Salt Tolerance Evolves More Frequently in C4 Grass Lineages

doi: 10.1111/jeb.12320

SHORT COMMUNICATION

Salt tolerance evolves more frequently in C4 grass lineages

L. BROMHAM & T. H. BENNETT Division of Ecology, Evolution and Genetics, Centre for Macroevolution and Macroecology, Research School of Biology, Australian National University, Canberra, ACT, Australia

Keywords: Abstract comparative; Salt tolerance has evolved many times in the grass family, and yet few cer- evolution; eal crops are salt tolerant. Why has it been so difficult to develop crops tol- halophyte; erant of saline soils when salt tolerance has evolved so frequently in PACMAD; nature? One possible explanation is that some grass lineages have traits that phylogeny; predispose them to developing salt tolerance and that without these back- Poaceae; ground traits, salt tolerance is harder to achieve. One candidate background salinity; trait is photosynthetic pathway, which has also been remarkably labile in trait lability. grasses. At least 22 independent origins of the C4 photosynthetic pathway have been suggested to occur within the grass family. It is possible that the evolution of C4 photosynthesis aids exploitation of saline environments, because it reduces transpiration, increases water-use efficiency and limits the uptake of toxic ions. But the observed link between the evolution of C4 photosynthesis and salt tolerance could simply be due to biases in phylogenetic distribution of halophytes or C4 species. Here, we use a phylogenetic analysis to investigate the association between photosynthetic pathway and salt tolerance in the grass family Poaceae. We find that salt tolerance is significantly more likely to occur in lineages with C4 photosynthesis than in C3 lineages. We discuss the possible links between C4 photosynthesis and salt tolerance and consider the limitations of inferring the direction of causality of this relationship.

descended from a few independent origins of salt toler- Introduction ance. Instead, salt tolerance has evolved frequently in a The amount of salt-affected land, currently over 6% of large number of different lineages. A recent study esti- the land surface area, is increasing through agricultural mated that there have been over 70 independent ori- practices and land clearance. Irrigated land, which pro- gins of salt tolerance in the grass family (Bennett et al., duces a third of the world’s food, is particularly prone 2013). Yet, although there is considerable benefit to to salinization: between 20 and 50% of the world’s irri- producing crop plants that can grow on salt-affected gation schemes are salt-affected (Flowers, 2004; Flowers land (Glenn et al., 1999; Rozema & Flowers, 2008), et al., 2010; Munns, 2011). Understanding the evolu- there have been few commercially viable salt-tolerant tion and maintenance of salt tolerance in plants may cereal crops produced (Flowers & Yeo, 1995; Flowers & help to develop strategies for utilizing and managing Flowers, 2005). salt-affected land. Why has it been so difficult to breed salt tolerance There are over 350 naturally halophytic (salt toler- into cereal crops when it has evolved so many times ant) grass species and subspecies. These halophytic within the grass family? There are several possible grasses are not clustered in clades of related species, all explanations (which are not mutually exclusive). Firstly, it may be that salt tolerance is a physiologically costly trait so that it is difficult to develop a productive Correspondence: L. Bromham, Division of Ecology, Evolution and crop plant that can produce commercially viable yields Genetics, Centre for Macroevolution and Macroecology, Research School of Biology, Australian National University, Canberra ACT 0200, while dealing with environmental salt. Secondly, salt Australia. Tel.: +61 2 61259545; fax: 61 2 61255573; tolerance is a genetically complex trait, which may not e-mail: [email protected] present an easy target for breeding programs or genetic

