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See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/260122490 Sulphur dioxide fumigation effects on stomatal density and index of non-resistant plants: implications for the stomatal palaeo-[CO2] proxy method. ARTICLE in REVIEW OF PALAEOBOTANY AND PALYNOLOGY · JANUARY 2012 Impact Factor: 1.94 CITATIONS READS 3 57 3 AUTHORS: Matthew Haworth Caroline Elliott-Kingston Italian National Research Council University College Dublin 23 PUBLICATIONS 395 CITATIONS 8 PUBLICATIONS 88 CITATIONS SEE PROFILE SEE PROFILE Jennifer C Mcelwain University College Dublin 93 PUBLICATIONS 2,262 CITATIONS SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately. Available from: Matthew Haworth Retrieved on: 09 November 2015 Review of Palaeobotany and Palynology 182 (2012) 44–54 Contents lists available at SciVerse ScienceDirect Review of Palaeobotany and Palynology journal homepage: www.elsevier.com/locate/revpalbo Research paper Sulphur dioxide fumigation effects on stomatal density and index of non-resistant plants: Implications for the stomatal palaeo-[CO2] proxy method Matthew Haworth a,⁎, Caroline Elliott-Kingston b, Angela Gallagher c, Annmarie Fitzgerald b, Jennifer C. McElwain b a b c CNR – Istituto di Biometeorologia (IBIMET), Via Giovanni Caproni 8, 50145 Firenze Italy School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland Department of Hydrology and Geo-environmental Sciences, Vrije Universiteit, De Boelelaan 1085–1087, 1081 HV Amsterdam, The Netherlands a r t i c l e i n f o Article history: Received 19 March 2012 Received in revised form 23 June 2012 Accepted 25 June 2012 Available online 4 July 2012 Keywords: stomata palaeo-[CO2] sulphur dioxide Triassic Jurassic Boundary Ginkgo Araucaria a b s t r a c t 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]. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The stomatal frequencies of fossil plants are increasingly being used as indicators of relative changes in palaeo-atmospheric levels of carbon dioxide (palaeo-[CO2]) through earth history (eg. Kürschner et al., 1996; Wagner et al., 1999; Kouwenberg et al., 2005; Self et al., 2006; Passalia, 2009; Beerling and Royer, 2011; Doria et al., 2011; Grein et al., 2011; Stults et al., 2011). The density of stomata on a leaf surface often reflects the atmospheric carbon dioxide concentration ([CO2]) during leaf development. This “snap-shot” of [CO2] permits the stomatal frequencies of fossil plants to reconstruct palaeo-[CO2] at a higher ⁎ Corresponding author. Tel.: +39 0553033711; fax: +39 055308910. E-mail addresses: [email protected] (M. Haworth), [email protected] (C. Elliott-Kingston), [email protected] (A. Gallagher), annmarie.fi[email protected] (A. Fitzgerald), [email protected] (J.C. McElwain). 0034-6667/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.revpalbo.2012.06.006 temporal resolution than other proxy methods (Beerling and Royer, 2002a,b; McElwain et al., 2002; Wagner et al., 2002; Haworth et al., 2005; Kouwenberg et al., 2005; Smith et al., 2010). This high temporal resolution is crucial to our understanding of geologically sudden perturbations to the environment and global carbon cycle that are commonly observed at mass extinction and ocean anoxic events (eg. Hesselbo et al., 2000; Beerling and Berner, 2002; Jenkyns, 2003; Hesselbo et al., 2007; Ruhl et al., 2009; Arango-Velez et al., 2011; Bacon et al., 2011; Ruhl and Kürschner, 2011; Schaller et al., 2011; Hermoso et al., 2012; Zeppel et al., 2012). These perturbations to the carbon cycle are marked by pronounced fluctuations in atmospheric [CO2] that are recorded in the stomatal numbers of fossil plants growing at the time (eg. McElwain et al., 1999; Beerling et al., 2002; McElwain et al., 2005; Lamarque et al., 2007; Bonis et al., 2010; Smith et al., 2010; Retallack et al., 2011; Steinthorsdottir et al., 2011). However, many of these extinction and ocean anoxic events are also characterised by the sudden release of large volumes of toxic gases such as sulphur dioxide (SO2) M. Haworth et al. / Review of Palaeobotany and Palynology 182 (2012) 44–54 and hydrogen sulphide (H2S) into the atmosphere from global scale volcanism and ocean euxina (Beerling and Berner, 2002; McHone, 2003; Grice et al., 2005; Kump et al., 2005; Self et al., 2006; Hori et al., 2007). It is therefore of great importance to understand the effect of toxic atmospheric gases on stomatal initiation to constrain and refine stomatal-based reconstructions of palaeo-[CO2] at these key intervals in earth history, which are crucial to our understanding and prediction of biodiversity and environmental responses to current rising [CO2] (Tanner et al., 2007; Haworth et al., 2010a). Stomatal frequency can be measured as stomatal density (SD: the number of stomata per mm 2), or as a ratio of the number of stomata to epidermal cells known as the stomatal index (SI) (Poole and Kürschner, 1999). The SI negates the effect leaf expansion may have on SD, however SI may still be positively influenced by light intensity (Kürschner, 1997; Kouwenberg et al., 2007; Casson et al., 2009). Many plant species exhibit similar proportional SD and SI reductions that serve to enhance plant water use efficiency in response to an increase in atmospheric [CO2] (Woodward, 1987; Kürschner et al., 1997; Beerling et al., 1998; Haworth et al., 2010b, 2011). Exposure to SO2 can induce a reduction in leaf expansion during development (Cowling and Koziol, 1978; Garsed et al., 1979; Saxe, 1983) due to impairment of photosynthesis (Zeigler, 1972) and leaf water relations (Black and Unsworth, 1979; Neighbour et al., 1988). Any decrease in leaf expansion following exposure to toxic atmospheric gases may therefore result in an increase in SD. Stomata are the site of entry for approximately 90% of atmospheric pollutants into the leaf (Unsworth and Black, 1981), it may therefore be expected that exposure to toxic gases might result in a change in stomatal number or conductance