Conifer species adapt to low-rainfall climates by following one of two divergent pathways

Timothy J. Brodribba, Scott A.M. McAdama, Gregory J. Jordana, and Samuel C.V. Martinsa,b

aSchool of Biological Sciences, University of Tasmania, Hobart, TAS 7001, Australia; and bDepartamento de Biologia Vegetal, Universidad Federal de Viçosa, 36570000 Viçosa, Brazil

Edited by Johanna Schmitt, University of California, Davis, CA, and approved August 19, 2014 (received for review May 1, 2014) Water stress is one of the primary selective forces in plant remaining in the soil (5, 16, 17). Although crucial in defining evolution. There are characters often cited as adaptations to water functional limits under water stress, neither xylem nor stomatal stress, but links between the function of these traits and characteristics have been used successfully to predict the mini- adaptation to drying climates are tenuous. Here we combine mum rainfall requirements for diverse species (18, 19). This can distributional, climatic, and physiological evidence from 42 species be explained by the fact that among woody plants, diversity in of conifers to show that the evolution of drought resistance water use, water acquisition, osmoregulation, growth rate, and follows two distinct pathways, both involving the coordinated plant hardiness interact to provide a wide range of viable sol- evolution of tissues regulating water supply (xylem) and water utions to survive and compete under different rainfall climates loss (stomatal pores) in leaves. Only species with very efficient (20–22). These strategies for regulating plant hydration form the stomatal closure, and hence low minimum rates of water loss, core of an empirical framework by which we understand plant inhabit dry habitats, but species diverged in their apparent survival “strategies” under water stress, yet a quantitative un- mechanism for maintaining closed stomata during drought. An derstanding of how water stress tolerance evolved or of which ancestral mechanism found in Pinaceae and Araucariaceae species metrics might allow prediction of species rainfall requirements relies on high levels of the hormone abscisic acid (ABA) to close remains elusive. stomata during water stress. A second mechanism, found in the Here we evaluate the proposition that adaptation to dry cli-

majority of Cupressaceae species, uses leaf desiccation rather than mates must involve coordinated evolution of stomatal and xylem EVOLUTION high ABA levels to close stomata during sustained water stress. systems and that, when considered together, characteristics of Species in the latter group were characterized by xylem tissues these tissues should reveal the adaptive pathway to survival in with extreme resistance to embolism but low levels of foliar ABA drying climates. Conifers (Pinales) provide the ideal-sized clade after 30 d without water. The combination of low levels of ABA under to address this fundamental evolutionary question. Living species stress with cavitation-resistant xylem enables these species to pro- of this ancient group are few in number (about 630 spp), yet they long stomatal opening during drought, potentially extending their are of great ecological and economic importance. Conifers photosynthetic activity between rainfall events. Our data dem- contain the oldest and largest living trees, but with 34% of co- onstrate a surprising simplicity in the way conifers evolved to cope “ ” with water shortage, indicating a critical interaction between xylem nifer species recently listed as threatened (International Union and stomatal tissues during the process of evolution to dry climates. for Conservation of Nature Red List), there is a strong imperative to understand their capacity to withstand environmental and cli- mortality | forest matic perturbations. In terms of stomatal and xylem evolution, conifers appear to be located in a critical part of the land plant phylogeny. Recent studies suggest that the stomatal regulation of ince plants first emerged from the early Paleozoic mud onto water loss in conifer species is functionally intermediate between Sdry land (1), their survival has hinged on finding a strategic balance between the imperative to grow and the risk of death by desiccation. Selection for growth rate favors a rapid uptake of Significance atmospheric CO2, but greater leaf porosity to CO2 comes at the cost of greater loss of water through transpiration (2, 3). Vas- A major determinant of plant species distribution on Earth is cular plants navigate between these competing demands by a specific tolerance to soil drying, yet there are currently no having sophisticated systems to regulate water supply (the xylem functional or anatomical characteristics that can predict spe- ’ network) and water loss (the stomata), which together maintain cies requirement for rainfall. This study examines the systems plant hydration (2, 4). Coordination between xylem and stomatal responsible for controlling water delivery and water loss in the tissues is a prerequisite for plants to function safely, in terms of leaves of conifers and finds functional evidence of how coni- avoiding hydraulic failure (5), and efficiently, in terms of resource fers have evolved in drying climates over the course of the last “ ” use (6), yet little is known about this coevolutionary process at the 150 million years. Two strategies for conserving water during core of plant adaptation to climate. water stress emerged. One group relied on the plant hormone Water stress is perhaps the most ubiquitous selective force abscisic acid to maintain stomata closed during sustained acting on woody plants, inflicting mortality from the arid zone to drought, and another, more derived group allowed leaves to dehydrate and resisted damage by producing a water trans- the understory of tropical rainforest (7), and particularly under port system capable of functioning under the extreme tension changing climatic conditions (8, 9). Plant exposure to water stress that develops in water-stressed plants. under conditions of soil drying is determined by static characters