ª 2014 THE AUTHORS. J. EVOL. BIOL. JOURNAL OF EVOLUTIONARY BIOLOGY ª 2014 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 1 2 L. BROMHAM AND T. H. BENNETT

manipulation (Roy et al., 2011). Thirdly, salt tolerance Here, we ask whether C4 photosynthesis is specifically may be more easily acquired with particular back- associated with the evolution of salt tolerance, in order grounds as starting points, and thus will evolve more to shed light on some of the factors that have allowed easily in certain lineages that already have these traits. some grass lineages to adapt to saline environments. It is this third possibility that we wish to examine in this study. Materials and methods One possible background trait that may enhance the capacity to evolve salt tolerance is photosynthetic path- A list of halophytic grasses was taken from Bennett way. The C4 mechanism of carbon fixation is a modi- et al. (2013). Most studies use a standard definition of a fied version of the ancestral (C3) photosynthetic halophyte as any species that can successfully complete pathway, and it has evolved independently over 60 its life cycle in saline conditions similar to those times in angiosperms (Sage et al., 2012), including an encountered in the natural environment, where saline estimated 22–24 gains within the grass family (Edwards conditions are defined as those where the soil solution & Smith, 2010; Grass Phylogeny Working Group II, has an electrical conductivity equivalent to ~80 mM 2012). By increasing the efficiency of carbon fixation, NaCl at saturation, following Aronson (1989). However, C4 plants can reduce photorespiration and thus allow this precise definition can rarely be applied in practice higher water-use efficiency and productivity. Therefore, as the exact level of tolerance is typically known only C4 photosynthesis has been assumed to have advanta- for species that have been closely studied in the labora- ges under conditions that promote photorespiration, tory (Flowers, 2004). In most cases, it is necessary to such as heat, drought, salinity and low atmospheric rely on reports of populations growing in saline condi- CO2 (Sage & Monson, 1999; Sage, 2004; Sage et al., tions in the field (see Bennett et al., 2013). For exam- 2012; Christin et al., 2013). ple, the eHALOPH database (http://www.sussex.ac.uk/ Plants with C4 photosynthesis are often found in salt- affiliates/halophytes) lists some species with specific affected areas, and taxa with C4 carbon fixation appear ranges of soil electrical conductivity, but other species to be overrepresented among halophytes (Aronson, according to discrete categories such as xerohalophyte 1989; Sage & Monson, 1999; Dajic, 2006; Eallonardo (e.g. inland salt desert species) or hydrohalophyte (e.g. et al., 2013). However, the association between photo- tidal swamp or salt marsh species). synthetic pathway and salt tolerance needs to be for- We used the molecular phylogeny published by mally tested within a phylogenetic framework in order Edwards and Smith (2010), which includes 2684 taxa to account for confounding factors (Christin et al., (approximately 20% of all grass species). Two hundred 2009; Osborne & Freckleton, 2009; Taylor et al., 2010). of these taxa were identified as halophytes, following C4 species are nonrandomly distributed in the grass Bennett et al. (2013). To test the generality of patterns, phylogeny (Table 1), with all known C4 species occur- all analyses were run both on the phylogeny of all Poa- ring in the large ‘PACMAD’ clade, which contains the ceae and also on a subtree containing the PACMAD subfamilies Panicoideae, Arundinoideae, Chloridoideae, clade only. Information on the photosynthetic path- Micrairoideae, Aristidoideae and Danthonioideae (Chri- ways of all grasses in the phylogeny was also taken stin et al., 2009, 2013). So even if a disproportionate from Edwards and Smith (2010). To test that our number of halophytic grasses use C4 photosynthesis, results are not the result of sampling bias in Edwards it is unclear whether this is due to a specific association and Smith data set, we also combined a list of all grass between the two traits, or because there is some other genera containing halophytes (see Bennett et al., 2013; feature of the PACMAD clade that increases the likeli- Table S2) with the complete genus-level phylogeny and hood of evolving salt tolerance (Edwards et al., 2007). photosynthetic pathway data set of Bouchenak-Khelladi et al. (2010). A randomization test was conducted to test whether more halophytes occur in C4 clades than expected by Table 1 The halophytes include in this study, as a proportion of chance. A null distribution of the expected number of the number of species represented in the phylogeny of Edwards halophytes occurring in C4 lineages was generated by and Smith (2010). There are proportionally more halophytes in randomly reassigning character states (200 halophytic/ the PACMAD clade (Panicoideae, Arundinoideae, Chloridoideae, 2484 nonhalophytic) across the tips of the phylogeny, Micrairoideae, Aristidoideae, Danthonioideae), which contains then counting the number of these that fell on C4 taxa. both C3 and C4 taxa, than there are in the BEP clade (Bambusoideae, Ehrhartoideae, Pooideae), which contains only The randomization was repeated 10 000 times. We then compared the observed number of halophytes in C3 lineages. C3 and C4 clades to this null distribution. The associa- Clade Species Halophytes Proportion tion between photosynthetic pathway and salt tolerance was deemed to be significantly different from BEP C only 1526 87 0.057 3 chance when the observed number of halophytes was PACMAD C3 and C4 1143 118 0.103 greater or less than in 95% of randomizations.

