SO2 on the stomatal frequencies of plant species with no evolutionarily acquired resistance to toxic gases in comparison to individuals grown under control conditions and atmospheres of elevated [CO2]. Fumigation with SO2 resulted in a general pattern of increased stomatal density (SD) values, but induced variability in the stomatal index (SI) responses of the plant species studied. Ginkgo biloba exhibited an increase in SI, whereas the araucariacean conifers Agathis australis and Araucaria bidwillii displayed reductions in SI that were indistinguishable from values observed under [CO2] enrichment. These results suggest that the presence of atmospheric SO2 may reduce the effectiveness of stomatal reconstructions of palaeo-[CO2] during intervals characterised by the release of large volumes of toxic gases into the atmosphere. However, exposure to SO2 induced significant increases in the SD/SI ratios of six of the seven species studied. Calculation of the SD/SI ratios of fossil plants may identify any stratigraphic horizons where the stomatal initiation responses of the fossil flora were affected by sudden fumigation with toxic gases, and thus influence palaeo-[CO2] estimates. Therefore the SD/SI ratios of fossil plants may serve as indicators of the effectiveness of stomatal reconstructions of palaeo-[CO2].

Calculated as stomatal ratios, the values generally tracked the CO2 variations predicted by a long-term carbon cycle model confirming the utility of this plant group to provide a reasonable measure of ancient atmospheric CO2 change

Figure 1–9
The lower epidermal characters of extant and fossil Ginkgo leaves. 1. Replica of lower epidermis on G. biloba leaf collected in 1924. Figs. 2–5. Ginkgo biloba (collected in 1998). 2. No significant differentiation between stomata zone and vein zone on a young leaf. 3. Significant differentiation between stomata zone and vein zone on a developed leaf. 4. Developing stomata on a young leaf. 5. Lower leaf epidermis on a developed leaf. Figs. 6–9. Fossil Ginkgo. 6. Lower leaf epidermis of G. coriacea. 7. Lower leaf epidermis of G. huttoni. 8. Lower leaf epidermis of G. yimaensis. 9. Lower leaf epidermis of G. obrutschewii. All scale bars = 100 μm

Assessing the potential for the stomatal characters of extant and fossil Ginkgo leaves to signal atmospheric CO2 change

Chen L.-Q., Li C.-S., Chaloner W. G., Beerling D. J., Sun Q. G., Collinson M. E., Mitchell P. L. (2001)

Li-Qun Chen, Cheng-Sen Li, William G. Chaloner, David J. Beerling, Qi-Gao Sun, Margaret E. Collinson, Peter L. Mitchell,


American Journal of Botany 88: 1309–1315 – https://doi.org/10.2307/3558342



The stomatal density and index of fossil Ginkgo leaves (Early Jurassic to Early Cretaceous) have been investigated to test whether these plant fossils provide evidence for CO2-rich atmosphere in the Mesozoic. We first assessed five sources of natural variation in the stomatal density and index of extant Gingko biloba leaves: (1) timing of leaf maturation, (2) young vs. fully developed leaves, (3) short shoots vs. long shoots, (4) position in the canopy, and (5) male vs. female trees. Our analysis indicated that some significant differences in leaf stomatal density and index were evident arising from these considerations. However, this variability was considerably less than the difference in leaf stomatal density and index between modern and fossil samples, with the stomatal index of four species of Mesozoic Ginkgo (G. coriacea, G. huttoni, G. yimaensis, and G. obrutschewii) 60–40% lower than the modern values recorded in this study for extant G. biloba. Calculated as stomatal ratios (the stomatal index of the fossil leaves relative to the modern value), the values generally tracked the CO2 variations predicted by a long-term carbon cycle model confirming the utility of this plant group to provide a reasonable measure of ancient atmospheric CO2 change.

Bennettitales pCO2 can be reconstructed based on cross-calibration of stomatal densities with those of co-occurring pCO2 responders, such as Ginkgoales

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. C. (2021)

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


GFF 143(2-3): 190-201 -Special Issue: Advances in Swedish palaeontology; the importance of fossils in natural history collections – The Department of Palaeobiology at the Swedish Museum of Natural History – https://doi.org/10.1080/11035897.2021.1895304



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.