of allometry in stem volume, leaf area, and root depth (10, 11), Author contributions: T.J.B. and S.A.M.M. designed research; T.J.B., S.A.M.M., and S.C.V.M. as well as the dynamic behavior of stomata and xylem in response performed research; T.J.B. and S.A.M.M. contributed new reagents/analytic tools; T.J.B., to increasing water stress (12–14). The timing of stomatal closure S.A.M.M., G.J.J., and S.C.V.M. analyzed data; and T.J.B. and S.A.M.M. wrote the paper. dictates how water reserves in the soil and plant are conserved The authors declare no conflict of interest. (14, 15), whereas the ability of the xylem water transport system This article is a PNAS Direct Submission. to sustain increasing tension in the water column without dys- 1To whom correspondence should be addressed. Email: [email protected]. function, because of cavitation or tissue distortion, defines the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lethal threshold at which leaves become cut off from water 1073/pnas.1407930111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1407930111 PNAS | October 7, 2014 | vol. 111 | no. 40 | 14489–14493 Downloaded by guest on September 25, 2021 the passively controlled ferns and the complex hormone-mediated behavior characteristics of flowering plants (23). Conifer xylem also exhibits enormous variation in resistance to damage under water stress, with some suggestion of evolution in xylem vul- nerability to climate change over the last 150 My (24). Thus, we focus here on conifers as a phylogenetically and functionally tractable group to investigate how coevolution of xylem and stomata has yielded successful functional configurations for survival in dry climates. Results Xylem Failure and Plant Death. Drying soil leads to increasing tension in the water transport system (quantified as water po- tential Ψ below zero; megapascals), and at a certain point, air becomes sucked into xylem tubes (tracheids), causing them to be disabled by cavitation and subsequent embolism (air) blockage (25). The vulnerability of the xylem to water stress is typically quantified by the leaf water potential (Ψl) at which 50% of the xylem is deactivated (Ψ50) (25). We first examined the degree of variation in this important stress-tolerance trait among extant conifers (SI Appendix, Table S1) by examining the vulnerability of leaf xylem to failure under water stress and comparing this with whole-plant resilience to water stress [defined here as the Ψl at the point of incipient (15–30%) leaf death]. A large range of resilience was found among conifer families, but clear trends were evident. Species belonging to Araucariaceae were the least tolerant of water deficit (Fig. 1A and SI Appendix, Fig. S1), whereas species of the dominant clades of Cupressaceae (Calli- troideae and Cupressoideae) survived very low Ψl without sus- taining damage to the leaves. The resilience of leaves to water stress was strongly correlated (r2 = 0.73), with the ability of the leaf/shoot xylem to resist failure (Fig. 1A, insert). This result supports previous findings for a small sample of conifers (17) and angiosperms (26) and verifies a general link between cavitation and plant mortality in tree species during acute episodes of water stress. Despite clear patterns in resilience and Ψ50 among spe- cies, neither character was correlated with species native rainfall climate (Fig. 1B). A similar lack of correlation was found re- cently for stem vulnerability (18) among 480 species (including 96 conifers), indicating that although xylem vulnerability pro- Fig. 1. Water stress tolerance in 42 species of conifers expressed as the vides an important index for when plant species will die on ex- water potential at incipient leaf damage during imposed soil drying (A). This posure to water stress (17), it is not predictive of where plants maximum tolerable water stress was very strongly correlated with the water ψ can survive in terms of rainfall. The inability of xylem vulnera- potential required to cause 50% loss of hydraulic conductance ( 50) in leaves bility to predict species minimum rainfall requirement (except (insert, R2 = 0.73) of all conifer families (Araucariaceae, green; basal among members of the Cupressaceae; Fig. 1B) suggests either Cupressaceae, black; derived Cupressaceae, red; Pinaceae, blue; Podocarpa- that conifer distributions are not limited by rainfall or that ceae, cyan; Sciadopityaceae, gray; Taxaceae, yellow). Despite its strong resisting cavitation in the xylem is only one possible strategy used predictive ability for lethal water stress, leaf xylem cavitation resistance was only significantly related to species rainfall limits (driest quarter rainfall by conifers to survive low rainfall. Assuming the latter hypothesis within each species’ natural distribution) in derived Cupressaceae (B). Mini- to be more likely (12), we examined the stomatal behavior of our mum leaf conductance to water vapor (gmin), in contrast, was found to conifer sample to determine whether there was evidence of an correlate (r2 = 0.51) with the mean dry quarter rainfall within each species interaction between xylem and stomatal characteristics that distribution (C). In each of the conifer families sampled, only species with < −2 −1 < might explain these observations. gmin 5 mmol m s were found to inhabit dry climates with 120 mm rain in the driest quarter of the year. Stomatal Closure During Water Stress. Unlike xylem vulnerability (Fig. 1B), the efficiency of stomatal closure during sustained (>30 d) water stress was correlated with the rainfall require- for plants growing in dry climates is of particular ecological ments of evergreen conifer species from each family except significance, as it provides the first quantitative character, to Araucariaceae (Fig. 1C). In all species, stomatal closure under the authors’ knowledge, linking plant function with rainfall conditions of sustained soil drying reduced transpiration to sta- requirement. Given that stomatal closure (in evergreen conifers) ble minimum rates 20–30 d after withholding water, but there is the only means of preserving water in the plant as drought was a 30-fold range in minimum leaf water vapor conductances continues, a link between the leakage of water from the leaf and (gmin) among the 42 conifer species examined. Species with a capacity to survive in dry climates is intuitive. Previous studies leaves that were leaky to water vapor under drought stress (high suggest variability in the leakiness of stomata (27, 28), and we gmin) were all native to regions of high rainfall (>120 mm of rain found in a subsample of seven species (from Araucariaceae, in the driest quarter), whereas species with low gmin were always Podocarpaceae, Cupressaceae, and Taxaceae) that between 50% native to drier environments (those experiencing <120 mm of and 95% of the leakage of water through leaves with closed rain in the driest quarter of the year) (Fig. 1C). The existence of stomata occurred through the stomatal surface, presumably −2 −1 an apparent threshold requirement for gmin < 5mmolm s through closed pores (SI Appendix, Table S2). The implication of

