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Damage structures in leaf epidermis and cuticle as an indicator of elevated atmospheric sulphur dioxide in early Mesozoic 1047298oras

C Elliott-Kingston a M Haworth b JC McElwain a

a School of Biology and Environmental Science University College Dublin Bel 1047297eld Dublin 4 Irelandb CNR mdash Institute for Plant Protection (IPP) Presso Area di Ricerca CNR Edi 1047297cio E Via Madonna del Piano 10 Sesto Fiorentino 50019 Firenze Italy

a b s t r a c ta r t i c l e i n f o

Article history

Received 9 May 2013Received in revised form 5 May 2014Accepted 7 May 2014Available online 23 May 2014

Keywords

Sulphur dioxideLeaf damageCryo-scanning electron microscopyPalaeo-volcanismFossil record

Volcanicepisodes are considered to be potentially important drivers of manymass extinction events in Earth his-

tory when a sharp decrease in the diversity and abundance of macroscopic organisms occurred Such events arehypothesised to be associated with volcanicsulphurdioxide (SO2) releaseThe effect of volcanism on atmospher-ic composition varies widely and is related to the magnitude and duration of the volcanic episode Currentlythere are limited methods for detecting the timing of SO2 release in the geological past In 1047297eld conditions thein1047298uence of SO2 on plants can be dif 1047297cult to separate from the effects of other volcanic emissions which entervia stomata and can affect plant physiology anatomy and morphology In order to assess the direct effects of SO2 associated with palaeo-volcanic episodes we conducted a six month growth chamber experiment growingplants under control (zero parts per million (ppm)) and continuous elevated (02 ppm) sulphur dioxide atmo-spheres Leaf morphological responses of ten species representative of Mesozoic gymnosperm and fern fossil1047298oras were examined using cryo-scanning electron microscopy Here we show that expanded fully matureleaves record unambiguous damage structures associated with injury from SO2 fumigationIn SO2 treated plantsleaves were smaller and did not persist distinct raised areas of cuticle surrounding stomata appeared surfacewaxes altered blistering of the cuticle occurred and the stomatal complex became distorted These resultshave clear implications for application to the fossil record as many of the observed damage structures have thepotential to be preserved in fossil plant cuticle and thus allow precise pinpointing of elevated SO2 episodes in

the geological past copy 2014 Elsevier BV All rights reserved

1 Introduction

11 Sources of sulphur dioxide

Volcanic episodes areconsidered to be importantdrivers of mass ex-tinction events in Earth history when the diversity and abundance of macroscopic organisms declined sharply (Courtillot and Renne 2003Ganino and Arndt 2009 van de Schootbrugge et al 2009 Wignall2011) These events are believed to be associated with the volcanic re-lease of sulphur dioxide (SO2) for example the PermianndashTriassic ex-tinction event of circa 252 Ma ago (Shen et al 2011) considered to bethe worst extinction event in Earth history (Benton and Twitchett2003) and the Triassicndash Jurassic extinction event of circa 201 Ma ago(McElwain et al 1999 Tanner et al 2001 Hesselbo et al 2002 vande Schootbrugge et al 2009 Whiteside et al 2010) Sulphur dioxideemitted by volcanic eruptions can be short-lived the atmospheric im-pacts are dependent on ejection volume and height Volcanoes may beforceful enough to project toxic gases including SO2 into the strato-sphere leading to global cooling (Rampino et al 1979 Rampino

2002 Tanner et al 2007) whereas effusive and 1047297ssure volcanoesmore frequently result in volcanic gases remaining in the troposphereleading to global warming (Gudmundsson 1996) Volcanic eruptionsalso discharge many other gases including hydrogen sulphide (H2S)which rapidly oxidises in the atmosphere to form SO2 (Brown 1982Kump et al 2005) carbon dioxide (CO2) hydrogen chloride (HCl) hy-drogen 1047298uoride (HF) and large amounts of water vapour (H2O) Thusvolcanic gases alone or in tandem damage plants Of all these gaseswe chose to concentrate on one of the major components of volcanicgases sulphur dioxide (Symonds et al 1994) Yet currently methodsfor detecting the timing and magnitude of release in the geologicalpast of sulphur dioxide are limited

Contemporary anthropogenic sources of SO2 result from the pro-cessing and combustion of fossil fuels containing sulphur Acid rain oc-curs when SO2 reacts in the atmosphere with water oxygen and otherchemicals to form a mild solution of sulphuric acid (H2SO4) (Grattan2005) which is then deposited on plants by wet or dry deposition de-pending on weather conditions (Kim et al 1997) A wealth of researchin the 1980s and 1990s into the effects of simulated acid rain on leavescon1047297rmsthat acid rain altersthe physical andchemical properties of cu-ticle and epicuticular waxes in some cases leading to deformed stoma-tal complexes during development of the cuticle (Haines et al 1985

Review of Palaeobotany and Palynology 208 (2014) 25ndash42

Corresponding author Tel+353 86 3917339E-mail address carolineelliottkingstongmailcom (C Elliott-Kingston)

httpdxdoiorg101016jrevpalbo201405001

0034-6667copy 2014 Elsevier BV All rights reserved

Contents lists available at ScienceDirect

Review of Palaeobotany and Palynology

j o u r n a l h o m e p a g e w w w e l s e v i e r c o m l o c a t e r e v p a l b o

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Percy and Baker 1987 1990 Turunen and Huttunen 1990 Tuomistoand Neuvonen 1993 Turunen et al 1995) Early greenhouse andplant growth chamber studies con1047297rmed that in addition to epicuticu-lar damage simulated acid rain composed of SO2 HCl and HF eitheralone or in combination resulted in the following effects permanentchromosomal and phenotypic changes in the offspring of HF-fumigated tomato seeds (Mohamed 1968) alterations in leaf areaand internode length in Citrus sinensis exposed to a mixture of SO2

and HF (Matsushima and Brewer 1972) leaf chlorosis with increasedleaf fall rate in Citrus unshu subjected to HF alone (Matsushima andBrewer 1972) and stomatal closure leading to reduced transpirationand raised leaf temperature in soybean (Glycine max) under HF fumiga-tion (Poovaiah and Wiebe 1973) Phytotoxic gases (atmospheric gasesthat inhibit plant growth or poison plants) can have major deleteriouseffects on plants growing near the source of a pollutant Plants thatgrow close to volcanic vents and are regularly subjected to phytotoxicgases such as SO2 may develop resistance to the pollutant (WinnerandMooney 1985 Haworth et al 2010) whereas those exposed to oc-casional and catastrophic amounts of toxic gases may succumb to foliarinjury and die (Haworthet al 2012)In a study onthe in1047298uence of vol-canic gases on Pinus halepensis Bartiromo et al (2012) showed degra-dation of epicuticular and epistomatal waxes as fusion of the waxstructures the presence of H2S in the fumarole gas likely caused thedamage In a further study Bartiromo et al (2013) undertook compre-hensive research into the effects of long-term exposure to volcanicgases on an angiosperm Erica arborea however it wasunclear whetherthe alterations in cuticle that occurred resulted from elevated CO2 orSO2 or both Regular exposure to volcanic gases has also been shownto modify leaf physiognomy (Bacon et al 2013) and change stomataldensityand its ratioto the stomatal index(Haworthet al 2012) There-fore development of leaf damage and changes in leaf structure andshape can provide a record of exposure to toxic gases If such plantswere subsequently preserved through fossilisation it may be possibleto detect signs of SO2 damage in the preserved cuticles and use this re-cord of leaf damage as a proxy for toxic gases in the geological pastHowever there are currently no studies where leaf-level SO2 damagehas been investigated and categorised in rigorously controlled experi-

mental conditions in plant growth chambers with a view to developinga palaeo-SO2 proxy

12 Effects of sulphur dioxide on plants

SO2 enters plants through minute epidermal pores called stomataWhen stomata open to allow exchange of carbon dioxide oxygen andwater vapour with the atmosphere sulphur dioxide enters by diffusiondueto thelower concentration gradient of SO2 insidetheplantStomataclose in response to low concentrations of atmospheric SO2 whereashigh concentrationsmay cause continuous stomatal opening as the epi-dermal cells surrounding the guard cells are damaged by SO2 and nolonger provide the structural support required for effective stomatalfunction (Black and Black 1979 Neighbour et al 1988 Robinson

et al 1998 McAinsh et al 2002) In addition SO2 can affect stomataby slowing their ability to close thus impairing stomatal control(McAinsh et al 2002) Once inside the aqueous environment of the cell SO2 is converted to sulphite (SO3