in order to reduce entry of these gases into the internal leaf (Mansfield and Majernik, 1970; Mansfield, 1973; Ayazloo et al., 1982; Ali et al., 2008). At present a consistent effect of fumigation with toxic atmospheric gases on SD and SI is not evident (Ayazloo et al., 1982; Bettarini et al., 1997; Tanner et al., 2007; Ali et al., 2008; Haworth et al., 2010a). Exposure to elevated levels of atmospheric [CO2] and [SO2] in isolation and combination have been proposed to induce a reduction in SI in the fern Nephrolepis exaltata collected from sites around the Kilauea volcano, Hawaii (Tanner et al., 2007). The legume Psoralea corylifolia exposed to episodic fumigation with SO2 from industrial emissions also displayed a reduction in SD (Ali et al., 2008). Higher stomatal resistance was found to confer greater tolerance to SO2 in the grasses Lolium perenne, Festuca rubra, Dactylis glomerata and Holcus lanatus, however this enhanced tolerance to fumigation with SO2 was not associated with reduced SD (Ayazloo et al., 1982). Reduced SD is also not associated with enhanced SO2 resistance in the Cucurbits Curcurbita pepo and Cucumis sativus (Bressan et al., 1978). The rush Scirpus lacustris and grass Agrostis canina collected from Italian volcanic degassing vents are resistant to fumigation with sulphurous gases, and exhibited no effects on stomatal number when exposed to H2S and SO2 respectively in controlled environment studies (Bettarini et al., 1997; Haworth et al., 2010a). This divergence in reported stomatal density and index responses to fumigation with SO2 may be the result of: 1) duration of exposure to the toxic gas (whether exposure is episodic or persistent); 2) the concentration of the toxic gas; 3) the level of SO2 resistance of the plant species, and; 4) differences in the functionality of stomata between different individuals and species (i.e. speed/tightness of stomatal closure) (Mansfield et al., 1988; Neighbour et al., 1988; Wellburn, 1994; Haworth et al., 2010a). It is these uncertainties that need to be resolved to fully understand the influence of toxic atmospheric gases on stomatal initiation over geological timescales. Plant species from volcanic degassing vents commonly grow in atmospheres enriched in [CO2], but are also exposed to toxic gases such as SO2 and H2S (Miglietta et al., 1993; Fordham et al., 1997; Raschi et al., 1997; Paoletti et al., 2005; Onoda et al., 2009). This constant exposure to toxic gases has led to the development of genetically 45 isolated populations resistant to fumigation with toxic gases through the exclusion of vulnerable genotypes over multiple generations (McNeilly, 1968; Ayazloo and Bell, 1981; Haworth et al., 2010a). Fumigation with H2S and SO2 has been shown not to affect stomatal initiation in resistant genotypes (Bettarini et al., 1997; Haworth et al., 2010a). This may suggest that those fossil plants exposed to chronic and persistent fumigation with toxic gases across multiple generations, such as those conditions experienced over geological timescales during the formation of large igneous provinces (LIPs) (Knight et al., 2004; Marzoli et al., 2004; Nomade et al., 2007; Van de Schootbrugge et al., 2009), will have experienced selection pressures favouring resistance and are unlikely to record any influence of these toxic gases in stomatal initiation (Haworth et al., 2010a). The development of resistance within a population caused by the selective pressures exerted by fumigation with toxic industrial gases has occurred within 200 years in Lolium perenne in Lancashire, UK (Bell and Mudd, 1976). However, stomatal initiation in non-resistant vegetation may still be affected by sudden or instantaneous fumigation with toxic sulphurous gases; possibly caused by a rapid increase in volcanic activity (Hames et al., 2000; Knight et al., 2004; Schaller et al., 2011), a shift in meteorological conditions bringing vegetation into contact with toxic gases (Grattan and Sadler, 1999; Hantemirov et al., 2000) or emission of toxic gases from ocean euxina (Grice et al., 2005; Kump et al., 2005). The natural fumigation of non-resistant vegetation with toxic gases may reduce the efficacy of high temporal resolution stomatal reconstructions of palaeo-[CO2] through these events (eg. McElwain et al., 1999; Beerling et al., 2002; McElwain et al., 2005; Lamarque et al., 2007; Bonis et al., 2010; Retallack et al., 2011; Steinthorsdottir et al., 2011). Sulphur dioxide and H2S form a major component of volcanogenic gases, and may even be emitted in larger volumes than CO2, with H2S rapidly oxidising in the atmosphere to form SO2 (Brown, 1982). It has been proposed that fumigation with SO2 may induce a reduction in stomatal initiation in developing leaves (Tanner et al., 2007) due to stomatal closure causing an increase in internal levels of [CO2] within the leaf (Unsworth and Black, 1981; Mansfield et al., 1988), thus inducing a systemic signal affecting stomatal initiation in developing leaves (Lake et al., 2001; Shimada et al., 2011). It is therefore of critical importance to understand the stomatal density and index responses of living plants with no evolutionarily acquired resistance to toxic gases to fumigation with SO2, and the potential impacts upon stomatal reconstructions of palaeo-[CO2]. This study aimed to grow relict plant species with ancient evolutionary histories in controlled environment conditions of elevated [CO2] and fumigation with SO2 in order to assess the effects of fumigation with SO2 on stomatal initiation, and the likely impacts on stomatal reconstructions of palaeo-[CO2] coinciding with sudden or instantaneous natural fumigation with toxic volcanic gases. Potential mechanisms to account for any effects of toxic volcanic gases on SD and SI values of fossil plants over geological timescales are also explored. 