Leaf epidermal cells of “living fossil” species are more sensitive than stomata to a doubling of CO2 concentration.

Screen Shot 2018-02-07 at 19.51.15
Fig. 1. Detail of a microscope observation of stomata and epidermal cells in a Sequoia sempervirens leaf abaxial surface.


Density and length of stomatal and epidermal cells in “living fossil” trees grown under elevated CO2 and a polar light regime

by Ogaya R., Llorens L., Penuelas (2011)

R. Ogaya a,*, L. Llorens b,1 , J. Peñuelas a

a Global Ecology Unit CREAF-CEAB-CSIC, CREAF (Center for Ecological Research and Forestry Applications), Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

b Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK


in Acta Oecologica 37: 381-385 –



During the Cretaceous and early Tertiary, when the climate was warm and the atmospheric CO2 concentration ([CO2]) was at least double that of the present-day, polar forests populated high latitude landmasses.

We investigated the density and length of stomata and other epidermal cells of two deciduous and three evergreen “living fossil” tree species representative of these ancient forests. These tree species were grown in a simulated Cretaceous high latitude environment at either ambient (400 ppmv) or elevated (800 ppmv) [CO2] during four years.

After 4 years growing at elevated [CO2], the leaf stomatal density and index (percentage of leaf epidermal cells that are stomata) of these plants were similar to those of their counterparts growing at ambient [CO2]. While the CO2 enrichment only modified the stomatal pore length in two of the five studied species, it increased significantly the overall length of the epidermal cells of all the species, reducing their density.

These results revealed that leaf epidermal cells of these “living fossil” species were more sensitive than stomata to an experimental doubling of atmospheric CO2 concentration.


Light intensity, temperature and stomatal morphology



The effect of light intensity and temperature changes on the stomatal and epidermal morphology of Quercus kelloggi: implications for paleoelevation reconstruction

by Kouwenberg L. L. R., McElwain J. C. (    )

Lenny L.R. Kouwenberg, Jennifer C. McElwain,



Recently a new method to reconstruct paleoelevation has been developed, based on the adjustment in number of stomata on leaves to the predictable decrease in CO partial pressure with altitude (McElwain, 2004). The first time 2 application of this method focuses on determining the altitude of growth of early Miocene Quercus pseudolyrata leaves from the northern Sierra Nevada. Calibration of the fossil stomatal numbers to altitude is based on the stomatal response of the modern day California black oak (Quercus kelloggi), whose leaves are indistinguishable from Q. pseudolyrata and grows in the same region. The clear correlation between increasing stomatal density and index vs. increasing altitude of growth found in Quercus kelloggii can for a large part be attributed to the predictable decrease in CO partial pressure. Many plants, 2 among which several Quercus species, show this increase in stomatal frequency with decreasing CO and CO is the only environmental parameter that globally and predictably changes with elevation. 2 2 However, several other climatic variables can also change with elevation and potentially influence stomatal density. The most important two are light intensity and temperature. In a natural setting, the relative influence of all environmental parameters on the stomatal change over altitude transect is extremely difficult to pry apart. Therefore, saplings of Quercus kelloggi were grown under controlled conditions in growth chambers, varying the levels of light and temperature. We will present preliminary data on the effect of both light levels and temperature regimes on stomatal frequency and epidermal cell morphology of Quercus kelloggii leaves.

Fossils and the evolution of stomatal function



Evolution of stomatal function: New perspectives and application to the fossil record

by McElwain J. C. (2012)


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


Presentation at New Phytologist Symposium Nr. 29 on Stomata 2012 –



Vascular plants have evolved a suite of different strategies to optimize carbon uptake against water loss via developmental, physiological and cellular level control of stomatal function.

Alterations to the development of stomatal number and size in response to atmospheric CO2 concentration enable some species to regulate stomatal conductance on time-scales of weeks to decades.

Other species demonstrate physiological control of stomatal aperture allowing for rapid control of stomatal function in response to abiotic factors on time scales of seconds to minutes.

Competing hypotheses have been proposed for the evolution of stomatal function in land plants with suggestions on one side that stomatal function is highly conserved across the plant phylogeny and the alternative view that stomatal function has increased incrementally across vascular plants from monilophytes to spermatophytes.