14490 | www.pnas.org/cgi/doi/10.1073/pnas.1407930111 Brodribb et al. Downloaded by guest on September 25, 2021 ABA and Stomatal Closure. We found two very distinct responses of foliar ABA accumulation as species were gradually desiccated by withholding water until incipient leaf death (over a mean period of 42 ± 11 d) (Fig. 2). Foliar ABA levels in all species increased after water was withheld (Fig. 2 and SI Appendix, Fig. S2), but in the “peaking-type” (P-type) response, these levels reached maxima over 7–20 d, after which they fell back to levels similar to or below those observed before withholding water. In all P-type − species, initial and final ABA levels were <1,000 ng g 1 fresh weight (FW). In contrast, in species found to have “rising- type” (R-type) responses, foliar ABA levels either continued to rise for the duration of the drought period or plateaued at high − levels. In all R-type species, final ABA levels were >1,500 ng g 1 FW (Fig. 2 and SI Appendix,Fig.S2). As a result of these con- trasting behaviors, foliar ABA levels after 1 month of imposed water stress were very different among species, ranging by more − than 100-fold from a minimum of 169 ng g 1 FW in the P-type − − Fig. 2. Examples of the two different types of foliar ABA dynamics (ng g 1 Juniperus chinensis to 17,660 ng g 1 FW in the R-type species FW) during sustained water stress. Peaking (P-type, red) responses were Cunninghamia lanceolata (SI Appendix,Fig.S2). There was observed in species of Cupressaceae (Cupressus cashmeriana, red triangles) a strong phylogenetic pattern in ABA dynamic response type, and Taxaceae (Cephalotaxus fortunei, red circles) (SI Appendix, Fig. S2). Fo- liar ABA levels in these species fell back to initial, unstressed levels as water with derived Cupressaceae and Taxaceae typically exhibiting stress approached lethal limits. Species with rising (R-type, blue) responses P-type ABA dynamics and low levels of ABA postdrought, and showed a steady increase (or a plateau) in foliar ABA level as water stress basal Cupressaceae, Pinaceae, Podocarpaceae, and Araucariaceae progressed and were observed in all Pinaceae (Cedrus deodara, blue dia- being R-type, with high levels of ABA at the conclusion of the monds), Araucariaceae (Araucaria heterophylla, blue squares), and Podo- imposed water stress (Fig. 3 and SI Appendix, Fig. S3). Ancestral carpaceae (SI Appendix, Fig. S2). All species could be characterized as one or state analysis (30) infers that the R-type ABA dynamic is the other of these two behavioral types.