2. Methods and materials 2.1. Controlled environment conditions and photosynthetic analyses The cycads Lepidozamia hopei and Lepidozamia peroffskyana grown from seed, six month-old specimens of the fern Osmunda regalis and two year-old specimens of gymnosperms Ginkgo biloba, Nageia nagi, Podocarpus macrophyllus, Araucaria bidwillii, and Aagathis australis were potted in 4 l square pots (15 × 15 × 23 cm) with 80% compost (2 kg m −3 15–10–20 N:P:K; Bord na Móna, Newbridge, County Kildare, Ireland), 20% vermiculite and 2.5 g l −1 slow release Osmocote fertilizer (15% N, 10% P2O5, 10% K2O, 2% MgO, plus trace elements; Scotts Co., Marysville, Ohio, USA). Plants were grown for 12 months in Conviron BDW-40 (Winnipeg, Manitoba, Canada) growth rooms in the Program for Experimental Atmospheres and Climate facility at Thornfield, under control atmospheric conditions (380 ppm CO2, no SO2), elevated [CO2] (1500 ppm CO2, no SO2) and 46 M. Haworth et al. / Review of Palaeobotany and Palynology 182 (2012) 44–54 fumigation with SO2 (380 ppm CO2, 0.2 ppm SO2). Atmospheric concentration of CO2 within the chambers was monitored by a PP-systems WMA-4 IRGA (PP-systems, Amesbury, MA, USA) and supplemented by compressed CO2 to increase [CO2] (BOC, Guildford, Surrey, UK). Sulphur dioxide fumigation was achieved by supplementing the chamber with compressed SO2 (BOC) to a concentration of 0.2 ppm. Atmospheric SO2 concentration was monitored by a Horriba APSA-370 Air Pollution Monitor (HORIBA Instruments Ltd, Northampton, UK). All other growth conditions remained constant, with plants experiencing 16 h of light per day in a simulated day/night program (5.00–6.00 dawn; 6.00–9.00 light intensity rises from 300 to 600 μmol m −2 s −1; 9.00–17.00 midday light intensity of 600 μmol m −2 s −1; 17.00–20.00 light intensity decreases 600 to 300 μmol m −2 s −1; 20.00–21.00 dusk), temperature regime (night time temperature of 18 °C rising to a midday peak of 28 °C), relative humidity of 80%, downward ventilation to ensure mixing of atmospheric gases and receiving 60 ml of water each day. In order to avoid mutual shading plants were randomised within areas of identical canopy height within the growth chambers (Hammer and Hopper, 1997; Sager and McFarlane, 1997). Chlorophyll-fluorescence measurements of plant photosynthetic performance were taken using a Hansatech PEA Fluorometer following 30 min of dark adaptation using a Hansatech dark adaptation leaf clip between 08.00 and 11.00 am on new leaf growth of a minimum of three plants in the control and SO2 fumigation growth chambers. Chlorophyll-fluorescence measurements provide an indication of the efficiency of Photosystem II (Fv/Fm); as expressed by the ratio of variable fluorescence (Fv) to the fluorescence maximum (Fm), where a ratio of 0.83 indicates a healthy plant, and values less than 0.83 suggest impairment of photosynthetic performance (Maxwell and Johnson, 2000). The number of dead reaction centres in Photosystem II (Fo/Fm) provides an indication of damage to the photosynthetic physiology and is calculated as the ratio the minimum fluorescence yield (Fo) to the Fm (Butler and Kitajima, 1975a, 1975b). In order to explore possible selective pressures experienced by non-resistant plants exposed to fumigation with SO2 during Earth history, plant species grown under glasshouse conditions of ambient [CO2] were selected (~ 400 ppm) with relatively high (Osmunda regalis: 247.5 ± 14.7 mmol m −2 s −1) and low (Ginkgo biloba: 18.0 ± 2.4 mmol m −2 s −1) stomatal conductance rates. These were then placed into the SO2 chamber (three replicates were used following two weeks acclimation to growth chamber conditions in the control chamber), and photosynthetic parameters recorded with increasing duration of exposure. Leaves were labelled using string treasury tags to permit the same leaf to be consistently sampled for chlorophyllfluorescence analysis. Analysis of photosynthetic physiology (photosynthetic light response and photosynthesis–internal leaf [CO2] curves) was performed using a PP-Systems Ciras-2 attached to a PLC6(U) leaf cuvette and LED light unit (PP-Systems, Amesbury, Massachusetts, USA) following the methods of Long and Bernacchi (2003). Photosynthetic light response curves allow the analysis of light use for photosynthesis in order to calculate the light compensation and saturation points and the apparent quantum efficiency. Photosynthetic rate– internal [CO2] curves permit calculation of the [CO2] compensation point, the efficiency of carbon uptake (Vcmax: carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase-oxygenase), maximum electron transport (Jmax) and maximum rate of photosynthesis (Amax) (Long et al., 1996; Long and Bernacchi, 2003). Due to the toxic effect of the SO2 damaging the foliage of O. regalis it was not possible to collect photosynthetic measurements from the fern. However, analysis was conducted on the same leaf of G. biloba for two weeks, until fissures appeared in the leaf making it impossible to produce a good seal between the leaf and the gaskets in the cuvette, or to discount the possibility of water vapour leaking from these areas of damage that would affect the infra-red gas analyser and calculations of photosynthesis and stomatal conductance (i.e. Long and Bernacchi, 2003). 