Research Ancestral stomatal control results in a canalization of fern and lycophyte adaptation to drought ScottA.M.McAdamandTimothyJ.Brodribb SchoolofPlantScience,UniversityofTasmania,PrivateBag55,Hobart,TAS,7001,Australia Summary Authorforcorrespondence: (cid:1) Littleisknownabouthowapredominantlypassivehydraulicstomatalcontrolinfernsand TimothyJ.Brodribb lycophytesmightimpactwateruseunderstress.Fernsandlycophytesoccupyadiversearray Tel:+61462261707 of habitats, from deserts to rainforest canopies, raising the question of whether stomatal Email:[email protected] behaviour is the same under all ecological strategies and imposes ecological or functional Received:29September2012 constraintsonfernsandlycophytes. Accepted:15January2013 (cid:1) Weexamined the stomatal response of a diversesample of fernand lycophyte species to bothsoilandatmosphericwaterstress,assessingthefoliarlevelofthehormoneabscisicacid NewPhytologist(2013)198:429–441 (ABA)overdroughtandrecoveryandthecriticalleafwaterpotential(Ψl)atwhichphotosyn- doi:10.1111/nph.12190 thesisindroughtedleavesfailedtorecover. (cid:1) The stomata of all ferns and lycophytes showed very predictable responses to soil and atmosphericwaterdeficitviaΨ,whilestomatalclosurewaspoorlycorrelatedwithchangesin Keywords: abscisicacid(ABA),drought, l ferns,lycophytes,stomata,vapourpressure ABA.Wefoundthatallfernsclosedstomataatverylowlevelsofwaterstressandtheirsur- deficit. vivalafterwardswaslimitedonlybytheircapacitanceanddesiccationtolerance. (cid:1) Ferns and lycophytes have constrained stomatal responses to soil and atmospheric water deficit as a consequence of a predominantly passive stomatal regulation. This results in a monotypicstrategyinfernsandlycophytesunderwaterstress. (Brodribb & McAdam, 2011; McAdam & Brodribb, 2012b) as Introduction well as molecular investigations into the function of key genetic Despite a relative stasis in stomatal morphology over >400mil- componentsessentialforstomatalsignalling(Chateretal.,2011; lionyr,thevegetativetissuebearingstomatahasevolvedimmense Ruszala etal., 2011). Particular focus in the investigation of the diversity and complexity in form and function, from the small evolution of stomatal control has been placed on comparing the bifurcating axes of the oldest fossilized stomatal-bearing plants stomata of the well-researched seed plants with nonseed plant (Batemanetal.,1998;Edwardsetal.,1998)tohighlyproductive groups. While complex metabolically driven stomatal control modern angiosperm leaves (Brodribb etal., 2009). In conjunc- predominates in the ecologically dominant and successful seed tionwiththismorphologicalevolution,thefunctionalbehaviour plants (Ache etal., 2010), fern and lycophyte stomata appear to of stomatal opening and closure has similarly radiated in com- be overwhelmingly regulated by a passive response to leaf water plexity, with significant implications for productivity and water status in the light (Brodribb & McAdam, 2011; McAdam & use (Doi etal., 2006; Doi & Shimazaki, 2008; Brodribb etal., Brodribb, 2012a). The predominance of a passive response of 2009; Brodribb & McAdam, 2011; Haworth etal., 2011, 2013; fernandlycophytestomataoccursdespitethepresenceandfunc- McAdam&Brodribb,2012a,b). tionofkeygeneticcomponentsintheselineagesthatareessential Extensive research has investigated the controls of stomatal for effective metabolic stomatal signalling (Chater etal., 2011; aperture in angiosperms (Raschke, 1975; Cowan & Farquhar, Ruszala etal., 2011). While these two lines of investigation 1977; Damour etal., 2010), with two processes responsible for appear contradictory, it seems most likely that, while the basal regulatingstomatalaperture,thepassivecontrolofguardcelltur- lineagesoflandplantsareinpossessionofanoperationalgenetic gor by leaf water status (Buckley & Mott, 2002; Buckley, 2005) frameworktoelicitmetabolicstomatalresponses,thepassivecon- and the active control of guard cell osmotic potential by the trol of stomata by water balance predominates in the regulation transport of ions across cell membranes (Schroeder etal., 2001; of stomatal behaviour. The most conclusive evidence for active Shimazaki etal., 2007; Lawson, 2009). While much work has control of fern and lycophyte stomata is a stomatal response to focused on the stomatal behaviour of angiosperms, our current red light that originates in the guard cells (Doi & Shimazaki, understanding of the evolution of stomatal control comes from 2008; McAdam & Brodribb, 2012b); there is also