ancestral condition in Pinophyta, a position supported by the EVOLUTION high levels of ABA and R-type dynamics in water-stressed Ginkgo biloba and Cycas (SI Appendix, Fig. S2). Although P-type this finding is that stomatal pores in the leaf continue to leak ABA dynamics are most common in species from the Calli- water vapor even as leaves are water stressed, but that some troideae and Cupressoideae clades of the Cupressaceae, there species are able to close stomata more efficiently than others. does not appear to be a monophyletic origin of this trait in this Stomatal closure in water-stressed conifers is driven by family because of the presence of clear P-type ABA dynamics in a combination of falling Ψ and a rising concentration of abscisic l the Taxaceae (Fig. 3). acid (ABA), both of which cause the turgor of guard cells sur- rounding stomatal pores to decline and close the pore (29). A Discussion recent study of two conifers from different families indicated Divergent Strategies for Surviving Water Stress. Conserving water a strong divergence in the way these drivers affected stomatal under drought conditions requires stomata to close tightly, a closure, with one species relying heavily on foliar ABA level and process that occurs as guard cell turgor approaches zero. Data Ψ the other on l to achieve stomatal closure under water stress here show efficient stomatal closure to be a prerequisite among (23). To determine whether these different modes of stomatal conifer species able to colonize dry environments, and that closure may explain different patterns of stomatal closure and among these conifers, two divergent means of achieving low drought sensitivity broadly among conifer species, we monitored guard cell turgor, and hence tight stomatal closure, have evolved. foliar ABA levels, Ψl, and transpiration in 42 species of conifers In one group, high levels of foliar ABA accumulated during (SI Appendix, Table S1) subjected to prolonged water stress. water stress exposure (R-type), enabling tight stomatal closure

Fig. 3. Maximum likelihood reconstructions (30) of foliar ABA dynamic type (42 species) and leaf xylem resistance to cavitation (or 50% loss of leaf hydraulic

conductance, ψ50; 58 species). An association between peaking-type ABA dynamics (red on the left) and resistant xylem (yellow and red on the right) is clearly apparent. The converse is also apparent, linking rising-type ABA dynamics (blue on the left) with cavitation vulnerable xylem (blue on the right). This latter syndrome is likely to be the ancestral state for conifers, with multiple transitions in ABA response type and xylem vulnerability, particularly in Taxaceae and Cupressaceae. Conifer phylogeny data were taken from ref. 38