2.2. Stomatal counts After full leaf development and expansion of new leaf growth under the atmospheric conditions, the uppermost leaves receiving full irradiance and not affected by self-shading were destructively sampled for stomatal counts. No new growth occurred in Osmunda regalis when grown under fumigation with SO2. Leaf cuticles were macerated using a 50:50 solution of glacial acetic acid and 30% H2O2 at 70 °C, stained using safranin-O solution and mounted in glycerol on glass slides. Cuticle images were taken under transmitted light using a Leica DM2500 microscope attached to a Leica DFC300FX camera and Syncroscopy Automontage (Syncroscopy, Cambridge, UK). Automontage was used to produce a “stacked image” where multiple images are collected as the focal depth passes through the z-axis of the plant cuticle; these images are then merged in order to ensure all details of relief represented within the final image are in focus. As an indicator of stomatal size, the stomatal pore length (SPL) (Wagner et al., 1996; Hetherington and Woodward, 2003) of ~ 20 stomata was measured using Automontage, with the average taken to represent the treatment value for a given species. A 0.09 mm 2 grid (Poole and Kürschner, 1999) was superimposed on the images for stomatal and epidermal counts using Syncroscopy AcQuis. Stomata and epidermal cells were counted on 837 images in total, with five stomata/epidermal cell counts performed on each of three leaves from a plant, with the average of 15 counts taken to represent the mean SD and SI of an individual plant (except for Lepidozamia peroffskyana and Lepidozamia hopei where nine counts were averaged). One-way ANOVAs using Bonferroni method were performed using SPSS 20 (IBM, New York, USA) to test whether SI, SD, and SPL values of the individuals grown under SO2 fumigation differed significantly to those grown under control atmospheric conditions (for further information on statistical analyses and results of post-hoc testing see Supplementary data). 3. Results All plant species produced new foliage following exposure to SO2 with the exception of the fern Osmunda regalis. Exposure to constant fumigation with 0.2 ppm SO2 resulted in a diverse range of SD, SI and SPL responses within those species that developed new leaf growth (Fig. 1 and Table 1). In all species sulphur dioxide fumigation resulted in a general pattern of increased SD values relative to plants grown in control or atmospheres of elevated [CO2], with the exception of the araucariacean conifer Araucaria bidwillii. However, statistically significant increases in SD relative to control atmospheres were only observed in Lepidozamia peroffskyana, G. biloba and P. macrophyllus, likely associated with reduced leaf expansion under SO2 fumigation (Fig. 1a). Nevertheless, exposure to SO2 did result in alteration of stomatal initiation in two of the seven species that produced new leaf growth under experimental atmospheric conditions (Fig. 1b). Ginkgo biloba exhibited a 53.8% increase in SI when exposed to SO2 in comparison to a 17.1% reduction when [CO2] was increased from 380 to 1500 ppm. In contrast, the broadleaved conifer Agathis australis displayed a 30.4% reduction in mean SI when fumigated with SO2, whereas growth in atmospheres enriched in CO2 induced a smaller 11.4% mean SI decrease (Fig. 1b). Podocarpus macrophyllus and A. bidwillii showed no significant effect of SO2 fumigation on stomatal initiation, but significant reductions in stomatal index of 12.5% and 28.4% respectively when grown in elevated [CO2]. However, the reductions in SI displayed by both araucariacean conifers A. australis and A. bidwillii when exposed to SO2 do not differ significantly from the reductions in SI values observed when grown under elevated [CO2]. The cycads Lepidozamia hopei and L. peroffskyana and broadleaved conifer Nageia nagi did not show any stomatal initiation response to an increase in [CO2], and did not exhibit any significant alteration of SI when fumigated with 0.2 ppm SO2 (Table 1). Exposure M. Haworth et al. / Review of Palaeobotany and Palynology 182 (2012) 44–54 47 a control SO2 fumigation elevated [CO2 ] b 250 Stomatal Density (mm2) b a a a a b 50 a a 100 a ab Lepidozamia peroffskyana a a a a 150 0 b b 200 a a a a a Lepidozamia hopei Ginkgo biloba Podocarpus macrophyllus Agathis australis Araucaria bidwillii Nageia nagi 25 b Stomatal Index (%) 20 a a 15 a a a ab b a b a a 10 5 a a a a a ab b a a 0 Lepidozamia peroffskyana c Lepidozamia hopei Ginkgo biloba Podocarpus macrophyllus Agathis australis Nageia nagi 20 b b 16 SD / SI Ratio Araucaria bidwillii a 12 b ab 8 c b b a a a a a b a a a a a a a 4 0 Lepidozamia peroffskyana Lepidozamia hopei Ginkgo biloba Podocarpus macrophyllus Agathis australis Araucaria bidwillii Nageia nagi Species Fig. 1. The effect of growth in atmospheres of elevated [CO2] (1500 ppm; no SO2) and fumigation with SO2 (380 ppm CO2; 0.2 ppm SO2) in comparison to control atmospheric conditions (380 ppm CO2; no SO2) on the stomatal characters of seven gymnosperm Mesozoic analogue plant species: a) stomatal density; b) stomatal index, and; c) the ratio of stomatal density to stomatal index values. Error bars indicate one standard deviation either side of mean. Mean sample values with different letters above histogram error bars are significantly different using Bonferroni method one-way ANOVA (F and P-values are given in Table 1 and post-hoc testing in Supplementary information). to SO2 did not induce a significant change in stomatal pore length in any of the plant species analysed (Table 1 and Supplementary data). Increases in the SD/SI ratio were observed in all of the species analysed, with the exception of Araucaria bidwillii, following exposure to SO2 (Fig. 1c). The SD/SI ratio increased significantly in new foliage developed under conditions of SO2 fumigation relative to foliage developed in control atmospheric conditions. A similar pattern was observed between the SD/SI ratios of foliage produced under SO2 fumigation and enhanced atmospheric [CO2], apart from those of Lepidozamia peroffskyana. Individuals of the grass species Agrostis canina collected from the Italian volcanic degassing vent of Mefite di Ansanto, Avellino, Italy, have been shown to possess an evolutionary acquired resistance to exposure to atmospheric SO2 (Haworth et al., 2010a). Fumigation with SO2 did not induce a reduction in SD or an increase in the SD/SI ratio of the Mefite A. canina in contrast to the responses observed in the non-resistant plants analysed (Table 1). 