inconclusive relativelyrecentinvestigationsintofunctionalstomatalbehaviour evidence of stomatal responses to CO (Mansfield & Willmer, 2 in modern representatives of extant lineages of vascular plants 1969; Doi & Shimazaki, 2008; Brodribb etal., 2009; Ruszala (cid:1)2013TheAuthors NewPhytologist(2013)198:429–441 429 NewPhytologist(cid:1)2013NewPhytologistTrust www.newphytologist.com New 430 Research Phytologist etal.,2011).Thebehaviouroffernandlycophytestomatainthe maximum photosynthetic rates (A) and stomatal conductances light is highly predictable, with guard cells passively linked to (g) (McAdam & Brodribb, 2012b) as well as a recent sugges- s changesintheturgorpressureoftheleaf(Brodribb&McAdam, tion of highly variable maximum xylem hydraulic resistances 2011; Brodersen etal., 2012; McAdam & Brodribb, 2012a). It and vulnerability in the stipe of different species (Pittermann hasbeensuggested(McAdam&Brodribb,2012b)thatanevolu- etal., 2011; Brodersen etal., 2012). Ecological diversity, geo- tionary transition in stomatal control towards a predominantly logical persistence and the capacity for physiological and mor- metabolic regulation of stomatal aperture occurred during the phological variability observed in ferns and lycophytes raise the radiations of seed plant lineages in drier late-Palaeozoic environ- question: do all ferns and lycophytes have the same predomi- ments (DiMichele & Aronson, 1992) and was instrumental in nance of passive control of leaf hydration by stomata and, if ensuring the competitive success and ecological dominance of so, what are the adaptations adopted by ferns and lycophytes seedplantsandthedemiseoffern-andlycophyte-dominatedfor- that allow them to survive with this stomatal control mecha- estsintotheMesozoic.Indeed,duringthePalaeozoic,lycophytes nism (Watkins & Cardelu(cid:1)s, 2012)? andfernsenjoyedarichperiodofecologicaldominance(Phillips To answer these questions we examined the physiological etal., 1985) and over their evolutionary history have included responsestowaterstress ofadiversity offernandlycophytespe- morphologicalrepresentativesofallterrestriallifeformscurrently cies,includingmesophyticterrestrialspecies,epiphytesandades- representedbyextantseedplants(Rothwell,1996). iccation-tolerant species. In particular, we quantified the Despite a reputation as mesic relicts, modern ferns and lyco- sensitivity of stomata to soil drought, critical water potentials at phytes are certainly not restricted to ever-wet environments. which plants died, and the role of ABA in the response to Evidence from the fossil record and extant taxa indicates that drought. Our results provide a standardized comparison of the ferns and lycophytes have repeatedly radiated into xeric, cold diversityofwatermanagementstrategiesinfernsandlycophytes. and variable habitats (DiMichele & Phillips, 2002; Hietz, 2010), with the ability to survive desiccation widely represented MaterialsandMethods (Hietz, 2010). The epiphytic growth habit, which exposes indi- viduals to fluctuating water availabilities (Hietz & Briones, Speciesexamined 1998; Watkins etal., 2007b; Watkins & Cardelu(cid:1)s, 2009), is an ecological diversification widely represented in ferns (Schneider To observe the diversity of physiological responses of ferns and etal., 2004; Schuettpelz & Pryer, 2009) and the lycophyte lycophytes to water deficit, six fern species and a lycophyte were genus Huperzia (Wikstr€om & Kenrick, 2000; Wikstr€om, specificallyselectedtospanawideecologicalrangeofspore-bear- 2001), concurrent with the rise of angiosperm-dominated for- ing vascular plants, including mesophytic, drought deciduous, ests. A number of extant fern genera are highly competitive terrestrial species, epiphytes and a desiccation-tolerant species and invasive, successfully out-competing seed plants and (Table1). dominating landscapes and vegetation types both in modern All species examined were represented by three identical-aged forests (e.g. Pteridium (Marrs & Watt, 2006) and Lygodium individuals grown either from spores or from rhizomes collected (Pemberton & Ferriter, 1998)) and throughout the Mesozoic inthefield.Plantsweregrownin1.3-lpotscontainingan8:2:1 (Wing etal., 1993). Associated with this ecological diversity mixofcompostedpinebark,coarseriversandandpeatmosswith within the ferns and lycophytes is a substantial variability in addedslow-releasefertilizer,andhousedintheglasshousesofthe Table1 Speciesusedtoassessthephysiologicaldiversityoffernsandlycophytesinresponsetowaterdeficit,andtheirdistinctivemorphologicalfeatures