Brodribb et al. PNAS | October 7, 2014 | vol. 111 | no. 40 | 14491 Downloaded by guest on September 25, 2021 (low gmin) (Fig. 4), presumably by maximizing the extrusion of anions from guard cells leading to very low guard cell turgor (31). A second group (those with P-type ABA responses) was found to produce tight stomatal closure despite low levels of foliar ABA during prolonged water stress. In the absence of high levels of ABA, we hypothesized that efficient stomatal closure in this group could only be achieved when guard cell turgor was pulled down by very negative Ψl (23). For these species to be able to reopen stomata and recover after relief from water stress would require leaves and xylem that are able to survive under the desiccated conditions necessary for achieving full stomatal closure. Supporting this hypothesis, we found that the dry forest species with low levels of foliar ABA under stress (P-type) also pos- sessed highly cavitation-resistant xylem and desiccation-tolerant leaves (Fig. 3). These two divergent strategies for enabling the persistence of conifer species in low-rainfall environments are likely to be as- sociated with different plant behaviors in terms of the dynamic responses of transpiration and photosynthesis during exposure to water stress (23). In particular, we found that species with P-type ABA responses also showed delayed stomatal closure during water stress, whereas R-type species closed stomata quickly over a very narrow range of water potentials (SI Appendix, Fig. S4). These different response trajectories of photosynthesis and transpiration during water stress [termed isohydric and aniso- hydric behavior (14)] are thought to represent contrasting eco- logical strategies of water and carbon maintenance under long- term water stress (12). Data here identify a probable origin of these different stomatal behaviors in conifers, mediated by dif- ferent characteristics of ABA metabolism in concert with xylem vulnerability to water stress-induced failure. Our analysis demonstrates how coevolution of xylem and stomata in the Pinales has yielded two distinct strategies for surviving in dry habitats. This marked evolutionary divergence in a group that is renowned for its evolutionary conservatism emphasizes the importance of considering multiple traits when Fig. 4. (A) Bubble plot showing foliar ABA levels in plants exposed to evaluating the potential of plant species to survive drought. One prolonged (>30 d) water stress, plotted against rainfall in the driest quarter ancient pathway in the Pinaceae, Podocarpaceae, Araucariaceae, for each species’ native distribution and minimum stomatal conductance and basal Cupressaceae uses high levels of ABA to close sto- (shown as balloon size). Two pathways lead to minimum water vapor leak- − − mata and maintain homeostasis in leaf water content during age from stomata <5 mmol m 2 s 1 required for survival in low-rainfall drought. Another, more recently evolved, strategy seen in de- environments (<120 mm rainfall in the driest quarter, yellow background). rived Cupressaceae and Taxaceae abandons this dependence The R-type adaptation (blue arrow) is associated with very high levels of on metabolically costly ABA and instead relies on leaf desicca- foliar ABA under severe water stress, whereas species with the P-type ad- tion to passively drive stomatal closure. Although the P-type aptation (red arrow) have low levels of foliar ABA under severe drought strategy also has attendant costs associated with building xylem stress. Leaf xylem vulnerability also makes up part of these two adaptive syndromes (B). Considering only the dry-adapted species (<120 mm dry tissue capable of functioning under extreme water stress (32), season rainfall), we find that P-type species (circled in red) separate very a glance at the dry limits of conifer distribution suggest this may be clearly from R-type species (circled in blue), having lower xylem vulnerability

the most effective strategy for survival in extremely water-limited to cavitation expressed as the ψl at a 50% loss of leaf hydraulic conductance environments. It remains to be seen whether this evolutionary (ψ50) and lower ABA levels after stress. pattern in conifers reflects their intermediate position between hydropassive stomatal control in ferns and hydroactive control of −2 −1 leaf hydration in angiosperms (33), or whether ABA signaling, quanta m s at the leaf surface during the day period) and 23 °C/15 °C xylem, and stress tolerance coevolve in all clades of seed plants. day/night temperatures. Relative humidity was maintained at 60% by a dehumidifier with integrated humidity sensors (ADH-1000, Airrex Portable Extending the ABA-Ψ50 framework to woody angiosperms in the future has the potential to provide a meaningful physiological ap- dehumidifier, Hephzibah Co Ltd). Temperature and humidity were logged for the duration of the experimental period using a HOBO Pro Series data proach for assessing the rainfall requirements of all woody species. logger (Onset). Materials and Methods Leaf hydraulic vulnerability was measured in 58 species of conifer from 46 genera, using sun-exposed branches collected from a range of sources in- Plant Material. Dynamic responses of water loss and ABA in response to cluding native stands, arboreta, or glasshouse-grown individuals (SI Ap- imposed water stress were measured in 42 species of conifer, including pendix, Table S1). Comparisons of material grown under different conditions representatives from 38 genera and all extant families, plus the gymnosperm are considered valid on the basis of a number of conifer studies (34, 35) Ginkgo biloba (SI Appendix, Table S1). All plants were saplings of similar age (3–20 y), ranging in size from 20 cm (shrub species) to 1.5 m. Plants were showing low plasticity in xylem P50 within species. grown in a medium 8:2:1 mix of composted pine bark, coarse river sand, and peat moss in pots sufficiently large to ensure plant survival over a period of Plant Gas Exchange, Foliar ABA Level, and Leaf Water Potential Monitoring 40 d without water (5–20 L, depending on plant size) in the glasshouses of Over Drought Stress. The pots of individuals from each species were dou- the School of Biological Sciences, University of Tasmania, Hobart, Australia. ble-bagged with a combination of plastic and aluminum foil to prevent Growth conditions were 16-h days (supplemented and extended in the evaporative water loss from the soil. Midday (11:30 AM to 13:30 PM) whole- morning and evening by sodium vapor lamps, ensuring a minimum 300–500 μmol plant water loss was quantified gravimetrically, and the collection of tissue