Observations of stomatal response to both instantaneous and long-term exposure to elevated atmospheric CO2 in UCD PÉAC (Programme for Experimental Atmospheres and Climate) do not completely support either competing hypothesis. Rather they suggest that the evolution of stomatal function is more complex and does not display a strong phylogenetic signature.

We show that loss of one aspect of stomatal function in response to an external stimulus can be readily compensated by improved functionality in another.

Experiments also show that there may be a trade-off between physiological control of stomatal conductance via stomatal aperture opening/closing and morphological control of conductance through developmental alteration to stomatal density and pore size.

This “stomatal trade- off hypothesis” is supported by the observation of a significant negative correlation between the magnitude of response of conductance to instantaneous change in CO2 and the magnitude and sign of response of stomatal density to long term elevated CO2 exposure. In other words, species which show rapid instantaneous changes in stomatal conductance do not alter stomatal density inversely under elevated CO2 and vice versa.

This has obvious and important implications for future development and application of the stomatal-CO2 proxy method which uses the inverse relationship between SD and CO2 to reconstruct palaeo-CO2 concentration in the geological past.

This will be discussed in relation to new estimates of palaeo-CO2 spanning the Eocene-Oligocene boundary between 40 and 25 million years ago when the Earth transitioned between an ice-free (greenhouse) and glaciated (icehouse) state.

How land plant life cycles first evolved

Science Magazine
Photo credit: Science – How plant life cycles evolved Plants that lived in the 407-million-year-old Rhynie Chert hot spring system had life cycles that were different from living species.


How land plant life cycles first evolved

by Kenrick P. (2017)

Paul Kenrick,

Department of Earth Sciences, The Natural History Museum, London SW7 5BD, UK



in Science 358(6370): 1538-1539 – DOI: 10.1126/science.aan2923 –


This year marks the 100th anniversary of the first in a series of papers on the biota of a 407-million-year-old hot spring system that opened a window onto early life on land (1). The site near the village of Rhynie in Aberdeenshire, Scotland, is exceptional because fossilization occurred in microcrystalline silica (chert), preserving organisms to the cellular level and shedding light on community structure and interactions among the plants, arthropods, fungi, algae, and cyanobacteria. Recent research on these remarkable fossils and advances in understanding plant developmental genetics are beginning to reveal how major changes in life cycle had an early influence on the direction of plant evolution.

When the Rhynie Chert fossils were first reported, the plants, in particular, caused quite a stir because they seemed to capture an early stage in the adaptation to life on land. Most lack key organs familiar in living species, such as leaves or roots. Moreover, they are small (<20 cm), with simple bifurcating axes that terminate in spore-bearing sacs. Later research has revealed that their life cycles also differed in important ways from those of living species.

Land plants inherited their biochemistry and cell biology from ancestral green algae, but their fundamental organs and tissues evolved on land. Their closest algal relatives have a haplontic life cycle, which typically features a simple multicellular haploid sexual phase (a gametophyte) and a unicellular diploid zygote (2). This implies that the ancestor of land plants was also haplontic, probably with a filamentous, weakly differentiated multicellular phase (3).

The transition to land entailed changes in life cycle in which the diploid zygote also became multicellular (4). This change was accompanied by substantial somatic development, resulting in the evolution of tissues and organs basic to plants, such as stems, leaves, roots, a vascular system, stomata (the minute pores that facilitate gaseous exchange), and sex and dispersal organs. The multicellular diploid phase evolved into a highly successful spore-producing dispersal unit (the sporophyte), laying the foundations of modern plant diversity. Changes in life cycle thus underpinned the early diversification of plants on land, but how such changes evolved remains a puzzle.

Life-cycle phases are known in varying degrees of detail for four of the six fossil plant species from the Rhynie Chert. Like ferns, the gametophytes and sporophytes lived as independent plants, but unlike any living land plants, tissues such as rooting structures, a vascular system, and stomata were expressed in both parts of the life cycle (6). Despite these similarities, sporophytes and gametophytes were not identical. Although both were axial and leafless, the gametophytes were smaller. Overall, habit and size are still poorly understood for several gametophytes, but one factor known to influence their size was the degree of development of their branching systems (1). Also, in several species, gametophyte axes terminated in an expanded cup-shaped structure that bore the sexual organs. These differences notwithstanding, the gametophyte bore much greater similarity to the sporophyte than it does in living species, and it developed tissues of greater diversity.