2minus) and hydrogen sulphite(HSO3

minus) sulphite is then partly oxidised to sulphate (SO42minus) (Zeigler

1972) A certain amountof sulphur canbe metabolised by plants butbe-yond a critical threshold level determined by the concentration of gas(Black and Black 1979) and duration of exposure (Ashenden 1979)damage occurs Sulphur oxides are acidic and at high concentrationsacids denature membrane-associated proteins embedded in the phos-pholipid bilayer of the plasma membrane that are essential for osmoticregulation(Heath1980) Membrane-associated calcium (mCa)ionsareimportant second messengers in signal transduction responses toenvironmental stimuli The calcium ions in1047298uence permeability by

stabilising membrane structure especially in conifers (DeHayes et al

1999) Hydrogen ions in acids such as sulphuric acid candisplace calci-um ions in the plasma membrane thus impairing physiological re-sponses to environmental stresses

Some SO2 permeatesthecuticle but most enters via thestomata dueto the lower resistance pathway The physical and chemical propertiesof some plant cuticles facilitate repellence of water and particles thatcould otherwise have negative effects on the plant (Neinhuis andBarthlott 1997Haworth and McElwain2008) Additionally wax struc-

tures on the cuticle re1047298

ect and scatter photosynthetically active and UV radiation therefore any phytotoxic gas that degrades plant cuticle in-duces increased absorbance of light and possible photo-oxidative dam-ageinleaves(Shepherd andWynne Grif 1047297ths2006) Thecomposition of cuticular waxes is known to be affected by many environmental factors(Holroyd et al 2002) Water stress or low nitrogen levels lead to in-creases in epidermal wax in Pinus palustris (Prior et al 1997) ozonepollution leads to a reduction in waxes in Populus tremuloides

(Mankovska et al 1998) whilst nitrogen oxides (NOx) and aerosolblack carbon from traf 1047297c pollution cause degradation of wax crystalstructure in Picea abies (Viskari et al 2000)

Once inside the plant atmospheric pollutants cause different typesof injury Prolonged exposure may cause chronic damage whilst sub-stantial episodic exposure can cause acute injury Physiological damageby SO2 includes down-regulation of photosynthesis (Noyes 1980Hallgren and Gezelius 1982 Haworth et al2012) Anatomical damageleads to disruption of water regulation as the epidermal and neighbourcells that provide structural support to the guard cells collapse leavingthe stomata permanently open (Neighbour et al 1988 Mans1047297eld1998 Robinson et al 1998) Morphological damage to the plant cuticlenegativelyimpacts cuticular waxes breaching the protectivebarrier be-tween the plant interior and exterior (Thompson and Kats 1978Bartiromo et al 2012) Kim et al (1997) found that differences in theuptake of SO2 also depend on plant type They showed that low uptakeofSO2 by gymnosperms (cab04 leaf sulphur content) ledto damagedcuticle whilst higher SO2 uptake damaged angiosperm cuticle (ca 04ndash06 leaf sulphur content) and very high levels of uptake were requiredto damage the cuticle of Ginkgo biloba (ca N07 leaf sulphur content)

Thepurpose of this workwas to analyse andcategorise theimpactof

continuous SO2 fumigation on plant epidermal and cuticle morphologyto determine if elevated SO2 resulted in universal damage type(s) thatwould be capable of detection in the fossil plant record

2 Methods and materials

21 Controlled environment conditions

Ten vascular plant species (three deciduous and seven evergreens)representative of Mesozoic gymnosperm and fern fossil 1047298oras(Table 1) were analysed for changes in epidermal and cuticle morphol-ogy when grown under continuous SO2 fumigation compared to a con-trol treatment Future work will include analysis of angiosperms Forplant growth methods see Haworth et al (2012) Plant species includ-

ed Lepidozamia hopei and Lepidozamia peroffskyana cycads grownfrom seed six month-old specimens of the fern Osmunda regalis andtwo year-old specimens of gymnosperms Ginkgo biloba Nageia nagiPodocarpus macrophyllus Araucaria bidwillii Agathis australis Taxodium

distichum and Wollemia nobilis All species were grown for a minimumof six months in CONVIRON BDW40 (Winnipeg Canada) walk-ingrowth rooms at the Programme for Experimental Atmospheres andClimate (PEacuteAC) facility in University College Dublin Six months wasdeemed an appropriate duration for the experiment as this allowed ad-equate time for each species to grow new leaves under experimentalconditions and for stabilisation of the plants physiological response toSO2 allowing us to measure long-term responses of plants to chronicSO2 fumigation in a simulated volcanic episode Plants were grownunder control atmospheric conditions (zero ppm SO2 209 O2

380 ppm CO2) or continuous fumigation with SO2 (02 ppm SO2

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209 O2 380 ppm CO2) We chose 02 ppm SO2 as the optimum con-centrationof atmospheric SO2 as this represents natural volcanic eventsover a wide geographical area and since SO2 has a short life-time inthe atmosphere it also represents a realistic concentration of chronicvolcanic SO2 present in the lower atmosphere where plants grow(Finlayson-Pitts and Pitts 1986 Brimblecombe 1996 Porter et al2002) Additionally we were obliged to take into account human safetyand SO2 exposure guidelines (European Union Council Directive 199930EC) Sulphur dioxidefumigation wasachieved by supplementing thechamber with compressed SO2 (BOC Gases Ireland Ltd) and monitoredby a Horriba APSA-370 AirPollution Monitor (HORIBA Instruments LtdNorthampton UK) Atmospheric SO2 was maintained at 0200 plusmn 0023(SD) ppm (n = 56486) for 215 days (full chamber data available onrequest) All other growth conditions remained constant with plantsexperiencing a 16-hour photoperiod 18 degC night to 28 degC middaytemperature 80 relative humidity and midday light intensity of 600 μ mol mminus2 sminus1 (see Haworthet al 2012 for detailed methodology)Allplant species produced newfoliage followingexposure to SO2 exceptthe fern O regalis Ginkgoalean G biloba and conifer W nobilis wheredamage to old growth leaves was investigated

22 Cryo-scanning electron microscopy

After six months mature fully-expanded new growth (exceptOsmunda regalis Ginkgo biloba and Wollemia nobilis see above) leaveswere chosenfor cryo-SEM microscopy from thetop of each plant receiv-ing full illumination and not affected by self-shading Leaves were col-lected using forceps cut to 05 times 05 cm and attached abaxial sidefacing upwards to an aluminium stub (1 cm diameter and 03 cmheight) using Sakura Finetek 4583 CRYO-OCT Compound (SakuraFinetek UK Thatcham UK) specimen holder adhesive Samples werethen pre1047297xed by immersion in slushy liquid nitrogen (following themethod of Umrath 1974) before being gold-plated for 120 s at 5 V under 2 mbar pressure in the presence of argon using an OxfordCT1500 Cryo Transfer System (Oxford Instruments Abingdon UK)Gold-coated specimens were then transferred for cryo-scanning elec-tron microscope analysis using a JEOL JSM-5410 LV microscope (JEOL UK Ltd Welwyn Garden City UK)

23 Image and data capture

In total 1016 leaves were imaged from both treatments (zero ppmSO2 and 02 ppm SO2) using cryo-scanning electron microscopy (seeTable 2) The entire area of each 05 times 05 cm leaf sample was scannedOn average 20 images per leaf sample were captured of which repre-sentative images are shown in Plates IndashX To avoid chamber effectplants were rotated between chambers halfway through the experi-ment (Hirano et al 2012) To avoid mutual shading plants wererandomised within areas of similar canopy height in the growth cham-

bers (Hammer and Hopper 1997 Sager and McFarlane 1997)

3 Results

A comparison of leaves from the zero (control) and elevated SO2

treatments con1047297rmed that after six months all ten species showed evi-dence of sulphur dioxide damageto their leaves including thefollowingeffects alteration of surface waxes (Plates IIndashX Table 1) blister-likelesions projecting above leaf surfaces (Plates I IV VI VII VIII IXTables1 3) folding andbursting of cuticle (Plates VX Table 1) sunkenleaf areas due to collapse of underlying epidermal cells (Plates I II IIIVIII Table 1) distortion of the stomatal complex (Plates I II IIITable 1) and decrease in stomatal wax plugs (Plates IV V IXTable 1) Control and SO2-treated leaves were subjected to identicalpreparation methods for cryo-scanning electron microscopy thereforethe observed damage structures on SO2 fumigated leaves were notcaused by the preparation methods as no damage structures were ob-served in control leaves (Plates IndashX Table 1)