48 M. Haworth et al. / Review of Palaeobotany and Palynology 182 (2012) 44–54 Table 1 One-way ANOVA results for stomatal index, stomatal density, stomatal pore length and SD/SI ratio values of plant species producing new leaf growth under fumigation with 0.2 ppm SO2 in comparison to plants grown in control and elevated [CO2] atmospheres. The SO2 resistant grass species Agrostis canina was collected from the volcanic degassing vent of Mefite di Ansanto, Italy (for details see Haworth et al., 2010a, 2010b). AB = abaxial leaf surface; AD = adaxial leaf surface. Additional detail and results of post‐hoc testing using Bonferroni method are summarised in Fig. 1 and provided in full in Supplementary data tables. Species Lepidozamia peroffskyana Lepidozamia hopei Ginkgo biloba Podocarpus macrophyllus Agathis australis Araucaria bidwillii Nageia nagi Agrostis canina (AB) Mefite Agrostis canina (AD) Mefite Stomatal density Stomatal index Stomatal pore length SD/SI ratio F2,6 P F2,6 P F2,6 P F2,6 P 9.019 0.493 153.254 24.742 1.534 2.999 20.284 0.523 0.245 0.016 0.634 0.00001 0.001 0.290 0.125 0.002 0.618 0.790 0.631 2.751 107.662 16.696 10.366 7.110 4.090 1.394 0.301 0.564 0.142 0.00002 0.004 0.011 0.026 0.076 0.318 0.751 2.909 16.004 1.847 4.283 1.049 4.306 4.259 0.046 4.883 0.131 0.004 0.237 0.061 0.394 0.069 0.071 0.955 0.055 7.654 17.841 11.952 15.467 10.512 0.046 41.774 0.080 0.061 0.022 0.003 0.008 0.004 0.011 0.955 0.0003 0.924 0.942 The resistance of a plant to the toxic effects of SO2 can be gauged by the analysis of the efficiency of Photosystem II (Fv/Fm) and the number of dead Photosystem II reaction centres (Fo/Fm) over time following exposure to SO2 (Haworth et al., 2010a). After six months the Fv/Fm values of new leaf growth under control atmospheric conditions and SO2 fumigation were recorded. Sulphur dioxide fumigation induced a reduction in mean Fv/Fm values of new leaf growth in all species with the exception of Ginkgo biloba. However, significant reductions in Fv/Fm were only observed in the cycad Lepidozamia hopei and conifers Podocarpus macrophyllus and Araucaria bidwillii (Table 2). Analysis of the photosynthetic performance of new leaf growth under control and SO2 fumigation suggests a degree of impairment of photosynthesis induced by exposure to SO2. Stomatal conductance (Gs) may play a significant role in plant resistance to SO2, however it was not possible to directly measure the effect of SO2 fumigation on the Gs of plants without impairing the effectiveness of the infra-red gas analyser due to the corrosive effects of SO2. However, previous studies have shown that resistance to SO2 is associated with reduced Gs. Following exposure to SO2 the foliage of Osmunda regalis with relatively high Gs rates quickly developed chlorotic lesions that rapidly became necrotic (Fig. 2a). This is co-incident with a severe decline in photosynthetic performance, as Fv/Fm values decreased from ~ 0.83 to ~ 0.27 after only 24 h exposure to SO2 (Fig. 2b). A similar pattern is observed in the damage to Photosystem II, where Fo/Fm values rapidly increased until the leaf was completely necrotic after three days of fumigation with 0.2 ppm SO2 (Fig. 2c). In contrast to O. regalis the foliage of Ginkgo biloba with low rates of Gs maintained photosynthetic performance for ~35 days, before rapidly declining with the onset of necrosis after 60 days of exposure to SO2 (Fig. 3a). The number of dead Photosystem II reaction centres followed an identical pattern, remaining constant for ~35 days, then declining rapidly after 60 days and indicating complete necrosis after 75 days of fumigation with SO2 (Fig. 3b). Analysis of photosynthetic and stomatal conductance rates of Osmunda regalis after 24 h of exposure to SO2 was not possible, suggesting complete impairment of C-uptake and gas-exchange (Fig. 2a). However, photosynthetic analysis of the same leaf of a Ginkgo biloba specimen following one week of fumigation with SO2 illustrated a decline in the efficiency of photosynthetic light use and carbon uptake. The light compensation point increased by 535.6%, while the light saturation point and apparent quantum efficiency decreased by 40.4% and 78.8% respectively (Fig. 3c). After one week of fumigation with SO2 the CO2 compensation point increased by 73.3%, indicative of sulphite competing for the active site of ribulose-1,5-bisphosphate carboxylase oxygenase (RubisCO) (Zeigler, 1972). The maximum rate of photosynthesis declined by 46.9%, consistent with reductions in the maximum carboxyation efficiency of RubisCO (the maximum rate of CO2 uptake) and maximum rate of electron transport in the regeneration of ribulose-1,5-bisphosphate (RuBP) of 43.2% and 20.8% respectively. The analysis of O. regalis and G. biloba indicate that SO2 impairs photosynthesis by causing damage to the photosynthetic apparatus, and that this was more apparent in O. regalis with a greater Gs rate (Figs. 2 and 3). 4. Discussion Fumigation with SO2 induced a degree of variability to SI values of the plant species studied. This may suggest that the presence of atmospheric SO2 during earth history may impair the efficacy of stomatal palaeo[CO2] reconstructions based upon fossil plants that did not possess an evolutionary acquired resistance to toxic gases (Tanner et al., 2007; cf. Haworth et al., 2010a). This variability in SI responses to SO2 fumigation is evident in the dramatic increase in SI values of Ginkgo biloba, a widely Table 2 Photosynthetic performance as indicated by efficiency of Photosystem II (Fv/Fm: ratio of variable fluorescence to the fluorescence maximum) of new leaf growth produced following six month's growth under control atmospheric conditions and fumigation with SO2. ± indicates one standard deviation. The relative change in Fv/Fm values induced by exposure to SO2 is indicated by ΔFm/Fv (%). One-way ANOVA results indicate significance levels of difference between mean Fv/Fm values of plants grown under control atmospheric conditions and SO2-fumigation. Plant species Control Fv/Fm Osmunda regalis Lepidozamia hopei Ginkgo biloba Nageia nagi Podocarpus macrophyllus Agathis australis Araucaria bidwillii 0.844 0.729 0.771 0.712 0.794 0.796 0.726 ± ± ± ± ± ± ± 0.008 0.044 0.018 0.073 0.028 0.010 0.045 SO2 Fv/Fm ΔFm/Fv (%) ANOVA F1,4 P – 0.620 0.780 0.689 0.747 0.774 0.564 −100.0 −15.0 1.2 −3.2 −5.9 −2.8 −22.3 – 