14492 | www.pnas.org/cgi/doi/10.1073/pnas.1407930111 Brodribb et al. Downloaded by guest on September 25, 2021 for assessment of Ψl and foliar ABA level was conducted as described by Hard as Nails, Sally Hansen) were applied to the abaxial (stomatal) surface of Brodribb and McAdam (23). Drought stress was initiated by withholding the leaf and water loss was remeasured after the varnish layers were dried Ψ water, and measurements of plant gas exchange, foliar ABA level, and l (g1). Finally, nail varnish was added to the adaxial leaf surface, and water were performed every 3 d until individuals showed the incipient signs of leaf loss (through the varnish layer) was remeasured (g2). Water loss through the death (an average of 42 ± 11 d standard deviation). To ensure plants were stomatal leaf surface was calculated by applying Fick’s law and assuming not killed by water stress, all plants were rewatered when ∼15% of leaves each leaf surface was composed of a series of unknown resistors to water were observed to have died (turned brown). Rewatering at this point typi- diffusion (ad-/abaxial surface resistance and leakiness of the varnish). An cally resulted in 15–30% leaf mortality observed 1 mo after rewatering. iterative solver (Microsoft Excel) was then used to determine gs of the ad- Droughting beyond this point is very often fatal. axial surface on the basis of known values of gmin, g1, and g2.

Leaf Hydraulic Vulnerability Measurements. Leaf hydraulic measurements Foliar ABA Level Quantification. Foliar ABA extraction, purification, and (Kleaf) were made according to the standard protocol outlined by Brodribb physicochemical quantification by ultra-performance liquid chromatography and Cochard (17). Detached shoots from at least three plants were bench- tandem mass spectrometry with an added internal standard were under- Ψ dried, and Kleaf was determined at intervals of 0.5 MPa ( l) by measuring the taken according to the methods of Brodribb and McAdam (23). rehydration flux of water into leaves. The relationship between Ψl and Kleaf was used to determine the P (Ψ , at 50% loss of K ) from a sigmoidal 50 l leaf Rainfall Estimations from Species Distributions. Geo-referenced collection curve fitted to the pooled data from each species. data for all conifer species were extracted from the Global Biodiversity In- formation Facility (www.gbif.org) and complemented by records from Farjon Minimal Stomatal Conductance Measurements. Whole-plant water loss mea- (36). Climate estimates were made for each point record, using Worldclim surements were converted to stomatal conductances (g ) by converting rel- s (37). The climate records for species were vetted, first by eliminating dupli- ative transpiration to g relative to maximum instantaneous g measured in s s cate records, then for consistency with species distribution descriptions (36), three leaves of each species before drought stress, using an infrared gas and then by comparing Worldclim estimates of altitude with altitudes pro- analyzer [Li6400, Li-Cor; conditions in the leaf cuvette of the infrared gas vided with each site record. Where Worldclim altitudes contrasted with al- analyzer were set to the regulated vapor pressure difference (VPD) of the titude descriptions by more than 300 m, we replaced these records with glasshouse = 1.2 kPa]. Transpiration in all species declined to stable minima estimates from nearby sites with altitudes consistent with the descriptions. (less than 0.5% change per day over 5 d), allowing a mean minimum tran- Tolerance of water stress under natural conditions was quantified as the spiration to be recorded over at least 10 d. This minimum whole-plant ’ transpiration was converted to a percentage of the predrought maximum mean precipitation of the driest quarter within each species natural distri- bution. Finally, we used the 25th percentile of driest quarter rainfall for each and converted to gs. Leaf temperature was assumed to be constant between instantaneous measures of g and whole-plant measures of transpiration. species as a means of biasing the dry end of each species distribution. s EVOLUTION In addition, we measured the distribution of water loss between stomatal and nonstomatal surfaces of leaves subjected to severe water stress to de- ACKNOWLEDGMENTS. The authors gratefully acknowledge John Ross for termine the percentage water loss resulting from stomatal “leakiness.” A advice and assistance in improving ABA extraction methods and Noel Davies for the operation of ultra-performance liquid chromatography equip- subsample of hypostomatic species with accessible bands of stomata were ment. T.J.B. and S.A.M.M. received funding from the Australian Research chosen (seven species from Araucariaceae, Taxaceae, Cupressaceae, and Council (DP120101868, FT100100237, and DE140100946). S.C.V.M. was funded Podocarpaceae), and water loss of excised leaves was measured gravimet- by a visiting scholarship to Tasmania from the Brazilian government rically until a gmin was reached (VPD and temperature were logged during [Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)/ measurements). At this point, multiple coats of clear nail varnish (Advanced Programa de Doutorado Sanduíche no Exterior (PDSE) (Program 6202/13-6)].

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