31 Changes in cuticular wax

Alterations in cuticularwax on SO2 treated leaveswere evident in allspecies that produced new leaf growth under the elevated SO2 treat-

ment conditions and on mature leaves of Ginkgo biloba and Wollemianobilis (Plates IIndashX Table 1) No difference in cuticular wax was ob-served on the mature leaves of Osmunda regalisbetween control and el-evated treatment chambers (Plate I Table 1) In general surface waxappeared thickened either as a result of excess wax production or per-haps because individual wax structures degraded andcombined into anunstructured mass Thick layersof waxappeared atopthe dome-shapeddamage structures in Lepidozamia hopei (Plate IX 2 4 Table 1) alongwith folds and twisted rolls of wax on the leaf surface (Plate IX 6 8Table 1) Epicuticular wax appeared slightly molten on Lepidozamia

peroffskyana leaves (Plate X 8 Table 1) covering some of the stomatalpores with a thin layer of wax Epicuticular wax quantity appears tohave lessened on SO2 treated G biloba leaves (Plate II 4 6 8 Table 1)

Table 1

Epicuticular damage features associated with sulphur dioxide leaf fumigation in plant species represented of Mesozoic gymnosperm and fern fossil 1047298oras

Plate no Species Division Changes insurface waxes

Raisedlesions

Blistered andburst cuticle

Collapsed epidermalcells

Stomatal complexdistortion

Decrease in stomatalwax plugs

I Osmunda regalis L Fern Deciduous

II Ginkgo biloba L Ginkgo

III Taxodium distichum (L) Rich Conifer

IV Agathis australis (D Don) Lindl Conifer Evergreen

V Araucaria bidwillii Hook Conifer

VI Nageia nagi (Thunb) Kuntze Conifer

VII Podocarpus macrophyllus (Thunb)Sweet Conifer

VIII Wollemia nobilis WG JonesKD Hill amp JM Allen

Conifer

IX Lepidozamia hopei (W Hill) Regel Cycad

X Lepidozamia peroffskyana Regel Cycad

Table 2

Number of cryo-scanning electron microscope images captured per treatment mdash control(zero ppm SO2) or sulphur dioxide fumigation (002 ppm SO2)

Plateno

Species Zero ppm SO2-treated leaves

02 ppm SO2-treated leaves

I Osmunda regalis L 49 53II Ginkgo biloba L 12 47III Taxodium distichum (L) Rich 46 78IV Agathis australis (D Don) Lindl 13 86V Araucaria bidwillii Hook 15 87VI Nageia nagi (Thunb) Kuntze 15 32VII Podocarpus macrophyllus (Thunb) Sweet 54 102VIII Wollemia nobilis WG Jones

KD Hill amp JM Allen21 66

IX Lepidozamia hopei (W Hill) Regel 40 71X Lepidozamia peroffskyana Regel 46 83

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Plate I Leaf epicuticular morphology of the fern Osmunda regalis (Osmundaceae) Scale bars 1 amp 2 = 200 μ m 3 amp 4 = 200 μ m 5 amp 6 = 50 μ m 7 amp 8 = 20 μ m

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Plate II Leaf epicuticular morphology of the conifer Ginkgo biloba (Ginkgoaceae) Scale bars 1 = 100 μ m 2 = 50 μ m 3 amp 4 = 20 μ m 5 amp 6 = 10 μ m 7 = 5 μ m 8 = 2 μ m

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209 O2 380 ppm CO2) We chose 02 ppm SO2 as the optimum con-centrationof atmospheric SO2 as this represents natural volcanic eventsover a wide geographical area and since SO2 has a short life-time inthe atmosphere it also represents a realistic concentration of chronicvolcanic SO2 present in the lower atmosphere where plants grow(Finlayson-Pitts and Pitts 1986 Brimblecombe 1996 Porter et al2002) Additionally we were obliged to take into account human safetyand SO2 exposure guidelines (European Union Council Directive 199930EC) Sulphur dioxidefumigation wasachieved by supplementing thechamber with compressed SO2 (BOC Gases Ireland Ltd) and monitoredby a Horriba APSA-370 AirPollution Monitor (HORIBA Instruments LtdNorthampton UK) Atmospheric SO2 was maintained at 0200 plusmn 0023(SD) ppm (n = 56486) for 215 days (full chamber data available onrequest) All other growth conditions remained constant with plantsexperiencing a 16-hour photoperiod 18 degC night to 28 degC middaytemperature 80 relative humidity and midday light intensity of 600 μ mol mminus2 sminus1 (see Haworthet al 2012 for detailed methodology)Allplant species produced newfoliage followingexposure to SO2 exceptthe fern O regalis Ginkgoalean G biloba and conifer W nobilis wheredamage to old growth leaves was investigated

22 Cryo-scanning electron microscopy

After six months mature fully-expanded new growth (exceptOsmunda regalis Ginkgo biloba and Wollemia nobilis see above) leaveswere chosenfor cryo-SEM microscopy from thetop of each plant receiv-ing full illumination and not affected by self-shading Leaves were col-lected using forceps cut to 05 times 05 cm and attached abaxial sidefacing upwards to an aluminium stub (1 cm diameter and 03 cmheight) using Sakura Finetek 4583 CRYO-OCT Compound (SakuraFinetek UK Thatcham UK) specimen holder adhesive Samples werethen pre1047297xed by immersion in slushy liquid nitrogen (following themethod of Umrath 1974) before being gold-plated for 120 s at 5 V under 2 mbar pressure in the presence of argon using an OxfordCT1500 Cryo Transfer System (Oxford Instruments Abingdon UK)Gold-coated specimens were then transferred for cryo-scanning elec-tron microscope analysis using a JEOL JSM-5410 LV microscope (JEOL UK Ltd Welwyn Garden City UK)

23 Image and data capture

In total 1016 leaves were imaged from both treatments (zero ppmSO2 and 02 ppm SO2) using cryo-scanning electron microscopy (seeTable 2) The entire area of each 05 times 05 cm leaf sample was scannedOn average 20 images per leaf sample were captured of which repre-sentative images are shown in Plates IndashX To avoid chamber effectplants were rotated between chambers halfway through the experi-ment (Hirano et al 2012) To avoid mutual shading plants wererandomised within areas of similar canopy height in the growth cham-

bers (Hammer and Hopper 1997 Sager and McFarlane 1997)

3 Results

A comparison of leaves from the zero (control) and elevated SO2

treatments con1047297rmed that after six months all ten species showed evi-dence of sulphur dioxide damageto their leaves including thefollowingeffects alteration of surface waxes (Plates IIndashX Table 1) blister-likelesions projecting above leaf surfaces (Plates I IV VI VII VIII IXTables1 3) folding andbursting of cuticle (Plates VX Table 1) sunkenleaf areas due to collapse of underlying epidermal cells (Plates I II IIIVIII Table 1) distortion of the stomatal complex (Plates I II IIITable 1) and decrease in stomatal wax plugs (Plates IV V IXTable 1) Control and SO2-treated leaves were subjected to identicalpreparation methods for cryo-scanning electron microscopy thereforethe observed damage structures on SO2 fumigated leaves were notcaused by the preparation methods as no damage structures were ob-served in control leaves (Plates IndashX Table 1)

31 Changes in cuticular wax

Alterations in cuticularwax on SO2 treated leaveswere evident in allspecies that produced new leaf growth under the elevated SO2 treat-

ment conditions and on mature leaves of Ginkgo biloba and Wollemianobilis (Plates IIndashX Table 1) No difference in cuticular wax was ob-served on the mature leaves of Osmunda regalisbetween control and el-evated treatment chambers (Plate I Table 1) In general surface waxappeared thickened either as a result of excess wax production or per-haps because individual wax structures degraded andcombined into anunstructured mass Thick layersof waxappeared atopthe dome-shapeddamage structures in Lepidozamia hopei (Plate IX 2 4 Table 1) alongwith folds and twisted rolls of wax on the leaf surface (Plate IX 6 8Table 1) Epicuticular wax appeared slightly molten on Lepidozamia

peroffskyana leaves (Plate X 8 Table 1) covering some of the stomatalpores with a thin layer of wax Epicuticular wax quantity appears tohave lessened on SO2 treated G biloba leaves (Plate II 4 6 8 Table 1)

Table 1

Epicuticular damage features associated with sulphur dioxide leaf fumigation in plant species represented of Mesozoic gymnosperm and fern fossil 1047298oras