6.187 1.044 0.212 10.785 3.565 10.795 – 0.042 0.325 0.655 0.011 0.088 0.013 ± ± ± ± ± ± 0.093 0.016 0.099 0.002 0.028 0.011 M. Haworth et al. / Review of Palaeobotany and Palynology 182 (2012) 44–54 49 Fig. 2. The effect of exposure to SO2 on Osmunda regalis: a) photographs illustrating the rapid onset of chlorosis and necrosis following SO2 fumigation; b) Fv/Fm values showing a rapid decline from optimal values of ~0.83 after SO2 fumigation, and; c) Fo/Fm values showing a rapid increase in the number of dead Photosystem II reaction centres after the onset of exposure to SO2. Error bars indicate one standard deviation either side of mean values of plants. used analogue species for stomatal palaeo-[CO2] reconstructions based upon fossil ginkgoaleans (eg. McElwain et al., 1999; Royer et al., 2001; Bonis et al., 2010; Smith et al., 2010; Steinthorsdottir et al., 2011). The a potential presence of volcanic sulphurous gases at key episodes of environmental change such as the Triassic–Jurassic boundary (TJB) mass extinction event may suggest that ginkgoalean based stomatal palaeo- b 1 0.8 0.8 Fo / Fm Fv / Fm 0.6 0.4 0.2 0.6 0.4 0.2 0 0 0 20 40 60 80 0 20 Time (days) d 12 week 0 10 8 6 4 2 0 Week 0 1 -2 -4 0 25 week 0 week 1 500 1000 Light CP Light SP AQE 12.0 842.1 0.033 64.3 502.0 0.007 1500 Photosynthetically Active Radiation (µmol m-2 s-1) 2000 Photosynthetic Rate (µmol m-2 s-1) Photosynthetic Rate (µmol m-2 s-1) c 40 60 80 Time (days) week 1 20 15 10 5 Week 0 1 0 -5 0 500 CO2 CP 60.0 104.0 1000 Vcmax 29.2 16.6 Jmax 37.1 29.4 1500 Amax 17.9 9.5 2000 Internal [CO2] Concentration (ppm) Fig. 3. The effect of exposure to SO2 on photosynthetic apparatus of Ginkgo biloba: a) Fv/Fm values following exposure to SO2 showing a decline from optimal levels after 30 to 40 days; b) Fo/Fm values after SO2 fumigation illustrating an increase in dead reaction centres occurring after 45 days, c) photosynthesis rate to photosynthetically active radiation response curve of the same G. biloba leaf following one week of fumigation to SO2, inset table shows photosynthetic light use efficiency parameters (Light CP = light compensation point, μmol m−2 s−1; Light SP = light saturation point, μmol m−2 s−1; AQE = apparent quantum efficiency, mol photon−1), and; d) photosynthetic rate to internal leaf [CO2] response curves of the same G. biloba leaf following one weeks exposure to SO2, inset table show photosynthetic parameters (CO2 CP = CO2 compensation point, ppm; Vc,max = maximum rate of carboxylation of ribulose-1,5-bisphosphate carboxylase oxygenase, μmol m−2 s−1; Jmax = maximum rate of electron transport in the regeneration of ribulose-1,5-bisphosphate, μmol m−2 s−1; Amax = maximum rate of photosynthesis, μmol m−2 s−1). Photosynthetic and light use efficiency parameters calculated using Long and Bernacchi (2003). 50 M. Haworth et al. / Review of Palaeobotany and Palynology 182 (2012) 44–54 [CO2] reconstructions at this interval (McElwain et al., 1999; Bonis et al., 2010; Steinthorsdottir et al., 2011) may underestimate palaeo-[CO2]. However, the araucariacean conifers Agathis australis and Araucaria bidwillii both exhibit reductions in SI values following fumigation with SO2, that do not differ significantly to the SI values exhibited under [CO2] enrichment (Fig. 1b). Carbon dioxide is the major plant substrate, with large components of photosynthetic and metabolic physiology utilised in its uptake and use. As such this physiology provides the means to detect fluctuations in [CO2], and co-ordinate physiological and morphological responses such as stomatal initiation (Gray et al., 2000; Lake et al., 2001; Hu et al., 2010). In contrast sulphur is not a major plant substrate (Hewitt and Smith, 1975; Legge et al., 1988; Hawkesford, 2000), and a similar mechanism is currently not evident for the detection of atmospheric SO2 and the signalling and co-ordination required to induce a direct shift in stomatal initiation. Nevertheless, in the seven plants studied, which did not possess an evolutionarily acquired resistance to toxic gases (cf. Haworth et al., 2010a), fumigation with SO2 induced significant changes in stomatal initiation in two of the species (Ginkgo biloba and Agathis australis) (Fig. 1b). This variability in stomatal initiation responses to SO2 fumigation may be the result of species specific differences in stomatal function (Heath, 1950; Franks and Farquhar, 2007; Doi and Shimazaki, 2008) and physiological tolerance to the toxic effects of sulphite (Zeigler, 1972; Ballantyne, 1973; Brychkova et al., 2007). Exposure to SO2 has been shown to induce both stomatal closure (Dytham, 2010; McAdam and Brodribb, 2012a) and opening (Black and Black, 1979; Neighbour et al., 1988; Bartiromo et al., 2012) possibly mediated by atmospheric humidity (Black and Unsworth, 1979), that would affect internal leaf [CO2] concentration (Unsworth and Black, 1981; Mansfield et al., 1988) and resultant systemic signalling of atmospheric [CO2] level (Lake et al., 2001), possibly accounting for the variability in stomatal initiation responses observed in this study. However, both A. australis and Araucaria bidwillii possess stomatal wax plugs that may prevent stomatal closure by physically obstructing the guard cells (Jeffree et al., 1971; Feild et al., 1998), reducing the likelihood of complete stomatal closure in these species, that would in turn induce an increase in internal leaf [CO2] (Tanner et al., 2007) and cause the reduction in stomatal initiation observed in both species. Disruption to the photosynthetic physiology, and its associated signalling and feedback mechanisms to monitor atmospheric [CO2] levels (Van Oosten et al., 1994; Mott and Woodrow, 2000; Hu et al., 2010), may also influence stomatal initiation. The degree of SI response to an increase in [CO2] is positively associated with transpiration rate in Populus trichocarpa (Miyazawa et al., 2006), possibly suggesting a role for root to shoot signalling in influencing stomatal initiation response to [CO2] (Miyazawa et al., 2006; Lake and Woodward, 2008). Fumigation with SO2 can induce a change in transpiration rates (Black and Unsworth, 1979; Neighbour et al., 1988), that may then alter stomatal [CO2] sensitivity and stomatal initiation in developing foliage. The potential disruptive effect of atmospheric SO2 on the efficacy of the SI–[CO2] relationship in plants without an adaptive tolerance to toxic gases may undermine the reliability of stomatal based reconstructions of palaeo-[CO2] during intervals of SO2 fumigation in earth history. During prolonged periods of global scale volcanism, such as the formation of large igneous provinces (eg. Marzoli et al., 1999; Schaller et al., 2011), vegetation may be exposed to persistent fumigation with toxic volcanic gases and develop an evolutionary acquired resistance to sulphurous gases (i.e. McNeilly, 1968; Bell and Mudd, 1976). Plants with such an evolutionary acquired resistance to the toxic effects of volcanic gases occur at volcanic degassing vents, where the vegetation has been exposed to harmful gases over multiple generations (Miglietta and Raschi, 1993; Raschi et al., 1997; Paoletti et al., 2005). Analysis of the stomatal responses of these plants suggests that individuals with an acquired adaptive resistance to toxic volcanic gases show no effect of fumigation with SO2 or H2S on stomatal initiation (Bettarini et al., 1997; Haworth et al., 2010a). Therefore, exposure to SO2 is only likely to influence stomatal initiation during episodes of sudden fumigation affecting plant populations that have not previously experienced selective pressures exerted by the toxic nature of these gases to develop SO2 resistance (Table 1). However, the plant species growing in closest proximity to the toxic gases emitted at these volcanic degassing vents are grasses (Fordham et al., 1997; Haworth et al., 2010a), that possess different stomatal morphologies (dumb-bell rather than kidney shaped guard cells) and physiological guard cell control mechanisms (Franks and Farquhar, 2007; Doi and Shimazaki, 2008; McAdam and Brodribb, 2012b) to the gymnosperms and early angiosperms upon which pre-Tertiary stomatal reconstructions of palaeo-[CO2] are based (eg. Passalia, 2009; Bonis et al., 2010; Steinthorsdottir et al., 2011). This raises the possibility that the SO2 resistance mechanisms and stomatal behaviour displayed by modern grass species at volcanic degassing vents may differ from the stomatal responses of more ancient plant lineages during SO2 fumigation events in earth history. Selective pressures exerted on plant species with less functional stomata (i.e. speed and tightness of stomatal opening / closing) (i.e. Mansfield and Meidner, 1966; Franks and Farquhar, 2007), may induce changes in stomatal initiation over geological timescales. The differential responses of Ginkgo biloba and Osmunda regalis to SO2 fumigation are likely associated with differences in Gs (Bressan et al., 1978; Winner and Mooney, 1980; Ayazloo et al., 1982). The relatively high Gs rate of O. regalis will have increased the entry of toxic SO2 into the internal leaf environment (Black and Unsworth, 1979; McHone, 2003) causing rapid chlorosis and necrosis of the foliage (Fig. 2a). In contrast, the lower Gs of G. biloba may have restricted the rate of entry of the toxic SO2 into the leaf, permitting the foliage of G. biloba to maintain photosynthetic performance for a greater period of time after the onset of fumigation (Fig. 3)(Mansfield and Majernik, 1970). During episodes of fumigation over multiple generations, this effect of Gs on an individual plants’ resistance to toxic volcanic gases may have become apparent in the SD and SI values of a fossil flora. Following the onset of global scale volcanism, selective pressures exerted by these harmful gases may have favoured those individuals within a population that possessed a genetic predisposition for lower SD and SI values (i.e. McNeilly, 1968). Across multiple generations, such as those observed over geological timescales such as at the TJB, this may become manifest in a progressive decline in SD and / or SI values to levels that would be considered limiting to C-uptake, or harmful in terms of energy dissipation and heat stress (McElwain et al., 1999; Steinthorsdottir et al., 2011). Further SO2 fumigation studies are therefore required in order to assess how intrinsic natural variability in SD may affect the selectivity of floral extinction observed at mass extinction events (eg. Van de Schootbrugge et al., 2009; Belcher et al., 2010; Mander et al., 2010) and induce genotypic changes in SD and/or SI over multiple generations. Additionally the role of acidic precipitation on the degradation of cuticular features and impairment of gas exchange (Haworth and McElwain, 2008; Bartiromo et al., 2012) and mobilisation of heavy metals within the soils such as calcium (Lawrence et al., 1999) that are important for stomatal function in plant stomatal responses to [CO2] (Webb et al., 1996) should be explored. The results of this study indicate that the presence of atmospheric SO2 may introduce an element of uncertainty to stomatal reconstructions of palaeo-[CO2] during episodes of perturbation to the global C-cycle through history. Nonetheless, SD and SI often track one another in terms of their proportional response to fluctuations in the level of atmospheric [CO2] (Woodward, 1987; Kürschner et al., 1997; Beerling et al., 1998; Haworth et al., 2010b, 2011). The relative difference between the SD and SI values therefore often remains constant with changing [CO2] (Fig. 1c). Exposure to SO2 results in a reduction in leaf expansion caused by disruption to leaf water control and impaired C-uptake (Cowling and Koziol, 1978; Garsed et al., 1979; Saxe, 1983) that is manifested in higher SD values (eg. Ali et al., 2008). Six of the seven non-resistant plant species analysed exhibited significant increases in their SD/SI ratio under SO2 fumigation (with M. Haworth et al. / Review of Palaeobotany and