andecologies(seeSupportingInformationFig.S1forimagesofeachspecies) Species Family Morphology Ecology Terrestrialferns Adiantumcapillus-venerisL. Pteridaceae Rhizomatousterrestrial,mesophytic Temperatetotropicalmoist, shadedhabitats DicksoniaantarcticaLabill. Dicksoniaceae Treefern,mesophytic Temperaterainforestunderstorey Pteridiumesculentum(G.Forst.) Dennstaedtiaceae Subterraneanrhizome,droughtdeciduous Cosmopolitan,fullsuntoopen Cockayne canopiedhabitats Epiphyticferns Pyrrosialingua(Thunb.)Farw. Polypodiaceae Epiphytic,rhizomatous Subtropicalcanopydwelling Rumohraadiantiformis(G.Forst.) Dryopteridaceae Epiphytic/lithophytic,rhizomatous Temperateclosedoropencanopy Ching Desiccation-tolerantfern CheilanthesmyriophyllaDesv. Pteridaceae Rhizomatousterrestrial,mesophytic Desiccation-tolerantfrom rockcrevices Lycophte Selaginellakraussiana(Kuntze) Selaginellaceae Terrestrial Subtropical,ever-wethabitats A.Braun NewPhytologist(2013)198:429–441 (cid:1)2013TheAuthors www.newphytologist.com NewPhytologist(cid:1)2013NewPhytologistTrust New Phytologist Research 431 SchoolofPlantScience,UniversityofTasmania,Hobart,Austra- overnight rehydrating through the cut end of the rachis, petiole lia.Noindividualhadexperiencedpreviousdroughtstress.Plants or stem to determine the viability of photosynthesis upon rehy- were grown under natural light conditions supplemented and dration. Rehydrating individual fronds or leaves simulated the extendedtoa16-hphotoperiodbysodiumvapourlamps,ensur- effect of soil rewetting without having to rewater whole plants, ing a minimum 300lmolquantam(cid:3)2 s(cid:3)1 at the leaf surface thereby allowing the three individuals to be tracked through an throughoutthedayperiod(maximumlightintensityoncloudless entire drought cycle. The following morning, leaf gas exchange days did not exceed 1100lmolquantam(cid:3)2s(cid:3)1 at leaf was measured on the rehydrated tissue, while the cut end level). Temperatures in the glasshouse were maintained at 22°C remained under water; this measurement was performed to during the day and 15°C at night. All plants were watered daily determine the Ψ at which different species sustained photosyn- l and fertilized with liquid nutrients weekly (unless undergoing thetic damage from water stress. Leaf recovery was defined drought). quantitatively as the recovery of photosynthesis following rehy- dration of tissue overnight; leaf death was defined as the Ψ at l which photosynthesis failed to recover above 0.5lmolm(cid:3)2s(cid:3)1 Drought,leafgasexchange,waterpotentialandfoliarABA following overnight rehydration (A ). Once photosynthesis in level 0 rehydrated leaves failed to recover in individual plants rehydra- Drought was initiated in three individuals per species by with- tion of leaves did not continue, in Adiantum capillus-veneris holding water. Over the course of the drought, once a day only, drought continued until plants reached (cid:3)6MPa. Follow- (reducing to once a week after 10d in more drought-tolerant ing leaf gas exchange measurement, the leaf area in the cham- species), between 12:00h and 13:00h, plants were transported ber was marked and the leaf or branch was taken for to the laboratory where leaf gas exchange, leaf water potential immediate Ψ measurement to assess effective rehydration of l (Ψ) andfoliarABAlevelwerequantifiedoneachindividual.Leaf the tissue (>(cid:3)0.2MPa). In the desiccation-tolerant species l gas exchange was measured using an infrared gas analyser (Li- Cheilanthes myriophylla, A recovered in rehydrated leaves when 6400;Li-Cor,Lincoln,NE,USA)onphotosynthetictissuefroma the plants were droughted beyond the limit of the pressure single leaf or stem (cuvette conditions: leaf temperature 22°C, chamber ((cid:3)10MPa); at this point, all three individuals were vapour pressure difference (VPD) maintained between 1.1 rewatered and leaf gas exchange, Ψ and ABA concentration l and 1.2kPa, 390lmol mol(cid:3)1 CO , light intensity 1000lmol were quantified daily until g had fully recovered to predrought 2 s quantam(cid:3)2s(cid:3)1, and flow rate 500mlmin(cid:3)1), and tissue was levels. In the two epiphytic fern species, Rumohra adiantiformis allowed to equilibrate to chamber conditions until stability was and Pyrrosia lingua, the three droughted individuals were rewa- reached(c.20min).Onthesameleaforstem,Ψ wasquantified tered at different stages to observe the response of g, A, Ψ and l s l ontissueexcisedandimmediatelywrappedindamppapertowel