Stomatal density and index of a fossil Platanus



Stomatal density and index data of Platanus neptuni leaf fossils and their evaluation as a CO2 proxy for the Oligocene

by Roth-Nebelsick A., Oehm C., Grein M., Utescher T., Kunzmann L., Friedrich J. P., Konrad W. (2014)

Anita Roth-Nebelsick, a  Christoph Oehm, a  Michaela Grein, b  Torsten Utescher, cd  Lutz Kunzmann, e  Jan-Peter Friedrich, a  Wilfried Konrad, f

State Museum of Natural History Stuttgart, Rosenstein 1, D-70191 Stuttgart, Germany
Übersee-Museum Bremen, Bahnhofsplatz 13, D-28195 Bremen, Germany
Steinmann Institute, Bonn University, Nuβallee 8, D-53115 Bonn, Germany
Senckenberg Research Institute, D-60325 Frankfurt, Germany
Senckenberg Natural History Collections Dresden, Königsbrücker Landstraβe 159, D-01109 Dresden, Germany
University of Tübingen, Department of Geosciences, Hölderlinstrasse 12, D-72074 Tübingen, Germany


in Rev. Palaeobot. Palynol. – 206: 1– 9 – DOI: 10.1016/j.revpalbo.2014.03.001 –



Stomatal data of Platanus neptuni leaves were used as CO2 proxy for the Oligocene.

CO2 calculation was based on a gas exchange model.

The data indicate quite a constant level of about 400 ppm throughout the Oligocene.


Platanus neptuni (Ettingshausen) Bůžek, Holý and Kvaček is a deciduous and preferentially azonal taxon of temperate to warm-temperate vegetation in Europe from the Late Eocene to the Late Miocene. The high fossilization potential of its leaves and easily identifiable stomata and epidermal cells make P. neptuni an excellent source of stomatal data that can be utilized as a CO2proxy. Moreover, it was found in former studies that CO2 data based on stomatal frequency data of P. neptuni overlapped to a high degree with CO2 results which are provided by other, contemporaneous taxa. In this study, the stomatal CO2 signal of P. neptuni is expanded to include the early Oligocene and is analyzed in more detail with three aims: 1) to evaluate the CO2 signal of P. neptuni stomatal data, 2) to check SI and SD data of P. neptuni for consistency, and 3) to contribute additional terrestrial CO2 data to the Oligocene record. During the Oligocene, full scale Antarctic glaciation occurred, punctuated by various distinct glaciation events. There is evidence that Oligocene glaciation was coupled to atmospheric CO2 level. Presently, the main proxy sources for Oligocene CO2 levels are alkenones and boron-isotope data, both obtained from marine sediments.

Since P. neptuni is an extinct taxon, CO2 was reconstructed by using an ecophysiological modeling approach to plant gas exchange which utilizes various other data in addition to stomatal density. Material was considered from sites which are dated to the following time intervals: early Oligocene — 33.9 to 32 Ma, late Oligocene — 27 to 26.2 Ma and 25.3 to 23 Ma, and latest Oligocene — around 24 Ma. Comparison of raw SI and SD data of P. neptuni revealed partially conflicting results, with the SD data indicating a decrease in CO2from the early to the late Oligocene whereas SI data indicate an increase. In contrast, CO2 results calculated with the gas exchange model indicate relatively stable CO2 for the considered time intervals, with levels of about 400 ppm. The reconstructed CO2 data points are similar to other proxy data and are consistent with the general climate development during the Oligocene.