Plate no Species Division Changes insurface waxes

Raisedlesions

Blistered andburst cuticle

Collapsed epidermalcells

Stomatal complexdistortion

Decrease in stomatalwax plugs

I Osmunda regalis L Fern Deciduous

II Ginkgo biloba L Ginkgo

III Taxodium distichum (L) Rich Conifer

IV Agathis australis (D Don) Lindl Conifer Evergreen

V Araucaria bidwillii Hook Conifer

VI Nageia nagi (Thunb) Kuntze Conifer

VII Podocarpus macrophyllus (Thunb)Sweet Conifer

VIII Wollemia nobilis WG JonesKD Hill amp JM Allen

Conifer

IX Lepidozamia hopei (W Hill) Regel Cycad

X Lepidozamia peroffskyana Regel Cycad

Table 2

Number of cryo-scanning electron microscope images captured per treatment mdash control(zero ppm SO2) or sulphur dioxide fumigation (002 ppm SO2)

Plateno

Species Zero ppm SO2-treated leaves

02 ppm SO2-treated leaves

I Osmunda regalis L 49 53II Ginkgo biloba L 12 47III Taxodium distichum (L) Rich 46 78IV Agathis australis (D Don) Lindl 13 86V Araucaria bidwillii Hook 15 87VI Nageia nagi (Thunb) Kuntze 15 32VII Podocarpus macrophyllus (Thunb) Sweet 54 102VIII Wollemia nobilis WG Jones

KD Hill amp JM Allen21 66

IX Lepidozamia hopei (W Hill) Regel 40 71X Lepidozamia peroffskyana Regel 46 83

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Plate I Leaf epicuticular morphology of the fern Osmunda regalis (Osmundaceae) Scale bars 1 amp 2 = 200 μ m 3 amp 4 = 200 μ m 5 amp 6 = 50 μ m 7 amp 8 = 20 μ m

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Plate II Leaf epicuticular morphology of the conifer Ginkgo biloba (Ginkgoaceae) Scale bars 1 = 100 μ m 2 = 50 μ m 3 amp 4 = 20 μ m 5 amp 6 = 10 μ m 7 = 5 μ m 8 = 2 μ m

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Plate I Leaf epicuticular morphology of the fern Osmunda regalis (Osmundaceae) Scale bars 1 amp 2 = 200 μ m 3 amp 4 = 200 μ m 5 amp 6 = 50 μ m 7 amp 8 = 20 μ m

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Plate II Leaf epicuticular morphology of the conifer Ginkgo biloba (Ginkgoaceae) Scale bars 1 = 100 μ m 2 = 50 μ m 3 amp 4 = 20 μ m 5 amp 6 = 10 μ m 7 = 5 μ m 8 = 2 μ m

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Plate II Leaf epicuticular morphology of the conifer Ginkgo biloba (Ginkgoaceae) Scale bars 1 = 100 μ m 2 = 50 μ m 3 amp 4 = 20 μ m 5 amp 6 = 10 μ m 7 = 5 μ m 8 = 2 μ m

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Plate III Leaf epicuticular morphology of the conifer Taxodium distichum (Cupressaceae) Scale bars 1 = 200 μ m 2 = 100 μ m 3 amp 4 = 20 μ m 5 amp 6 = 20 μ m 7 amp 8 = 5 μ m

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Plate IV Leaf epicuticular morphology of the conifer Agathis australis (Araucariaceae) Scale bars 1 amp 2 = 200 μ m 3 amp 4 = 100 μ m 5 amp 6 = 20 μ m 7 amp 8 = 10 μ m

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Plate V Leaf epicuticular morphology of the conifer Araucaria bidwillii (Araucariaceae) Scale bars 1 amp 2 = 200 μ m 3 amp 4 = 50 μ m 5 amp 6 = 20 μ m 7 amp 8 = 20 μ m

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Plate VI Leaf epicuticular morphology of the conifer Nageia nagi (Podocarpaceae) Scale bars 1 amp 2 = 200 μ m 3 amp 4 = 100 μ m 5 amp 6 = 50 μ m 7 amp 8 = 20 μ m

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Plate VII Leaf epicuticular morphology of the conifer Podocarpus macrophyllus (Pocarpaceae) Scale bars 1 amp 2 = 200 μ m 3 amp 4 = 100 μ m 5 amp 6 = 20 μ m 7 amp 8 = 10 μ m

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Plate VIII Leaf epicuticular morphology of the conifer Wollemia nobilis (Araucariaceae) Scale bars 1 amp 2 = 200 μ m 3 amp 4 = 50 μ m 5 amp 6 = 20 μ m 7 amp 8 = 20 μ m

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Plate IX Leaf epicuticular morphology of the cycad Lepidozamia hopei (Zamiaceae) Scale bars 1 amp 2 = 100 μ m 3 amp 4 = 20 μ m 5 amp 6 = 20 μ m 7 amp 8 = 20 μ m

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Plate X Leaf epicuticular morphology of the cycad Lepidozamia peroffskyana (Zamiaceae) Scale bars 1 amp 2 = 200 μ m 3 amp 4 = 200 μ m 5 amp 6 = 10 μ m 7 amp 8 = 5 μ m

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Thick long rods of surface wax lying1047298at alongthe leaf surface above thevascular bundles and on the epidermal cells can be clearly seen in thecontrol superimposed by wax crystals (Plate II 5 Table 1) Thesethick rodsof wax could not befoundin any ofthe SO2 treated leaves al-though the superimposed wax crystals had altered little In Agathis

australis wax appeared thickened on top of the raised circular damagestructures but not on theremainder of theleaf (Plate IV 246 Table 1) Araucaria bidwillii epidermal cell structure was clearly delineated in thecontrol leaves but this delineation diminished under SO2 fumigation(Plate V 4 6 8 Table 1) indicating that surface wax structures haddisintegrated and merged 1047297lling the gaps between the cells makingthe leaf surface appear 1047298atter and less well-de1047297ned Wax accumulatedon the raised damage structures in Nageia nagi (Plate VI 6 8 Table 1)but the remainder of the leaf showed little evidence of alteration in cu-ticular waxes In Podocarpus macrophyllus folds of wax could clearly beseen (Plate VII 4 Table 1) similar to those on L hopei (Plate IX 6 8Table 1) and thick wax accumulation appeared on the dome-shapeddamage structures (Plate VII 2 6 8 Table 1) Taxodium distichum con-trol leaves possessed a large amount of epicuticular wax in very distinct

individual wax structures such as rods and plates (Plate III 7 Table 1)but in the SO2 treated leaves these had merged into an unstructuredwax agglomeration (Plate III 4 6 8 Table 1) Thick wax coveringthe leaf surface of W nobilis in the control treatment changed underSO2 treatment into smaller individual wax structures (Plate VIII 4Table 1) these were not joined in a continuous layer of wax as theywere in the control plants Wax also appears homogenised in structureand thickened on top of the raised circular damage structures(Plate VIII 4 6 8 Table 1)

32 Lesions Raised areas of damage on leaf surfaces

Themost distinctive feature associated with SO2 fumigation was the

appearanceof raised circular areas on theleaf surface which we refer tohere as lesions These damagestructureswere found on thefern Osmun-

da regalis (Plate I 4 6 8 Tables 1 3) on one cycad Lepidozamia hopei

(Plate IX 2 4 Tables 1 3) and on four of the six conifers Agathis

australis (Plate IV 2 4 6 Tables 1 3) Nageia nagi (Plate VI 2 4 6 8Tables 1 3) Podocarpus macrophyllus (Plate VII 2 4 6 8 Tables 1 3)and Wollemia nobilis (Plate VIII 4 6 8 Tables 1 3) Without exceptionstomata were seen on the top of each dome-shaped lesion indicatingthat thestructures were not just raisedcuticle butraisedabaxial epider-mal tissue as this is where stomata are located Entry of phytotoxic SO2

mayhave occurred through thestomatalporedamaging theunderlyingand surrounding tissue Stomata on top of the lesions were open inmany cases Cracks appeared in the top of the lesions in N nagi (PlateVI26)and W nobilis (Plate VIII 6) Circular craters or cavities of a sim-

ilar size to thelesions also appeared in N nagi (Plate VI24 Tables1 3)

possibly indicating the subsequent collapse of a raised damage struc-ture Since these bowl-shaped cavities were below the surface of theleaf the tissue beneath was likely degraded resulting in collapse

33 Blistered and burst cuticle

Sulphur dioxide had a deleterious impacton leaf cuticlein onecycadand one conifer Circular holes were evident in the cuticle of Lepidozamia peroffskyana(Plate X 4 Table 1) indicating that thecuticlemay have burst The same circular holes in the leaf cuticle were seen in Araucaria bidwillii (Plate V 8 Table 1) in addition to large variouslyshaped lsquobubblesrsquo of cuticle (Plate V 2 4 Table 1) It is clear that theseblisters and bubbles were raised areas of cuticle and did not containepidermal cells as observed in the dome-shaped lesions describedabove (eg Plates I IV VI) as the cuticle blisters did not have stomata lo-cated on them In contrast to the lesions stomata were clearly seenbelow the raised and burst cuticle level with the leaf surface (egPlate V 4)