foliar ABA concentration over recovery. The first individual usingaScholanderpressurechamberandmicroscopetoprecisely was rewatered on the first day on which stomata closed (stoma- measurethexylembalancepressurewithoutthelossofanywater tal closure was defined as stomatal conductance <20% that of fromtheleaftissue.FoliarABAwasextracted,purifiedandphysi- initial fully hydrated stomatal conductance), and the second cochemically quantified using ultra-high-performance liquid and third individuals were rewatered over the extended period chromatography tandem mass spectrometry (UPLC-MS/MS) that followed when leaves were losing water but not declining withanaddedinternalstandard(aphytohormonequantification in Ψ or showing signs of leaf death. l methodfirstusedbyKojimaetal.,2009)accordingtothemeth- odsofMcAdam&Brodribb(2012a),withthefollowingmodifi- Turgorlosspointandvolumeofavailablewateruntilleaf cations for improving sample purification for UPLC-MS/MS death analysis. Following extraction and reduction under vacuum at 35°C to <1ml, the sample was taken up in 391-ml washes of Pressure–volume (PV) analysis was performed on at least five weak aqueous sodium hydroxide (pH 8). These washes were foliage samples, each from different plants, for each species to loaded on to a solid-phase extraction 600-mg SAX cartridge determine turgor loss point (Ψ ) (Tyree & Hammel, 1972) as tlp (Maxi-CleanTM;GraceDavisonDiscoverySciences,Deerfield,IL, well as the volume of water in the leaf that was available until USA), preconditioned with 10ml of weak aqueous sodium plants reached lethal relative water content (RWC). The night hydroxide (pH 8). The loaded sample on the cartridge was beforemeasurements,foliagewasbaggedandexcisedunderwater washed with 10ml of methanolandABA waselutedwith 15ml to ensure that Ψ was high (>(cid:3)0.05MPa). First leaf weight l of2%aceticacidinmethanol(v/v). ((cid:4)0.0001g; Mettler-Toledo, Melbourne, Australia) followed immediatelybyΨ wasperiodicallymeasuredovergradualdesic- l cation in the laboratory; care was taken to ensure that no water Assessingrecoveryusingrehydratedleavesandplants was lost from the petiole of the stem during Ψ assessment. PV l Following leaf gas exchange measurements, a pinnule, leaf or curveswereconstructedbyplottingΨ againstRWCofanaggre- l stem in closest proximity to the tissue sampled for gas exchange gationofpointsfromeachofthefiveleavesandΨ determined tlp was excised in water and bagged. Over the course of the by the inflection point of the relationship (Supporting Informa- imposed drought, no individual suffered defoliation in excess tion Fig. S2); RWC was determined according to the following of 20% of the original leaf area. The excised tissue was left equation: (cid:1)2013TheAuthors NewPhytologist(2013)198:429–441 NewPhytologist(cid:1)2013NewPhytologistTrust www.newphytologist.com New 432 Research Phytologist ðFW (cid:3)DWÞ Results RWC¼ Eqn 1 ðTW (cid:3)DWÞ Stomatalresponsetoleafwaterpotential (FW,thefreshweight(mass)oftissue;DW,thedryweight(mass) The stomata of a diverse sample of fern and lycophyte species oftissue;TW,theweight(mass)offullyhydrated,turgidtissue.) closed over a relatively small window of Ψ between (cid:3)0.6 and In all species, the volume of water available, as capacitance, l (cid:3)2.1MPawhendroughted(Fig.1).Asignificantlinearrelation- betweenstomatalclosureandleafdeathwascalculatedbyextrapo- ship between Ψ atstomatalclosureandΨ wasobserved when latingfromthePV relationshiptheRWCat whichleavesdiedby l tlp all species were compared (P<0.05; R2=0.616; data not using the Ψ at which A in rehydrated leaves failed to recover. In l shown).Duringtheimpositionofwaterstressandrecoveryfrom thedesiccation-tolerantC.myriophylla,threeadditionalindividuals stress,g ofallfernandlycophytespecieswasstronglyinfluenced were droughted to determine an accurate RWC and time until A s by Ψ, with no hysteresis in g observed in individuals that were in rehydrated leaves failed to recover and leaves died. The FW of l s rehydrated(Fig.1). leavesatthislethalRWCwasthenusedtocalculatethevolumeof wateravailableuntilleafdeathaccordingtothefollowingformula: Asmallmarginbetweenstomatalclosureandleafdeathin Available waterðmmol/m2Þ¼ðWW (cid:3)ðFW (cid:3)DWÞÞ fernsandlycophytes death (cid:5)M=LA Terrestrial fern and lycophyte species typically had a small Ψ l Eqn 2 marginbetweenstomatalclosureandleafdeathfromwaterstress, except for the dessication-tolerant species C. myriophylla, which (WW, the weight (mass) of