Stomatal density and stomatal index of fossil Buxus and CO2 concentration



Stomatal density and stomatal index of fossil Buxus from coprolites of extinct Myotragus balearicus Bate (Artiodactyla, Caprinae) as evidence of increased CO2 concentration during the late Holocene.

by Rivera L., Baraza E., Alcover J. A., Bover P., Rovira C. M., Bartolomé J. (2014)

Rivera Leidy, Baraza Elena, Alcover Josep A., Bover Pere, Rovira Carla M., Bartolomé Jordi,


in Holocene 24, 876–880 –  doi: 10.1177/0959683614530445 –

CrossRef Full Text | Google Scholar –



Stomatal density of plants may vary depending on environmental factors, such as CO2concentration. Under the current atmospheric conditions, it is expected that leaves have different stomatal density than they had hundreds or thousands of years ago, due to the rise in CO2 in the atmosphere.

Coprolites of the extinct Myotragus balearicus from Cova Estreta (Pollença, Mallorca), with a radiocarbon age of 3775–3640 cal. bc, have been used to study the diet of this bovid. A significant amount of epidermal fragments of Buxus was found in the faecal material. Three coprolites were used to estimate the stomatal density and stomatal index of Buxus epidermal fragments from this period.

Samples of the endangered Buxus balearica, the sole species of Buxus currently present on Mallorca, and samples of the Buxus sempervirens, present in the nearest mainland, were also collected in different locations. Leaves were examined using microscopy to determine and compare the stomatal density and stomatal index between current plant material and coprolite material.

The results indicated a higher value for stomatal index (12.71) and stomatal density (297.61 stomata/mm2) in leaves from the coprolites versus leaves of the living B. balearica and B. sempervirens species (7.99 and 227.77 stomata/mm2 respectively).

These results could provide a palaeobotanic evidence of a change in atmospheric CO2 concentration since mid-Holocene in the Mediterranean basin.

Stomatal frequency-based palaeo-CO2 reconstructions

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Bennettitalean leaf cuticle fragments (here Anomozamites and Pterophyllum) can be used interchangeably in stomatal frequency-based palaeo-CO2 reconstructions

by Steinthorsdottir M., Bacon K. L., Popa M. E., Bochner L., McElwain J. C. (2011)


1 Department of Geological Sciences, Stockholm University, SE-10691 Stockholm, Sweden;

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

3 Faculty of Geology and Geophysics, University of Bucharest, 1, N. Balcescu Ave., 010041, Bucharest, Romania

4 Department of Geology and Environmental Geosciences, Lafayette College, Easton, PA, USA



in Palaeontology 54: 867–882 –



Bennettites are an abundant and frequently well- preserved component of many Mesozoic fossil floras, often playing an important ecological role in flood plain vegetation communities.

During a recent study focusing on stomatal indices of Triassic–Jurassic fossil plants, it became evident that the leaf fragments of two bennettite genera Anomozamites Schimper (1870) emend. Harris (1969) and Pterophyllum Brongniart (1825) display a significant overlap of leaf shape as well as cuticular characters.

Owing to the preference of recognition of single taxa (ideally species) for the stomatal method, we use a database of 70 leaf fragments of Anomozamites and Pterophyllum compressions from five isotaphonomic Late Triassic sedimentary beds of Astartekløft in East Greenland to test whether leaf and cuticle fragments of the two genera can be separated using a range of quantitative and qualitative morphological and statistical analyses.

None of the observed characters – including stomatal frequencies – could be applied to separate the fragments of the two genera into well-defined groups. Our results therefore indicate that fragmented material and dispersed cuticles cannot be utilized to distinguish between Anomozamites or Pterophyllum at the genus level, but that instead these cuticle fragments may be used interchangeably as stomatal proxies.

Classification of fossil leaves into either of these genera is thus only possible given adequate preservation of macro-morphology and is not possible based solely on cuticle morphology.

We suggest that this large inter- and intra-generic morphological variation in both leaf and cuticle traits within Anomozamites and Pterophyllum may be related to the bennettites’ role as understory plants, experiencing a range of micro-environmental conditions, perhaps depending mainly on sun exposure.

Based on the results obtained in this study, we conclude that Anomozamites and Pterophyllum cuticle fragments can be employed interchangeably in palaeo [CO2] reconstructions based on the stomatal method, thus potentially annexing a plethora of bennettitalean fossil plant material as CO2 proxies, including dispersed cuticles.