Stomata in Podozamites harrisii, Pseudotorellia resinosa and Pseudotorellia palustris

Details: A) Podozamites harrisii; incomplete leafy shoot showing three attached leaves (left). Detail of stomatal band from abaxial leaf cuticle showing cell outlines of transversely oriented, paracytic (monocyclic) stomata (top right). Detail from adaxial leaf cuticle showing rectangular epidermal cell outlines (bottom right). B) Pseudotorellia resinosa; two isolated multiveined leaves (left). Detail of stoma showing lateral subsidiary cells and inner periclinal walls of guard cells (top middle). Detail of outer surface of abaxial cuticle showing stomatal pit with stomatal aperture (bottom middle). C) Pseudotorellia palustris; two isolated multiveined leaves (left). Detail of stoma showing lateral subsidiary cells (top right). Detail of outer surface of abaxial cuticle showing a stoma with papillae (bottom right). Scale bars: A= 1cm, 20μm, 20μm respectively; B= 5mm, 40μm, 40μm respectively; C= 1cm, 20μm, 20μm respectively. Tevshiin Govi lignite, Mongolia, ca. 125 million years before present.

Strap shaped leaf fossils from the Tevshiin Govi locality

Oak Spring Garden Foundation (2021)



May 19, 2021OSGF

The Tevshiin Govi fossil locality is a small coal mine, located approximately 220 km south of Ulaanbaatar, Mongolia. Superbly preserved plant fossils occur three dimensionally, little altered from their original shape and size. Individual plant parts can be extracted whole from the soft lignitic matrix that surrounds them. The Tevshiin Govi locality has yielded a variety of fossil plants, some similar to living conifers and filmy ferns, but others that have no close living relatives.

 Details:  TheTevshiin Govi locality (left); compressed log removed from the sediment (center); cone of Krassilovia compressed in the sediment (upper right); compressed leaf remains preserved as lignite (lower right). Tevshiin Govi lignite, Mongolia, ca. 125 million years before present.

Novel genes in the first land plants led to the single origin of stomata, but the stomatal closure of seed plants resulted from later gene expansions

How ancient plants began using water when they moved on to land

Bowles A., Paps J., Bechtold U. (2022)

Alexander M. C. Bowles, Jordi Paps, Ulrike Bechtold,

The Conversation – New Phytologist – https://doi.org/10.1111/nph.17981




  • The origin of land plants and their descendants was marked by the evolution of key adaptations to life in terrestrial environments such as roots, vascular tissue and stomata. Though these innovations are well characterized, the evolution of the genetic toolkit underlying their development and function is poorly understood.
  • We analysed molecular data from 532 species to investigate the evolutionary origin and diversification of genes involved in the development and regulation of these adaptations.
  • We show that novel genes in the first land plants led to the single origin of stomata, but the stomatal closure of seed plants resulted from later gene expansions. By contrast, the major mechanism leading to the origin of vascular tissue was cooption of genes that emerged in the first land plants, enabling continuous water transport throughout the ancestral vascular plant. In turn, new key genes in the ancestors of plants with true leaves and seed plants led to the emergence of roots and lateral roots.
  • The analysis highlights the different modes of evolution that enabled plants to conquer land, suggesting that gene expansion and cooption are the most common mechanisms of biological innovation in plant evolutionary history.

Stomata in Psilotum

The Earliest Known Land Flora

Bower F. (1920)

Nature 105: 712–714 – https://doi.org/10.1038/105712a0



II. COMPARISON of these four fossil species from Rhynie with other fossils already known from the early Devonian period shows that a very homogeneous flora existed at that time, consisting chiefly of leafless and rootless land-living plants. These and other characters, such as their large, distal, sometimes solitary, and often forked sporangia, stamp these plants as exceptionally primitive. Among living plants the nearest of kin to them are clearly the Psilotaceæ, a family which has long presented a problem in morphology and classification. It comprises two living genera, Psilotum and Tmesipteris. Both genera are rootless. Their imperfect morphological differentiation is shown by the fact that botanists are not yet agreed whether their lateral appendages are to be held as truly foliar or not. Psilotum is native throughout the tropics, and is represented by two well-marked species. The commonest, P. triquetrum, has upright and shrubby aerial shoots, with radial construction and frequent bifurcations. These spring from leafless underground rhizomes, profusely bifurcated. They are covered with rhizoids, and contain a myccrhizic fungus. On the lower part of the aerial shoots simple spine-like leaves are borne, but towards the distal ends these are replaced by forked spurs, between the prongs of which a synangium, usually with three loculi, is seated. The aerial shoot is traversed by a vascular strand consisting of xylem in the form of a hollow many- rayed star, with sclerotic core, and branch-strands run out to the appendages. The whole is covered by epidermis with stomata, and the cortex provides the photosynthetic tissue. Tmesipteris is represented by only one species, limited to Australasia. It grows usually among the massed roots that cover the stems of tree-ferns, but some times upon the ground. Its general form is like that of Psilotum, but the underground rhizomes are longer and the appendages larger, while only two loculi are usually present in each synangium. Clearly the form and vascular structure of these plants are generally like those of the Rhynie flora.