Palynology 182 (2012) 44–54 the exception of Araucaria bidwillii) (Fig. 1c). This suggests that the SD/ SI ratio of fossil plants may be employed to establish the likelihood that any inferred shift in palaeo-[CO2] derived from the SI values of the fossil plant is the result of the palaeo-atmospheric [CO2] in which that leaf developed, and not fumigation of non-resistant plants with toxic volcanic gas such as SO2. The absence of any significant change in the SD/SI ratio values of SO2 resistant Agrostis canina (Table 1) may suggest that SO2 resistant fossil plants would be unlikely to exhibit any effect of the presence of toxic atmospheric gas on stomatal initiation. Any shift in the SD/SI ratio of fossil plant species through a stratigraphic section may suggest that the fossil floras did not possess an evolutionary acquired resistance to toxic gases and were exposed to geologically instantaneous fumigation with noxious volcanic gases that may have affected SI values in a particular horizon. An increase in the ratio of SD to SI values may also be the result of abiotic stresses other than SO2, such as environmental or physiological aridity (eg. Tichà, 1982). Therefore SD/SI ratios should be employed in a stratigraphic context alongside palaeo-environmental indicators of water availability (eg. Webb et al., 1996; Ufnar et al., 2004), palaeo-botanical indicators of 100 51 SO2 induced cuticlar damage (eg. Bartiromo et al., 2012) and geochemical indicators of sulphur (eg. Williford et al., 2009). Analysis of the SD/SI ratios of fossil Ginkgoaceae from the TJB fossil flora of Astartekløft, East Greenland (data from Steinthorsdottir et al., 2011), identify a particular stratigraphic horizon where SD/SI values are significantly different from those displayed in the section (Fig. 4). This may suggest that plant fossils deposited directly above the transition between the Triassic and Jurassic periods may have been affected by the presence of atmospheric SO2, or another abiotic stress associated with a change in facies, that may have influenced stomatal initiation in the fossil plants, and the resulting reconstructed palaeo-[CO2] value for that particular stratigraphic horizon (see Supplementary data). The results of this study suggest that SD/SI ratios of fossil plants should be considered stratigraphically using a one-way or nested design ANOVA, when conducting stomatal reconstructions of palaeo-[CO2], in order to identify horizons where the efficacy of the SI–[CO2] relationship may be impaired. This study utilised continuous SO2 exposure, further work is required to assess the effect of intermittent fumigation on stomatal initiation, and also the effect of mixtures of atmospheric pollutants when combined with atmospheres of enhanced [CO2] that are more likely to be representative of atmospheric conditions that occurred during episodes of global climate change and disruption to the global carbon cycle. 9 8 90 70 Height (m) 60 Early Jurassic (Hettangian) 80 5. Conclusion This study has shown contrasting responses of stomatal initiation to fumigation with SO2 in plant species that do not possess an evolutionary acquired resistance to toxic gases. Exposure to SO2 induced a general increase in SD associated with reduced leaf expansion. However, new foliage developed after exposure to SO2 exhibited reductions, increases and no change in the SI values of the species analysed. This variability in stomatal initiation induced by exposure to harmful SO2 may be the result of disruption to the photosynthetic physiology and water control mechanisms that likely influence stomatal formation in developing leaves. The effect of atmospheric SO2 on the SI values of the plants analysed in this study raises uncertainties surrounding the efficacy of stomatal based reconstructions of palaeo-[CO2] during intervals of earth history where vegetation experienced sudden fumigation with toxic gases. However, exposure to SO2 also resulted in increases in the ratio of SD to SI values in six of the seven species analysed. Therefore we propose that future stomatal reconstructions of palaeo-[CO2] during episodes of global scale volcanism or ocean anoxia employ changes in the SD/SI ratios of fossil plants to identify any stratigraphic horizons where the vegetation may have been exposed to sudden fumigation with toxic gases that could have affected stomatal initiation. 7 6 5.5 50 5 4 40 30 20 10 Late Triassic (Rhaetian) 3 2 1.5 1 0 Acknowledgements 0 0 10 20 30 SD / SI ratio Mudstone Conglomerate Leaf fossils Sandstone Ironstone Shell concretions Limestone Cross-bedding Rootlets Fig. 4. SD/SI ratio values of fossil Ginkgoaceae collected from Triassic–Jurassic deposits of Astartekløft, East Greenland. Stratigraphy from Hesselbo et al. (2002), SD/SI data from Steinthorsdottir et al. (2011). LSD ANOVA indicates that variance of SD/SI ratio values of fossil plants within bed 6 are significantly different to all other fossil plant beds through the section. This may indicate possible SO2 fumigation during the interval represented by fossil plant bed 6, however a shift in depositional environment from crevasse splay to swamp may also account for the apparent shift in SD/SI values relative to the rest of the section. Fossil plant bed 5 is considered to represent the transition between the Triassic and Jurassic periods based upon the last occurrence of Triassic Lepidopteris ottonis and first appearance of Jurassic Thaumatopteris brauniana (Harris, 1937) and pollen Cerebropollenites thiergartii (Mander et al., 2010). We thank the following for scientific discussion and technical assistance: Antonio Raschi (CNR-IBIMET), Ray O'Haire, Emma Reeves, Liam Kavanagh, Bredagh Moran (UCD, Ireland), Aidan Blake, Kelly Krause, Matthew Gilroy, Craig Berg (CONVIRON, Canada), Michael Doyle, Chris Bergweiler (PP-Systems, USA). The comments of Lawrence Tanner (Le Moyne College, USA) and an anonymous reviewer significantly improved this manuscript. 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