leaf water at 100% RWC (g); could survive extremely low Ψ (Figs1, 2). In the mesophytic l FW , the fresh leaf weight (mass) at the lowest recoverable lycophyteSelaginellakraussiana,theΨ marginbetweenstomatal death l RWC of leaves (g); DW, the dry leaf weight (mass) (g); M, the closureandleafdeathwasonly0.01MPa,whileintheterrestrial molarmassofwater(gmol(cid:3)1);LA,leafarea(m2).) fern species this margin ranged from 0.19MPa in A. capillus- As a consequence of slow equilibration of Ψ in droughted veneris to 0.4MPa in Pteridum esculentum (Fig.2). The two l leaves of the two epiphytic fern species (R.adiantiformis and epiphytic fern species had slightly larger safety margins between P.lingua), PV curves were constructed using a modified method stomatal closure and irreversible photosynthetic damage, with to observe the relationship between RWC and Ψ over the 0.42 and 0.92MPa for P.lingua and R.adiantiformis, respec- l extended period of desiccation. Following the inflection point of tively(Fig.1). therelationshipbetweenRWCandΨ,leavesweresealedinabag There was very little difference between the Ψ values when l l containing damp paper towel to maintain high humidity and drought-stressed leaves died in all fern species (c. 0.8MPa lower allowed to equilibrate overnight. The following morning leaf in the epiphytic species; Fig.1). In terrestrial fern and lycophyte weight and Ψ were concurrently measured and the leaves were species, >80% of leaves died as a result of drought at a Ψ l l then allowed to desiccate on the bench for c. 10h before leaf between (cid:3)1.2MPa (A.capillus-veneris and S.kraussiana) and weight and Ψ were again measured. The leaf was then bagged (cid:3)2.2MPa (P.esculentum), while the leaves of the epiphyte l andallowedtoequilibrateovernight.Thiscyclewascontinuedfor R.adiantiformis perished when droughted to –2.2MPa and 5doruntilΨ inequilibratedleavesfailedtorecover. >50% of leaves of P.lingua died when droughted to (cid:3)3MPa l (Figs1, 2). Importantly, there was no evidence of a feedback in stomatalcontrolbetweenAandg,wherebyreducedordamaged Stomatalresponsetovapourpressuredifference s Aasaresultofdroughtcausedstomatatoremainclosedonrehy- Theresponse of stomatato atmospheric waterstress(asopposed dration; in recovered leaves with depressed photosynthesis, g s tosoilwaterstress)wasinvestigatedbyexaminingtheresponseof remained relatively high (Fig. S3). This resulted in highly ineffi- g to step-wise transitions in VPD in well-watered individuals. cientlossofwaterinplantsrecoveringfromdroughtdamage. s Three individuals of each species were acclimated to laboratory conditionsovernightandthefollowingdaytheresponseofg toa s Thestomatalresponseoffernsandlycophytestodrought sequence of VPD transitions (1–2–1kPa) was measured on isnotmediatedbyABA photosynthetic tissue using an infrared gas analyser (Li-6400; Li-Cor); leaf cuvette conditions were maintained at 22°C, and In all species, ABA levels were augmented in response to VPDwasregulatedattherequiredkPabyadjustingthehumidity water deficit, but in most species this augmentation did not in the inlet air by bubbling the incoming air through water and correlate with changes in g (Fig.3), instead occurring after s adjusting the amount passing through a desiccant column stomatal closure and Ψ ; following Ψ , the levels of ABA in tlp tlp containingcalcium sulphate (390lmol mol(cid:3)1CO ,light inten- the leaves of the terrestrial and desiccation-tolerant species 2 sity1000lmolquantam(cid:3)2s(cid:3)1andflowrate500mlmin(cid:3)1).All increased to levels exceeding 1000ngg(cid:3)1 FW (Fig.4). Similar conditions in the chamber, including A and g, were automati- relationships between g, Ψ and ABA level were observed s s l callyloggedeveryminute.Leaveswereacclimatedfor20minfol- when ABA levels were expressed in terms of DW calculated lowingeachVPDstep. from the relationship between RWC and Ψ determined from l NewPhytologist(2013)198:429–441 (cid:1)2013TheAuthors www.newphytologist.com NewPhytologist(cid:1)2013NewPhytologistTrust New Phytologist Research 433 (a) (b) (c) (d) (e) (f) (g) Fig.1 Therelationshipbetweenstomatalconductanceandleafwaterpotentialpooledfromthreeindividualsofthreemesophyticterrestrialfernspecies (a–c)andalycophyte(d),twoepiphyticfernspecies(e,f)andadesiccation-tolerantfernspecies(g)whendroughtedbywithholdingwater(circles). Solidverticallinesrepresenttheturgorlosspoint(Ψ )(seeSupportingInformationFig.S2)anddashedredverticallinesrepresenttheleafwaterpotential tlp (Ψ)atwhichtheassimilationratedidnotrecoverinrehydratedleaves(A ).Squaresymbolsrepresentmeasurementsmadeonindividualsthatwere l 0 rehydrated. PV curves (Figs S4, S5). Only two species showed an overlap Predictableresponsesoffernandlycophytestomatato between the phase of ABA rise and stomatal closure. In atmospherichumidity P.lingua, this relationship occurred over extremely low foliar ABA levels (<20ngg(cid:3)1 FW) (Fig.3) and in R.adiantiformis All fern and lycophyte stomata were highly sensitive to atmo- the recovery of g when droughted plants were rewatered was spheric humidity (Fig.5). Following an increase in VPD from 1 s not significantly influenced by the ABA level in the leaf to2kPa,thefernandlycophytestomataclosedfollowingasingle (Fig.1). exponential decay function, reaching a new steady-state g in s (cid:1)2013TheAuthors NewPhytologist(2013)198:429–441 NewPhytologist(cid:1)2013NewPhytologistTrust www.newphytologist.com New 434 Research Phytologist (a) (b) (c) (d) (e) (f) (g) Fig.2 Theassimilationrateofexcisedleavesrehydratedovernighttofullhydration(>-0.2MPa)fromknowndroughtedleafwaterpotentials,inthree mesophyticterrestrialdroughtdeciduousferns(a–c),alycophyte(d),twoepiphyticferns(e,f)andadesiccation-tolerantfern(g).Solidverticallines representtheleafwaterpotentialatwhichstomataclose(definedas<20%ofinitialstomatalconductance). <20min, with no hydropassive, wrong-way response (Fig.5). Alimiteddiversityoffernandlycophytestrategyin The degree of stomatal closure in response to this increase in responsetowaterstress VPD varied between species, with the stomata of C.myriophylla closing by only 30.95(cid:4)3.65% while the stomata of There was a large variability in the minimum RWC that the S.kraussiana closed by 66.26(cid:4)4.37% (Table2). Stomatal leaves of fern and lycophyte species could survive (Fig.6). An responses to a subsequent decrease in VPD from 2 to 1kPa increaseindesiccationtoleranceincreasedthevolumeofinternal resulted in stomata reopening that followed the same exponen- leaf water available to the plant during drought (Fig.6). The tial trajectory with no evidence of hydropassive closure (Fig.5, leavesofterrestrialfernandlycophytespecieswereunabletosur- Table2). The stomatal responses of ferns and lycophytes to vivedesiccationtoanRWC<85%andhadonlyaminimalvol- changes in atmospheric humidity were highly predictable ume of water available before leaf death, ranging from (Fig. S6). 46.4mmol H O m(cid:3)2 in A.capillus-veneris to 492.1mmol H O 2 2 NewPhytologist(2013)198:429–441 (cid:1)2013TheAuthors www.newphytologist.com NewPhytologist(cid:1)2013NewPhytologistTrust New Phytologist Research 435 (a) (b) (c) (d) (e) (f) (g) Fig.3 Therelationshipbetweenfoliarabscisicacid(ABA)level(ngg(cid:3)1freshweight)andstomatalconductancebeforestomatalclosureinthree mesophyticterrestrialfernspecies(a–c),alycophyte(d),twoepiphyticfernspecies(e,f)andadesiccation-tolerantfernspecies(g)whendroughted (circles)andrecovered(squares).Significantregressions(solidlines)andR2areshownwherepresent. m(cid:3)2inS.kraussiana(Fig.6).Theleavesoftheleastresistantspe- volume of available water in the leaf before death as a result of cies, Dicksoniaantarctica, were unable to survivean RWCbelow drought (Fig.6). The desiccation-tolerant fern species 97.2%,whiletheleavesofthelycophyteS.kraussianasurvivedto C.myriophyllawasabletosurviveextremelylowRWCs(c.10%) an RWC of only 87.3% (Fig.6). The limited tolerance of leaf and,likethetwoepiphyticspecies,hadalargevolumeofinternal desiccation was a trait characteristic of the mesophytic terrestrial leafwater(3824.8mmolH Om(cid:3)2(cid:4)44.07)availablebeforeleaf 2 speciesonly(Fig.6).Theleavesofthetwoepiphyticfernspecies death (Fig.6). The size of the internal leaf water buffer was a were characterized by a tolerance of low RWC, with leaves of product of leaf volume and desiccation tolerance and was highly R.adiantiformis surviving to an RWC of 41.7% and those of correlated with the duration of drought that could be tolerated P.linguato26.7%;thistoleranceoflowRWCresultedinalarge (P=0.001;R2=0.902;Fig.6). (cid:1)2013TheAuthors NewPhytologist(2013)198:429–441 NewPhytologist(cid:1)2013NewPhytologistTrust www.newphytologist.com New 436 Research Phytologist (a) (b) (c) (d) (e) (f) (g) Fig.4 Therelationshipbetweenfoliarabscisicacid(ABA)level(ngg(cid:3)1freshweight)andleafwaterpotential(–MPa)showingaclearrelationshipbetween