34 Collapsed leaf tissue and distortion of stomatal complexes

Interveinal leaf tissuecollapsedin thethree deciduous species underinvestigation Osmunda regalis Ginkgo biloba and Taxodium distichumand in one of the evergreen conifers Wollemia nobilis Leaf interveinaltissue contains epidermal and mesophyll cells since scanning electronmicroscopy only shows leaf surface details it is unclear whether theepi-dermal cells alone collapsed or whether the underlying mesophyll cellswere also damaged The leaf vascular bundles remained intact how-ever leaving the veins standing above the rest of the leaf surface inthe SO2 damaged leaves The epidermal cells did not collapse in thecontrol leaves of these species (eg Plate I 3 Plate II 1 Plate III 1 3Plate VIII 1) con1047297rming that cellular collapse in the SO2 fumigated

leaves did not result from cryo-scanning electron microscopy whichdoes not lead to plant tissue desiccation but instead produces imagesof fully hydrated cells The subsidiary cells of G biloba are generally pa-pillate (Denk and Velitzelos 2002) (Plate II 1 3) The subsidiary cellscollapsed as a result of SO2 fumigation but the papillae did not andremained clearly visible (Plate II 2 4 Table 1) However the lack of structural support to the guard cells following degradation of the sub-sidiary cells or neighbour cells caused some distortion of the stomatalcomplex in all three deciduous species (Plate I 8 Plate II 4 6 8Plate III 2 Table 1) In G biloba for example twisting of both ends of the guard cells away from the long axis of aperture is clearly visible(Plate II 6) whereas in O regalis the guard cells themselves have col-lapsed (Plate I 8) In contrast in the evergreen W nobilis despite thefact that the interveinal tissue collapsed the guard cells remained un-

changed and the stomatal complex was not distorted that is the ends

Table 3

Leaf tissue lesions associated with sulphur dioxide fumigation are dome-shaped raised structures with epidermal cells and stomata located on their surface

Species No of leaves Mean lesion size(mm2)

Mean lesion area(mm2)

Length Width

Osmunda regalis 1 076 112 085Ginkgo biloba ndash Not observed Not observed ndash

Agathis australis 2 037 plusmn 003 (se) 042 plusmn 003 (se) 016 plusmn 002 (se) Araucaria bidwillii 2 026 plusmn 016 (se) 030 plusmn 018 (se) 008 plusmn 009 (se)Nageia nagi 4 035 plusmn 001 (se) 038 plusmn 003 (se) 013 plusmn 001 (se)Podocarpus macrophyllus 1 014 014 002Taxodium distichum ndash Not observed Not observed ndash

Wollemia nobilis ndash Not observed Not observed ndash

Lepidozamia hopei 1 026 027 007Lepidozamia peroffskyana ndash Not observed Not observed ndash

Mean lesion size in fern 076 112 085Mean lesion size in gymnosperms 028 plusmn 004 (se) 030 plusmn 005 (se) 008 plusmn 002 (se)

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of the guard cells were not twisted away from the long axis of the sto-matal aperture (Plate VIII 4 Table 1)

35 Reduction in stomatal wax plugs

In six of the ten species investigated stomatal pores are occluded bywax plugs The three deciduous species and oneof the evergreen cycadspecies Lepidozamia peroffskyana do not possess stomatal plugs Wax

plugs were unaffected by continuous SO2 fumigation in two speciesNageia nagi (Plate VI 2 4 6 Table 1) and Podocarpus macrophyllus

(Plate VII246 Table 1) and altered to varying degrees in the remain-ing four Lepidozamia hopei (Plate IX 4 6 8 Table 1) Agathis australis

(Plate IV 2 4 8 Table 1) Araucaria bidwillii (Plate V 2 4 6 Table 1)and Wollemia nobilis (Plate VIII 4 Table 1) Stomatal pores in L hopei

(Plate IX 4 6 8 Table 1) contained some wax in the control treatmentbut none in the SO2 treated plants The biggest change occurred in A australis (Plate IV 248 Table 1) stomatal cavities were completely1047297lled with wax in the control treatment but no wax remained in anystoma following treatment with SO2 Wax in the stomata of A bidwillii

(Plate V 2 4 6 Table 1) degraded somewhat when exposed to persis-tent SO2 fumigation and wax appears to have lifted out of some of theporesThe thick plugof waxcoveringeach stoma in W nobilisdegradedwax still occluded the pore but appeared as individual wax structuresrather than as a plug (Plate VIII 4 Table 1)

4 Discussion

41 SO 2 damage surrounding stomata

One very distinctive SO2 damage response found on leaves was thepresence of lesions circular areas of raised tissue surrounding (usuallyopen) stomata in one deciduous and 1047297ve evergreen species (Plates IIV VI VII VIII IX) indicating that open stomata were the entry pointfor sulphur dioxide The SO2 subsequently damaged the underlyingcells leading to uplifting of epidermal and possibly mesophyll tissueIt is currently unclear what is inside these dome-shaped lesions on liv-ing leaves subjected to SO2 fumigation They may be 1047297lled with liquid

water or gases including water vapour Alternatively the lesions maybe 1047297lled with swollen plant tissues Cell walls grow irreversibly as a re-sult of turgor pressure (Cosgrove 2005) Loss of osmotic control withinthe leaf mesophyll tissue may have led to irreversible cell wallstretching and the lesions may be 1047297lled with larger than normal meso-phyll cells

Exposure to SO2 has been shown to induce both stomatal openingand closing (Black and Black 1979 Neighbour et al 1988 Robinsonet al 1998 McAinsh et al 2002) depending on the concentration of gas Mans1047297eld(1998) suggested that increases in stomatal conductanceoccur when SO2 damages the epidermal cells surrounding guard cellsremoving structural resistance to the guard cells and preventing guardcell closure However when the guard cells themselves are damagedby SO2 they lose turgor and the stomatalpore closes In this study per-

sistent fumigation with SO2 likely resulted in less effective controlof stomata in the deciduous but not evergreen species because the epi-dermal cells surrounding the guard cells of the three deciduous speciesOsmunda regalis Taxodium distichum and Ginkgo biloba all collapsed inelevated SO2 (Plate I 2 Plate II 2 4 Plate III 2 4) removing structuralsupport for the stomata and initially allowing the guard cells to openwide (Mans1047297eld 1998) Sulphur dioxide then entered the stomatal cav-ity through the open pores damaging both the underlying mesophylltissue and the guard cells themselves which subsequently collapsedand closed (Plate I 8 Plate II 6 Plate III4 8)(Mans1047297eld 1998)Incon-trast stomata in theseven evergreen species do not appearto have col-lapsed due to guard cell damage (Plates IV ndashX) demonstrating anobvious visible difference in SO2 damage between deciduous and ever-green species Nonetheless stomatal effectiveness may still be compro-

mised in the evergreen species as it is not possible to see whether the

guard cells are open or closed under wax that occludes the pores Inthe case of evergreen Wollemia nobilis the interveinal tissue collapsedindicating that the underlying mesophyll cells and possibly the epider-mal cells were damaged but the guard cells remained unchanged andthe stomatal complex was not distorted This may be due to a thickerleaf cuticle in this evergreen species compared to the three deciduousspecies (Burrows and Bullock 1999 Balsamo et al 2003 Hill 2003)the thicker cuticle allowed the shape of the stomatal pore to be main-

tained despite collapse of the surrounding epidermal cells42 SO 2 damage to cuticle and cuticular waxes