ª 2014 THE AUTHORS. J. EVOL. BIOL. doi: 10.1111/jeb.12320 JOURNAL OF EVOLUTIONARY BIOLOGY ª 2014 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY Salt tolerance evolves more frequently in C4 grasses 3

The correlation between salt tolerance and photosyn- kappa parameter was selected with the best fit to equal thetic pathway was tested using Pagel’s (1994) correla- branch lengths (see Bennett et al., 2013). The rate of tion analysis for discrete characters, as modified by evolution of salt tolerance in C3 and C4 clades was Maddison and Maddison (2006). We analysed the tran- compared using the estimated rates of these transitions. sition rate between two states for the two characters that is between salt tolerant and salt sensitive, and Results between C3 and C4. This method estimates the fit of a Markov model where the rate of change in each char- The phylogenetic distribution of salt tolerance and C4 acter is independent of the state of the other and com- photosynthesis is shown in Fig. 1. pares it to the fit of a correlated, state-dependent Significantly more halophytes occur in C4 lineages model, where the rate of transition from salt sensitive than if salt tolerance was random with respect to pho- to salt tolerant is dependent on the type of photosyn- tosynthetic pathway, both across the whole Poaceae, thetic pathway. If the state-dependant model fits signif- and within the PACMAD clade (Table 2). For example, icantly better, then this suggests that the evolution of there are only three identified C3 halophytes in the the two traits is correlated. The significance of the like- PACMAD clade of the species-level phylogeny, where lihood difference between the models is estimated by we would expect 20–60 halophytes to occur in the C3 comparison with simulated data. We used the ‘Pagel94 lineages of the PACMAD if salt tolerance was randomly correlation analysis’ function of mesquite, optimizing distributed on the phylogeny (Fig. 2). likelihoods with ten iterations and estimating signifi- Because not all grass species are included in our cance from 1000 simulations (Maddison & Maddison, analysis, we checked that our results were not an arte- 2006) using a maximum likelihood omnibus test as fact of undersampling halophytes in C3 clades. Using described by Pagel (1994). The optimal scaling of the the list of all grass genera containing halophytes

Fig. 1 Distribution of halophytes (salt-tolerant species) on the grass family, mapped onto the evolutionary pattern of C4 photosynthesis estimated by Edwards and Smith (2010).

ª 2014 THE AUTHORS. J. EVOL. BIOL. doi: 10.1111/jeb.12320 JOURNAL OF EVOLUTIONARY BIOLOGY ª 2014 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 4 L. BROMHAM AND T. H. BENNETT

Table 2 Results of the correlation analyses conducted on both the whole grass family (Poaceae) and on the PACMAD clade, comparing the ﬁt of a model where the evolution of C4 and salt tolerance is correlated with a model to one where they are uncorrelated. The estimates are log likelihood (ÀlnL) of the correlated and uncorrelated models; difference in likelihoods (DlnL) of the two models; P values derived from 1000 simulations; estimated rate of gain of salt tolerance in C3 and C4 clades.