waterstressandABAlevels,pooledfromthreeindividualsofthreemesophyticterrestrialfernspecies(a–c),alycophyte(d),twoepiphyticfernspecies(e,f) andadesiccation-tolerantfernspecies(g)whendroughtedbywithholdingwater(circles).Solidverticallinesrepresenttheleafwaterpotential(Ψ)when l stomataclosed(seeFig.1)anddashedredverticallinesrepresentΨ atwhichtheassimilationratedidnotrecoverinrehydratedleaves(A ).Square l 0 symbolseachrepresentmeasurementsmadeonindividualsthatwererehydrated. Discussion controlled by a passive response to leaf water status (Brodribb & McAdam, 2011). Such uncomplicated regulation of plant Predominantlypassivestomatalcontrolinfernsand hydration appears to result in a very simple drought adaptation lycophytes strategy whereby ferns and lycophytes modify water storage The stomatal behaviour among fern and lycophyte species with and desiccation tolerance as a means of extending drought sur- diverse growth habits and ecologies was found to be remark- vival time (Fig.6). This canalized adaptive pathway in response ably conservative, with the stomata of all species predominantly to drought must have significant ramifications for the ability of NewPhytologist(2013)198:429–441 (cid:1)2013TheAuthors www.newphytologist.com NewPhytologist(cid:1)2013NewPhytologistTrust New Phytologist Research 437 ferns and lycophytes to compete with seed plants, which have to survive for a long period of time with stomata closed before complex stomatal control and diverse water management strat- leavesdied,unlikethemesophyticterrestrialspecies(Fig.6).This egies (Tardieu & Simonneau, 1998). The complexity of a increasedtoleranceofepiphyticfernspeciestodroughtstresswas largely metabolic regulation of stomatal aperture in seed plants a result of the combination of a physiological tolerance of low can be recognized by the variability in stomatal responses to RWC and anatomical modification of leaves to increase the changes in VPD, from highly sensitive plants that elicit ‘feed- amount of water available before leaf death during drought forward’ responses to increases in VPD, to plants that are rela- (Fig.6).Epiphyticfernsarecapableofdevelopmentallyadapting tively insensitive (Farquhar, 1978; Franks & Farquhar, 1999; arelianceoneitherofthesetwostrategiestosurvivewaterstress, Mott & Peak, 2012). This diversity in response is unlike the with Asplenium auritum being desiccation-tolerant as a young predictable responses of fern and lycophyte stomata to changes sporophyte and maturing into a drought-avoiding, high-capaci- in VPD which are regulated by a passive response to leaf water tanceplantassizeincreases(Testo&Watkins,2012).Epiphytic status (Fig.5) (Brodribb & McAdam, 2011; McAdam & ferns are known to have large water storage capabilities in leaves Brodribb, 2012a). (as in the genus Pyrrosia with specialized hydrenchyma cells in theleaf(Ongetal.,1992))aswellaswater-storingrhizomesand stems (Dubuisson etal., 2009). Available water, as defined here Physiologicalandmorphologicaladaptationsaloneare bytheleafcapacitancemultipliedbytheviablerangeofleafwater essentialforfernsandlycophytestosurvivedry content after stomatal closure, gives a volume of water that can environments supplytheslowleakageofwaterfromplantswithclosedstomata Many epiphytic fern and lycophyte species are exposed to dry assuming hydraulic isolation from the soil (Linton & Nobel, and/or fluctuating environments, which raises the question of 1999).Thesignificantrelationshipwefoundbetweenthetimeto how these plants compete with seed plants when their stomata leaf death and leaf available water highlights the fundamental are predominantly regulated passively by leaf water status (Wat- importance of theinteraction between thestorage and partition- kins & Cardelu(cid:1)s, 2012). Only a small amount is known about ingofleafwaterandthetimetoleafdeathasaresultofdrought. the physiological adaptations of epiphytic fern species. These Epiphytic ferns areknowntomaintainaΨ lowenoughtoclose l includealowerleafhydraulicconductancecomparedwithterres- stomata for an extended period of time before leaves die (Ong trial ferns (Watkins etal., 2010), desiccation-tolerant gameto- etal., 1992). The combination of these two strategies, a physio- phytes(Watkinsetal.,2007a)andoccasionallyCrassulaceanacid logical tolerance of low RWC and partitioning and isolation of metabolism (CAM) (Ong etal., 1986; Holtum & Winter, largevolumesofwaterfromthestomata,isacommonfeatureof 1999).Interestingly,theadaptationofCAMintropicalepiphytic epiphyticfernsandlycophytes,whicharelargelycharacterizedby fernsisnotassociatedwithanenhancedtolerancetolongperiods succulent