Sulphur dioxide is not a systemic poison injury is local damagedleaves abscise and new leaves develop normally (Thomas 1951) Inthis study new leaves developed to replace SO2 damaged leaves in allspecies except the deciduous fern Osmunda regalis whose leaves didnot persist for more than one week (Haworth et al 2012) and in theevergreen conifer Wollemia nobilis Ginkgo biloba initiated new leavesbut these did not develop nor expand Another obvious effect of SO2 fu-migation was the alteration in cuticular waxes observed in all speciesthat produced new leaves in SO2 treatment conditions and in oldgrowth leaves of G biloba and W nobilis Cuticular waxes are formedin epidermal cells and transported within and above the cuticle(Samuels et al 2008) Exposure to SO2 and other toxic gases results inthe degradation of structural surface waxes into amorphous wax(Huttunen 1994 Kaipiainen et al 1995 Kupcinskiene and Huttunen2005) In this study individual wax structures on SO2 treated leaves ap-pear to have degraded and become less structured giving an appear-ance of 1047298at layers of wax on the leaf surfaces including on top of thelesions surrounding the open stomata Due to the hydrophobic proper-ties of epicuticular wax surface waxes determine leaf wettability(Neinhuis and Barthlott 1997) Thus wax degradation increases leaf wettability as watercontact anglesdecreasewith negative implicationsfor foliar uptake of inorganic ions and leaching of nutrient cations par-ticulate contamination that inhibits photosynthesis and increases leaf temperature and attack by pathogenic organisms that require waterfor germination (Haines et al 1985 Percy and Baker 1987 1990

Turunen and Huttunen 1990 Neinhuis and Barthlott 1997) Otherstudies have measured the impact of phytoxic gases on a limited num-ber of plant species Our research goes further by investigating the ef-fects of SO2 on a wide range of taxa including one fern oneginkgophyte two cycads and six coniferous species under controlledenvironment conditions Wax damage observed in nine of the ten spe-cies (Plates IIndashX) may have been followed by cuticle damage therebybreachingthe protectivebarrier between plantinterior and atmospherethat allowed SO2 to enter the leaf and water to exit which led to in-creased tissue desiccation compromised tissue tension and hastenedleaf abscission

The form of delivery of SO2 to the plant is an important determinantof injury Kim et al (1997) found Ginkgo biloba leaves to be resistant togaseous dry deposition but susceptible to acid rain In a growth experi-

ment study on theeffect of high [SO2] on leaf macromorphology under-taken in the same environmental conditions Bacon et al (2013)showed that G biloba was the most severely affected species of 1047297venearest living equivalent (NLE) taxa selected as analogues for abundantTriassicndash Jurassic fossil taxa In this study G biloba subjected to persis-tent SO2 by gaseous dry deposition incurred acute leaf damage (PlateII Table 1) Collapsed epidermal cells led to folding of tissue on theleaf surface of G biloba (Plate II 2 4 Table 1) and Taxodium distichum

(Plate III 2 4 6 Table 1) It is possible that these folds and the twistedrolls of wax seen on the leaf surfaces of Podocarpus macrophyllus (PlateVII 4 Table 1) and Lepidozamia hopei (Plate IX 6 8 Table 1) would beeasily observable in fossil cuticles Lesions on six of the ten species(Plates I IV VI VII VIII IX Table 1) may also be observable in the fossilrecord depending on the method of preservation of the fossil cuticle

for example permineralisation may preserve the structures Useful

39C Elliott-Kingston et al Review of Palaeobotany and Palynology 208 (2014) 25ndash42

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analytical tools for observation of fossil cuticles include scanning elec-tron microscopy and non-destructive atomic force microscopy

43 Towards the development of an SO 2 proxy

This study con1047297rmsthat persistentexposureto 02ppmSO2 resultedin a range of damage types in the ten species studied (Plates IndashXTable 1) includingalterations in cuticular wax in nine of the ten species

(Plates IIndashX Table 1) characteristic dome-shaped lesions in six of theten species (Plates I IV VI VII VIII IX Tables 1 3) interveinal cell col-lapse in four species (Plates I II III VIII Table 1) that resulted in distor-tionof thestomatal complexes in all three deciduous species (Plates I IIIII Table 1) a decrease in stomatal waxplugs in three species (Plates IVVIX Table 1) andblisteredand burst cuticlein twospecies (PlatesVXTable 1) None of theexperimentalplants had been subjected to SO2 ex-posurepriorto theexperimentIn thegeological past taxa that acquiredresistance over time may have persisted through SO2 events whilstnon-resistant taxa may have become extinct (Haworth et al 2010) If some of the unambiguous SO2 damage structures are found togetherin fossil leaf cuticle such as the dome-shaped lesions (Plates I IV VIVII VIII IX Tables 1 3) changes in leaf surface waxes (Plates IV VIVII VIII IX Table 1) and folding and twisting of cuticle (Plates II III)

this may signify SO2 fumigation to the leaves at the time of fossilisationbut potentially other destructive acids also The potential effects on liveleaf tissue of other volcanically released acids such as HCl and HF mustbe ruled out with further experiments However we think it is unlikelythat these acids would cause similar epicuticular damage types as SO2

since both acids are commonly used in the extraction of fossil cuticlefrom sediments and no similar damage structures to those induced bySO2 have been observed Sulphur dioxide responses can be grouped ac-

cording to leaf life-span with deciduous species typically showing col-lapsed epidermal cells in combination with altered leaf surface waxesand evergreen species typically showing raised lesions and alterationsin surface waxes

44 Implications for the fossil record

As fossil plant cuticle representsthe external morphological featuresof the preserved plant it may be possible to detect evidence for thepre-cise timing of SO2 eventsassociated with intense episodes of past volca-nic activity which are considered as potentially important driversof some mass extinction events such as those that occurred at thePermianndashTriassic and Triassicndash Jurassic boundaries The results fromthis research have implicationsfor theinterpretation of thefossil record

Fig 1 Integration of cryo-scanning electron microscopy with existing palaeobotanical indicators of palaeo-SO2 and volcanic gases Used in conjunction these methods can be applied to

fossil leafcuticles andmacrofossil leaves to pinpointthe timingof palaeo-SO2 episodesin thefossilrecord andpermittestingof therole of SO2 as a hypothesiseddriverin extinctionevents

40 C Elliott-Kingston et al Review of Palaeobotany and Palynology 208 (2014) 25ndash42

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of the guard cells were not twisted away from the long axis of the sto-matal aperture (Plate VIII 4 Table 1)

35 Reduction in stomatal wax plugs

In six of the ten species investigated stomatal pores are occluded bywax plugs The three deciduous species and oneof the evergreen cycadspecies Lepidozamia peroffskyana do not possess stomatal plugs Wax

plugs were unaffected by continuous SO2 fumigation in two speciesNageia nagi (Plate VI 2 4 6 Table 1) and Podocarpus macrophyllus

(Plate VII246 Table 1) and altered to varying degrees in the remain-ing four Lepidozamia hopei (Plate IX 4 6 8 Table 1) Agathis australis

(Plate IV 2 4 8 Table 1) Araucaria bidwillii (Plate V 2 4 6 Table 1)and Wollemia nobilis (Plate VIII 4 Table 1) Stomatal pores in L hopei

(Plate IX 4 6 8 Table 1) contained some wax in the control treatmentbut none in the SO2 treated plants The biggest change occurred in A australis (Plate IV 248 Table 1) stomatal cavities were completely1047297lled with wax in the control treatment but no wax remained in anystoma following treatment with SO2 Wax in the stomata of A bidwillii

(Plate V 2 4 6 Table 1) degraded somewhat when exposed to persis-tent SO2 fumigation and wax appears to have lifted out of some of theporesThe thick plugof waxcoveringeach stoma in W nobilisdegradedwax still occluded the pore but appeared as individual wax structuresrather than as a plug (Plate VIII 4 Table 1)

4 Discussion

41 SO 2 damage surrounding stomata

One very distinctive SO2 damage response found on leaves was thepresence of lesions circular areas of raised tissue surrounding (usuallyopen) stomata in one deciduous and 1047297ve evergreen species (Plates IIV VI VII VIII IX) indicating that open stomata were the entry pointfor sulphur dioxide The SO2 subsequently damaged the underlyingcells leading to uplifting of epidermal and possibly mesophyll tissueIt is currently unclear what is inside these dome-shaped lesions on liv-ing leaves subjected to SO2 fumigation They may be 1047297lled with liquid

water or gases including water vapour Alternatively the lesions maybe 1047297lled with swollen plant tissues Cell walls grow irreversibly as a re-sult of turgor pressure (Cosgrove 2005) Loss of osmotic control withinthe leaf mesophyll tissue may have led to irreversible cell wallstretching and the lesions may be 1047297lled with larger than normal meso-phyll cells