Clade ÀlnL (uncorrelated) ÀlnL (correlated) DlnL P C3 Rate C4 Rate

Poaceae 822.2 798.5 23.7 < 0.001 0.023 0.074 PACMAD 466.0 438.0 28.0 < 0.001 0.010 0.075

(Bennett et al., 2013) and the complete genus-level approach, we show that halophytic grasses are signifi- phylogeny and photosynthetic pathway data set of cantly more likely to occur in lineages with C4 photo- Bouchenak-Khelladi et al. (2010), we found only one synthesis than expected if salt tolerance was random C3 halophytic species from the PACMAD that was not with respect to photosynthetic pathway. Salt tolerance included in our species-level analysis (Rytidosperma appears to have evolved repeatedly within many C4 rufum), but many more C4 halophytes not in the spe- grass clades, with salt tolerance arising at a more fre- cies-level phylogeny. This suggests that undersampling quently in C4 lineages than in C3 groups. In fact, salt of C3 halophytes has not biased this analysis. tolerance has evolved relatively rarely in C3 lineages If the overrepresentation of halophytes in the PAC- outside of the ‘core’ Pooideae (Edwards & Smith, MAD clade was due to some feature of PACMAD other 2010). But the observation of this significant correlation than photosynthetic pathway, then we would expect to does not, by itself, establish a direct causal connection observe many salt-tolerant C3 species in the PACMAD. between the two traits, nor the direction of causality. However, there are significantly fewer C3 halophytes in Does salt tolerance favour the evolution of C4 photo- the PACMAD (observed = 3) than expected on the synthesis, does C4 promote the evolution of salt toler- basis of chance (expected > 20: Fig. 2). Instead, almost ance, or are both traits linked indirectly, for example, all C3 halophytes on the phylogeny are found in the by tending to co-occur in taxa adapted to open and arid BEP clade (Bambusoideae, Ehrhartoideae, Pooideae), habitats? particularly in the core Pooideae. Therefore, we can Taken at face value, our results could be interpreted conclude that PACMAD lineages with C4 photosynthe- as evidence that, within the grass family, C4 lineages sis are more likely to contain salt-tolerant species than have been more likely to develop salt tolerance than C3 C3 lineages in the PACMAD, a pattern confirmed by lineages. There are far more origins of salt tolerance the correlation analyses. within the grasses (around 70) than origins of C4 The correlation analyses indicated a significant associ- (around 20), and the gains of salt tolerance tend to be ation between salt tolerance and C4 photosynthesis on distributed near the tips of the phylogeny and are the species-level phylogeny of grasses (Table 2). The therefore relatively recent (Bennett et al., 2013). This correlated model, which allows the rate of transition pattern is compatible with the hypothesis that the from salt sensitive to salt tolerant to vary according to adoption of C4 photosynthesis allowed expansion into the state of the photosynthetic pathway, fits the arid and saline habitats (Stromberg, 2011); therefore, data significantly better than the uncorrelated model, C4 lineages were more likely to produce halophytic spe- for both the whole Poaceae family (DlnL = 23.7, cies (Osborne & Freckleton, 2009). C4 lineages may be P < 0.001) and for the PACMAD clade (DlnL = 28.0, an advantageous starting point for the evolution of salt P < 0.001). For the phylogeny of all Poaceae, the best- tolerance, given that the greater water-use efficiency of fitting model gave an estimate of the rate of gain of salt C4 photosynthesis lowers the flux of water and salts tolerance in C4 clades that was approximately three through the plant per growth unit, which can reduce times higher than the estimated rate of gain of salt tol- the amount of salt that a plant has to exclude, compart- erance in C3 clades. The analysis on only the PACMAD mentalize, or secrete for a given amount of carbon clade gave even more pronounced results, with an esti- fixation (Sage, 2001). mated rate of gain of salt tolerance in C4 clades approx- An alternative explanation of this link between pho- imately seven times higher than the estimated rate of tosynthetic pathway and salt tolerance is that lineages gain of salt tolerance in C3 clades (Table 2). adapted to saline environments may be more likely to evolve C4 photosynthesis. It has been argued that the Discussion adaptation to harsh environments, such as arid or saline habitats, has promoted selection for C4 photosyn- Although it has often been proposed that plants with thesis, by favouring traits that reduce ionic stress C4 photosynthesis are more likely to be able to adapt to through decreasing transpiration rates. For example, live in saline habitats, this hypothesis has not been Kadereit et al. (2012) estimated that there have been 10 robustly tested before. Using a broad-scale comparative origins of C4 photosynthesis in the Chenopodiaceae,