leaves or rhizomes and can survive in environments of drought as seen in angiosperm CAM species; rather, CAM in with highly sporadic water availability (Watkins & Cardelu(cid:1)s, epiphytic ferns improves carbon and water balance over short 2012). This apparent dependence on water storage and desicca- (diurnal)periodsofwaterstress(Ongetal.,1986). tion tolerance contrasts with seed plants which, with a predomi- While we found that the function of stomata in a diversity of nantly metabolic regulation of stomatal control, have the fernandlycophytespecieswasthesame,speciesfromdrierenvi- opportunityofmodifyingstomatalbehaviourthroughhormones ronments (epiphytes and desiccation-tolerant species) were able suchasABAduringdroughtstress(Wilkinson&Davies,2002). HighphysiologicaltoleranceofleavestolowRWCiscommon Table2 Themeanpercentagereductioninstomatalconductance(g) in (fern) epiphytes and exemplified by desiccation-tolerant and s followinganincreaseinvapourpressuredeficitfrom1to2kPaandthe poikilohydric species (Hietz, 2010). Within ferns, desiccation meangs(asapercentageofaninitialgsat1kPa)afteradecreaseinvapour toleranceiswidelyrepresented,occurringinmostmajorlineages, pressuredifference(VPD)from2to1kPainsevenfernandlycophyte withestimatesrangingfrom5to10%offernspeciesbeingdesic- species(n=3) cation-tolerant or poikilohydric, while in the lycophytes two of %stomatal %g ofinitial the three extant familieshave desiccation-tolerant species (Hietz, s Species closure(1–2kPa) (cid:4)SE (2–1kPa) (cid:4)SE 2010). The over-representation of desiccation tolerance in ferns andlycophytesrelativetoseedplantlineages(Oliveretal.,2000) Adiantumcapillus 47.50 4.68 105.45 3.81 is probably a reflection of the limited options of water -veneris Cheilanthes 30.95 3.65 99.85 0.63 management strategy provided by a predominantly passive sto- myriophylla matalcontrol.Thestomatalresponsesofdesiccation-tolerantand Dicksonia 33.53 3.82 100.30 3.26 epiphytic ferns are the same as those of mesophytic terrestrial antarctica ferns. Pteridium 35.37 5.31 102.65 2.60 esculentum Pyrrosialingua 32.89 5.70 98.04 1.02 Implicationsfortheevolutionoflandplants Rumohra 42.60 9.87 106.75 1.53 adiantiformis In ferns and lycophytes, the passive stomatal control of leaf Selaginella 66.26 4.37 100.23 1.85 hydration still provides an efficient means of regulating leaf kraussiana hydration in response to changes in water stress (Brodribb & (cid:1)2013TheAuthors NewPhytologist(2013)198:429–441 NewPhytologist(cid:1)2013NewPhytologistTrust www.newphytologist.com New 438 Research Phytologist (a) (b) (c) (d) (e) (f) (g) Fig.5 Representativeresponsesofstomatalconductance(molm(cid:3)2s(cid:3)1)tostep-wisetransitionsinvapourpressuredeficit(kPa)insixfernspecies(a–f)and alycophyte(g). McAdam,2011)(Fig.5).Yetthismechanismofstomatalcontrol seedplantsadynamic,stomatal-mediatedresponsetoepisodesof in ferns andlycophytesdoes notofferplantstheabilitytoenter- soil drought that occurs over relatively brief periods (hours to tainadiversityofresponsestosoilwatercontentthroughchanges days). The predominance of a metabolic regulation of stomata inmetabolicregulationofstomatalikeseedplants(Tardieu&Si- particularly by ABA in seed plants additionally allows the leaves monneau,1998).Itispossiblethattheevolutionofanincreased of species that do not have physiological and morphological regulation of stomata by ABA in seed plants may have been adaptations to dry environments to survive periods of soil driven bytheselective pressureforastomatalstrategy thatcould droughtbymodifyingthesensitivityofstomatatosmallchanges enhance the survival of plants over both short- and long-term in Ψ (Umezawa etal., 2004; Fujita etal., 2005), unlike the l periodsofsoilwaterstress;thiswouldofferanadvantageoverthe mesophyticterrestrialfernsandlycophytes(Fig.1).Adoptingthe changesinmorphologyordesiccationtolerancerequiredbyferns strategy of ABA-regulated stomatal control in seed plants is also andlycophytes(Fig.6).Thismighthavebeenparticularlyadvan- likelytobeimportantbothforoptimizingwateruseduringfluc- tageous during the drying climate of the early Permian (DiMic- tuatingsoilwateravailability(Chavesetal.,2003)andduringthe hele & Aronson, 1992). A heightened sensitivity of stomata to recoveryfromstressinfacilitatingtherepairofxylemtissueafter increased ABA concentrations, augmented by drought, offers drought(Lovisoloetal.,2008). NewPhytologist(2013)198:429–441 (cid:1)2013TheAuthors www.newphytologist.com NewPhytologist(cid:1)2013NewPhytologistTrust