Exposure to SO2 has been shown to induce both stomatal openingand closing (Black and Black 1979 Neighbour et al 1988 Robinsonet al 1998 McAinsh et al 2002) depending on the concentration of gas Mans1047297eld(1998) suggested that increases in stomatal conductanceoccur when SO2 damages the epidermal cells surrounding guard cellsremoving structural resistance to the guard cells and preventing guardcell closure However when the guard cells themselves are damagedby SO2 they lose turgor and the stomatalpore closes In this study per-

sistent fumigation with SO2 likely resulted in less effective controlof stomata in the deciduous but not evergreen species because the epi-dermal cells surrounding the guard cells of the three deciduous speciesOsmunda regalis Taxodium distichum and Ginkgo biloba all collapsed inelevated SO2 (Plate I 2 Plate II 2 4 Plate III 2 4) removing structuralsupport for the stomata and initially allowing the guard cells to openwide (Mans1047297eld 1998) Sulphur dioxide then entered the stomatal cav-ity through the open pores damaging both the underlying mesophylltissue and the guard cells themselves which subsequently collapsedand closed (Plate I 8 Plate II 6 Plate III4 8)(Mans1047297eld 1998)Incon-trast stomata in theseven evergreen species do not appearto have col-lapsed due to guard cell damage (Plates IV ndashX) demonstrating anobvious visible difference in SO2 damage between deciduous and ever-green species Nonetheless stomatal effectiveness may still be compro-

mised in the evergreen species as it is not possible to see whether the

guard cells are open or closed under wax that occludes the pores Inthe case of evergreen Wollemia nobilis the interveinal tissue collapsedindicating that the underlying mesophyll cells and possibly the epider-mal cells were damaged but the guard cells remained unchanged andthe stomatal complex was not distorted This may be due to a thickerleaf cuticle in this evergreen species compared to the three deciduousspecies (Burrows and Bullock 1999 Balsamo et al 2003 Hill 2003)the thicker cuticle allowed the shape of the stomatal pore to be main-

tained despite collapse of the surrounding epidermal cells42 SO 2 damage to cuticle and cuticular waxes

Sulphur dioxide is not a systemic poison injury is local damagedleaves abscise and new leaves develop normally (Thomas 1951) Inthis study new leaves developed to replace SO2 damaged leaves in allspecies except the deciduous fern Osmunda regalis whose leaves didnot persist for more than one week (Haworth et al 2012) and in theevergreen conifer Wollemia nobilis Ginkgo biloba initiated new leavesbut these did not develop nor expand Another obvious effect of SO2 fu-migation was the alteration in cuticular waxes observed in all speciesthat produced new leaves in SO2 treatment conditions and in oldgrowth leaves of G biloba and W nobilis Cuticular waxes are formedin epidermal cells and transported within and above the cuticle(Samuels et al 2008) Exposure to SO2 and other toxic gases results inthe degradation of structural surface waxes into amorphous wax(Huttunen 1994 Kaipiainen et al 1995 Kupcinskiene and Huttunen2005) In this study individual wax structures on SO2 treated leaves ap-pear to have degraded and become less structured giving an appear-ance of 1047298at layers of wax on the leaf surfaces including on top of thelesions surrounding the open stomata Due to the hydrophobic proper-ties of epicuticular wax surface waxes determine leaf wettability(Neinhuis and Barthlott 1997) Thus wax degradation increases leaf wettability as watercontact anglesdecreasewith negative implicationsfor foliar uptake of inorganic ions and leaching of nutrient cations par-ticulate contamination that inhibits photosynthesis and increases leaf temperature and attack by pathogenic organisms that require waterfor germination (Haines et al 1985 Percy and Baker 1987 1990

Turunen and Huttunen 1990 Neinhuis and Barthlott 1997) Otherstudies have measured the impact of phytoxic gases on a limited num-ber of plant species Our research goes further by investigating the ef-fects of SO2 on a wide range of taxa including one fern oneginkgophyte two cycads and six coniferous species under controlledenvironment conditions Wax damage observed in nine of the ten spe-cies (Plates IIndashX) may have been followed by cuticle damage therebybreachingthe protectivebarrier between plantinterior and atmospherethat allowed SO2 to enter the leaf and water to exit which led to in-creased tissue desiccation compromised tissue tension and hastenedleaf abscission

The form of delivery of SO2 to the plant is an important determinantof injury Kim et al (1997) found Ginkgo biloba leaves to be resistant togaseous dry deposition but susceptible to acid rain In a growth experi-

ment study on theeffect of high [SO2] on leaf macromorphology under-taken in the same environmental conditions Bacon et al (2013)showed that G biloba was the most severely affected species of 1047297venearest living equivalent (NLE) taxa selected as analogues for abundantTriassicndash Jurassic fossil taxa In this study G biloba subjected to persis-tent SO2 by gaseous dry deposition incurred acute leaf damage (PlateII Table 1) Collapsed epidermal cells led to folding of tissue on theleaf surface of G biloba (Plate II 2 4 Table 1) and Taxodium distichum

(Plate III 2 4 6 Table 1) It is possible that these folds and the twistedrolls of wax seen on the leaf surfaces of Podocarpus macrophyllus (PlateVII 4 Table 1) and Lepidozamia hopei (Plate IX 6 8 Table 1) would beeasily observable in fossil cuticles Lesions on six of the ten species(Plates I IV VI VII VIII IX Table 1) may also be observable in the fossilrecord depending on the method of preservation of the fossil cuticle

for example permineralisation may preserve the structures Useful

39C Elliott-Kingston et al Review of Palaeobotany and Palynology 208 (2014) 25ndash42

7262019 1-s20-S0034666714000645-main11

httpslidepdfcomreaderfull1-s20-s0034666714000645-main11 1618

analytical tools for observation of fossil cuticles include scanning elec-tron microscopy and non-destructive atomic force microscopy

43 Towards the development of an SO 2 proxy

This study con1047297rmsthat persistentexposureto 02ppmSO2 resultedin a range of damage types in the ten species studied (Plates IndashXTable 1) includingalterations in cuticular wax in nine of the ten species

(Plates IIndashX Table 1) characteristic dome-shaped lesions in six of theten species (Plates I IV VI VII VIII IX Tables 1 3) interveinal cell col-lapse in four species (Plates I II III VIII Table 1) that resulted in distor-tionof thestomatal complexes in all three deciduous species (Plates I IIIII Table 1) a decrease in stomatal waxplugs in three species (Plates IVVIX Table 1) andblisteredand burst cuticlein twospecies (PlatesVXTable 1) None of theexperimentalplants had been subjected to SO2 ex-posurepriorto theexperimentIn thegeological past taxa that acquiredresistance over time may have persisted through SO2 events whilstnon-resistant taxa may have become extinct (Haworth et al 2010) If some of the unambiguous SO2 damage structures are found togetherin fossil leaf cuticle such as the dome-shaped lesions (Plates I IV VIVII VIII IX Tables 1 3) changes in leaf surface waxes (Plates IV VIVII VIII IX Table 1) and folding and twisting of cuticle (Plates II III)

this may signify SO2 fumigation to the leaves at the time of fossilisationbut potentially other destructive acids also The potential effects on liveleaf tissue of other volcanically released acids such as HCl and HF mustbe ruled out with further experiments However we think it is unlikelythat these acids would cause similar epicuticular damage types as SO2

since both acids are commonly used in the extraction of fossil cuticlefrom sediments and no similar damage structures to those induced bySO2 have been observed Sulphur dioxide responses can be grouped ac-

cording to leaf life-span with deciduous species typically showing col-lapsed epidermal cells in combination with altered leaf surface waxesand evergreen species typically showing raised lesions and alterationsin surface waxes

44 Implications for the fossil record

As fossil plant cuticle representsthe external morphological featuresof the preserved plant it may be possible to detect evidence for thepre-cise timing of SO2 eventsassociated with intense episodes of past volca-nic activity which are considered as potentially important driversof some mass extinction events such as those that occurred at thePermianndashTriassic and Triassicndash Jurassic boundaries The results fromthis research have implicationsfor theinterpretation of thefossil record

Fig 1 Integration of cryo-scanning electron microscopy with existing palaeobotanical indicators of palaeo-SO2 and volcanic gases Used in conjunction these methods can be applied to

fossil leafcuticles andmacrofossil leaves to pinpointthe timingof palaeo-SO2 episodesin thefossilrecord andpermittestingof therole of SO2 as a hypothesiseddriverin extinctionevents

40 C Elliott-Kingston et al Review of Palaeobotany and Palynology 208 (2014) 25ndash42

7262019 1-s20-S0034666714000645-main11

httpslidepdfcomreaderfull1-s20-s0034666714000645-main11 1618

analytical tools for observation of fossil cuticles include scanning elec-tron microscopy and non-destructive atomic force microscopy

43 Towards the development of an SO 2 proxy

This study con1047297rmsthat persistentexposureto 02ppmSO2 resultedin a range of damage types in the ten species studied (Plates IndashXTable 1) includingalterations in cuticular wax in nine of the ten species