Stomata in Araucaria fossils

Fig. 2C. Cuticle of Araucaria nathorstii Dusén. (18-26). (18) Stomata of adaxial cuticle, showing four subsidiary cells. NSM PP-12155. Scale bar = 50 m. (19) Abaxial cuticle, showing elongate rectangular normal cells and regular stomatal rows. NSM PP-12155. Scale bar = 100 m. (20) Details of stomatal rows of abaxial cuticle. Stomatal apparatus in same row separated by 1-2 regular epidermal cells. NSM PP-12155. Scale bar = 50 m. (21) Stomata in abaxial cuticle showing four subsidiary cells. NSM PP-12155. Scale bar = 50 m. (22) Sinuous anticlinal walls of abaxial epidermal cells. NSM PP-12155. Scale bar = 50 m. (23) Stomata of adaxial cuticle, and four subsidiary cells. NSM PP-12164. Scale bar = 50 m. (24) Adaxial cuticle, showing three stomatal rows. Stomatal rows separated by 3-5 rows of elongate rectangular cells. NSM PP-12164. Scale bar = 100 m. (25) Abaxial cuticle, showing stomatal rows, with stomata of variable orientation. NSM PP-12164. Scale bar = 100 m. (26) Close-up of (25). Stomatal apparatus monocyclic, with 4-6 subsidiary cells. NSM PP-12164. Scale bar = 50 m. 

Araucarian leaves and cone scales from the Loreto Formation of Río de Las Minas, Magellan Region, Chile 1

Asakawa T., Yabe A., Yamada T., Uemura K., Terada K., Leppe M., Hinojosa F., Nishida H. (2016)

Takeshi Asakawa, Chiba University

Atsushi Yabe, National Museum of Nature and Science

Toshihiro Yamada, Osaka City University

Kazuo Terada, Fukui Prefectural Dinosaur Museum, Japan

Marcelo Leppe, Instituto Antartico Chileno

Felipe Hinojosa, University of Chile

Harufumi Nishida, Chuo University


Botany 94(9): 805-815 – DOI: 10.1139/cjb-2016-0059



Fig. 2D. Araucaria sp. (27-33). (27) Shoot and leaves. NSM PP-12166 a,b. Scale bar = 10 mm. (28) Close-up of (27), showing leaf. NSM PP-12166a. Scale bar = 5 mm. (29) Close-up of (27), showing sections of expanding leaves. NSM PP-12166b. Scale bar = 5 mm. (30) Adaxial cuticle consisting of quadrangular to irregular epidermal cells and few stomata (white arrowhead). Not all the stomata are recognized owing to poor preservation. NSM PP-12165. Scale bar = 100 m. (31) Abaxial cuticle, showing irregular rows of stomata, separated by 1-3 rows of irregular or rectangular epidermal cells. NSM PP-12166. Scale bar = 100 m. (32) Abaxial cuticle showing variably oriented stomatal apparatus. NSM PP-12165. Scale bar = 100 m. (33) Close-up of (32), showing four subsidiary cells. NSM PP-12165. Scale bar = 50 m. [Colour online.] 

Cone scales and leaves of the Araucariaceae are reported from the Loreto Formation in Río de Las Minas, Punta Arenas, Chile. Two types of cone scales including one new species, Araucarites alatisquamosus are recognized. They are similar to Araucaria section Eutacta. Two types of leaves are assigned to Araucaria nathorstii Dusén, and one new type is distinguished in the specimens from the type locality of A. nathorstii. A narrow leaf type is identical to Dusén’s “Blätter der sterilen Zweige (leaves of sterile shoot)” of A. nathorstii, and a broad leaf type is identical to his “Blätter der fertilen Zweige (leaves of fertile shoot)”. Both types show characters of leaves of section Araucaria as suggested by earlier studies. A smaller leaf type differs from the original description of A. nathorstii, and consists of small appressed leaves with the obtuse apex and stomatal orientation that are the characteristic of the section Eutacta. The occurrence of leaves and cone scales similar to section Eutacta suggest the presence of araucarians close to this section in southernmost South America during the Eocene-early Oligocene, and provide evidence for the wide distribution and diversity of genus Araucaria relatives in South America during the Paleogene.