(Plates IIndashX Table 1) characteristic dome-shaped lesions in six of theten species (Plates I IV VI VII VIII IX Tables 1 3) interveinal cell col-lapse in four species (Plates I II III VIII Table 1) that resulted in distor-tionof thestomatal complexes in all three deciduous species (Plates I IIIII Table 1) a decrease in stomatal waxplugs in three species (Plates IVVIX Table 1) andblisteredand burst cuticlein twospecies (PlatesVXTable 1) None of theexperimentalplants had been subjected to SO2 ex-posurepriorto theexperimentIn thegeological past taxa that acquiredresistance over time may have persisted through SO2 events whilstnon-resistant taxa may have become extinct (Haworth et al 2010) If some of the unambiguous SO2 damage structures are found togetherin fossil leaf cuticle such as the dome-shaped lesions (Plates I IV VIVII VIII IX Tables 1 3) changes in leaf surface waxes (Plates IV VIVII VIII IX Table 1) and folding and twisting of cuticle (Plates II III)

this may signify SO2 fumigation to the leaves at the time of fossilisationbut potentially other destructive acids also The potential effects on liveleaf tissue of other volcanically released acids such as HCl and HF mustbe ruled out with further experiments However we think it is unlikelythat these acids would cause similar epicuticular damage types as SO2

since both acids are commonly used in the extraction of fossil cuticlefrom sediments and no similar damage structures to those induced bySO2 have been observed Sulphur dioxide responses can be grouped ac-

cording to leaf life-span with deciduous species typically showing col-lapsed epidermal cells in combination with altered leaf surface waxesand evergreen species typically showing raised lesions and alterationsin surface waxes

44 Implications for the fossil record

As fossil plant cuticle representsthe external morphological featuresof the preserved plant it may be possible to detect evidence for thepre-cise timing of SO2 eventsassociated with intense episodes of past volca-nic activity which are considered as potentially important driversof some mass extinction events such as those that occurred at thePermianndashTriassic and Triassicndash Jurassic boundaries The results fromthis research have implicationsfor theinterpretation of thefossil record

Fig 1 Integration of cryo-scanning electron microscopy with existing palaeobotanical indicators of palaeo-SO2 and volcanic gases Used in conjunction these methods can be applied to

fossil leafcuticles andmacrofossil leaves to pinpointthe timingof palaeo-SO2 episodesin thefossilrecord andpermittestingof therole of SO2 as a hypothesiseddriverin extinctionevents

40 C Elliott-Kingston et al Review of Palaeobotany and Palynology 208 (2014) 25ndash42

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httpslidepdfcomreaderfull1-s20-s0034666714000645-main11 1718

Mass extinction events regularly coincided with the formation of largeigneous provinces (LIPs) during Earth history (Leckie et al 2002Courtillot andRenne 2003 Ganinoand Arndt 2009) Theplant damagestructures observed in our experiment offer a means of detecting SO2

release into the atmosphere due to intrusion of LIPs into high sulphur-containing rock such as evaporites and pyrite in shales and limestoneAnother possible application includes testing hypotheses of H2S releaseassociated with oceanic anoxic events (OAEs) (Kump et al 2005 Knoll

et al 2007) Ocean euxiniaanoxia occurs when the ocean becomes an-oxic andcertain bacteriaeg sulphur bacteriaproduce large volumes of toxic H2S gas that is released into the troposphere H2S reacts with O2 tobecome SO2 (Kump et al 2005) Based on these hypotheses and our ob-servations distinct cuticle damage structuresshould be observed in fernand gymnosperm plant cuticles spanning OAEs such as OAE2 in theCenomanian and other OAEs of similar and greater magnitude Tapho-nomic processes such as transport dehydration microbial degradationandor compression are unlikely to result in similar damage structuresto those induced by elevated SO2 because previous studies haveshown that when leaf cuticle is preserved it shows little chemical alter-ation from its pristine state and has undergone little microbial degrada-tion (Moumlsle et al 1997) Furthermore transport of any distance resultsin mechanical damage structures such as tearing andor shredding(Gastaldo 2007) neither of which can alter the micromorphology of leaf cuticle Similarly we have not observed twisting and folding of cu-ticle similar to that induced by SO2 (Plates II III Table 1) following leaf dehydration (McElwain pers obs) Finally we found no signi1047297cant ef-fect of elevated SO2 on post leaf abscission degradation processes (asmeasured by loss of leaf area over time) that could mask or alter theSO2 damage structures induced when the leaf was still attached to theplant (Gallagher et al unpublished) Although at this stage the SO2 in-duced damage structures identi1047297ed here are qualitative and can onlybe used to demonstrate the presence of SO2 in the atmosphere futurework aims to develop a more quantitative proxy Used in conjunctionwith other recently identi1047297ed palaeobotanical indicators of palaeo-SO2

such as quanti1047297ed X-ray transmission electron microscope and scan-ning electron microscope cuticle analysis (Bartiromo et al 20122013) leaf shape changes (Bacon et al 2013) and shifts in the ratio of

stomatal density to stomatal index values (Haworth et al 2012) theuse of cryo-SEM to identify the epidermal and epicuticular SO2 damagestructures described in this study will provide an additional valuabletool for directly pinpointing the timing of SO2 episodes in the fossil re-cord (see Fig 1) and for the 1047297rst time permit testing of the role of SO2 as a hypothesised driver of mass extinction

5 Conclusions

Persistent sulphur dioxide fumigation resulted in leaf damage to allSO2 fumigatedspeciesDistinct raised areasof tissue(lesions)surround-ing usually open stomata were observed epicuticular and epistomatalwaxes altered twisting and folding of leaf surface occurred where epi-dermal cells collapsed and cuticle blistered and burst We suggest

that where preservation permits these distinctive SO2 damage struc-tures could now be used as an SO2-proxy to pinpoint important pertur-bations in atmospheric SO2 concentration in the fossil record

Acknowledgements

We thank the following for scienti1047297c discussion and technical assis-tance Dr Cormac OConnell and Dr David C Cottell (Electron Micro-scope Laboratory UCD Ireland) Ms Bredagh Moran Mr Ray OHaireMr Liam Kavanagh (UCD Ireland) Mr Matthew Gilroy (ConvironUK) and Mr Aidan Blake (Aaron Refrigeration Ireland) We thankDr Karen L Bacon for helpful discussion on the manuscript We appre-ciate the comments and suggestions of two anonymous reviewerswhich improved the quality of this manuscript We gratefully acknowl-

edge funding from an EU Marie Curie Excellence Grant(MEXT-CT-2006-

042531) an IRCSET Embark scholarship (R10679) an EU Marie CurieIntra-European Fellowship (PEA-IEF-2010-275626) a European Re-search Council grant(ERC-279962-OXYEVOL) and a Science FoundationIreland PI grant (SFI-PI1103)

References

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Bartiromo A Guignard G Barone Lumaga MR Barattolo F Chiodini G Avino RGuerriero G Barale G 2012 In1047298uence of volcanic gases on the epidermis of Pinushalepensis Mill in Campi Flegrei southern Italy a possible tool for detecting volca-nism in present and past 1047298oras J Volcanol Geotherm Res 233ndash234 1ndash17

Bartiromo A Guignard G Barone Lumaga MR Barattolo F Chiodini G Avino RGuerriero G Barale G 2013 The cuticle micromorphology of in s itu Erica arboreaL exposed to long-term volcanic gases Environ Exp Bot 87 197ndash206

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Haworth M Gallagher A Elliott-Kingston C Raschi A Marandola D McElwain JC2010 Stomatal index responses of Agrostis canina to CO2 and sulphur dioxide impli-cations for palaeo-[CO2] using the stomatal proxy New Phytol 188 845ndash855

Haworth M Elliott-Kingston CGallagherA Fitzgerald AMcElwain JC 2012 Sulphurdioxidefumigation effects on stomatal density and index of non-resistant plants im-plications for the stomatal palaeo-[CO2] proxy method Rev Palaeobot Palynol 18244ndash54

Heath RL1980 Initial eventsin injury to plantsby airpollutantsAnnu Rev Plant Physiol31 395ndash431

Hesselbo SP Robinson SA Surlyk F P iasecki S 2002 Terrestrial and marine extinc-tion at the Triassicndash Jurassic boundary synchronized with major carbon-cycle pertur-bation a link to initiation of massive volcanism Geology 30 (3) 251ndash254

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Prior SA Pritchard SG Runion GB Rogers HH Mitchell RJ 1997 In1047298uence of atmo-spheric CO2 enrichment soil N and water stress on needle surface wax formation inPinus palustris (Pinaceae) Am J Bot 84 (8) 1070ndash1077

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