ª 2014 THE AUTHORS. J. EVOL. BIOL. doi: 10.1111/jeb.12320 JOURNAL OF EVOLUTIONARY BIOLOGY ª 2014 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY Salt tolerance evolves more frequently in C4 grasses 5

(a) Expected number of a clade of the Amaranthaceae containing many halo- C4 halophytes in grass Observed = 118 phytes, but probably only one ancestral origin of salt phylogeny P < 0.0001 tolerance. However, as with this study, inference of direction of causality may be conflated with differences in lability between traits. 2000 2500 3000 Although we can be confident of a significant evolutionary link between salt tolerance and C4 photosyn-

Frequency thesis, the direction of the relationship cannot be easily inferred from the phylogenetic pattern alone. This is because different patterns of trait lability could create the false impression of a directional causal relationship.

0 500 1000 1500 If salt tolerance is relatively labile in grasses (see Ben- nett et al., 2013), then although most extant halophytic 30 40 50 60 70 80 lineages have relatively recent origins, we can expect (b) Expected number of that salt tolerance has been gained and lost throughout C4 halophytes in the history of the grasses. Because we cannot directly PACMAD clade Observed = 118 P < 0.0001 reconstruct past evolution and loss of salt tolerance, we cannot rule out that C4 photosynthesis has typically arisen in lineages growing under saline conditions, then some of those C4 lineages lose salt tolerance or move to different habitats. So the order of acquisition may be an artefact of trait lability: C4 photosynthesis may appear Frequency deeper in the tree, and therefore to have been gained ﬁrst, because it evolves less often and is lost less often than salt tolerance. Regardless of the direction of causality of the link

0 500 1000 1500 between photosynthetic pathway and salt tolerance, these two hypotheses – C4 promotes evolution of salt 60 70 80 90 tolerance vs salt tolerance promoting evolution of C4 – (c) Expected number of are not mutually exclusive. Ongoing adaptation to C3 halophytes in allow exploitation of open, arid and saline habitats may PACMAD clade have resulted in the promotion of both salt tolerance Observed = 3 and C4 photosynthesis. Although responses to salinity P < 0.0001 are distinct from responses to aridity, mechanisms of tolerance to these stresses have much in common (Munns, 2002; Des Marais & Juenger, 2010), so it is possible that adaptation to aridity provides enabling conditions that promote salinity tolerance (or vice Frequency versa). It is also possible that an indirect link between C4 and salt tolerance could arise through ecological prefer- ence or biogeographic patterns. C4 grasses in the PAC- MAD are more frequently found in open and arid 0 500 1000 1500 habitats than C3 PACMAD species (Osborne & Freckl- 20 30 40 50 eton, 2009; Pau et al., 2012). As highly saline soils do not generally support closed-canopy vegetation (man- Fig. 2 Distribution of expected numbers of halophytes in each grove forests being a notable exception), halophytes clade if salt tolerance occurred independently of photosynthetic will also tend to occur in open habitats. Saline soils are pathway. In each case, the observed value is not contained in the distribution of expected values, so the null model of chance also particularly prevalent in arid and semi-arid regions, association between salt tolerance and photosynthetic pathway so the large number of halophytes in C4 clades may be can be rejected. The test was repeated on both the whole grass explained by their inhabiting the general areas where phylogeny and on the subclade containing the PACMAD families salinity is more prevalent. Edwards and Donoghue

(see Materials and methods for details). Because all C4 halophytes (2013) point out that although the biased frequencies occur within the PACMAD clades, the observed number of C4 of transitions to C4 across the grasses may be due to halophytes is the same for both the whole family and the anatomical enablers, because the large bundle sheath PACMAD. cells of PACMAD grasses could give them a natural advantage over in evolving C4 photosynthesis, it may

ª 2014 THE AUTHORS. J. EVOL. BIOL. doi: 10.1111/jeb.12320 JOURNAL OF EVOLUTIONARY BIOLOGY ª 2014 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 6 L. BROMHAM AND T. H. BENNETT

be also the case that Pooid grasses tend to be distributed C4 photosynthesis. There is a growing effort to engineer in cooler climates, and there is less advantage to evolv- the C4 pathway into C3 crop species to increase their ing C4 photosynthesis than there is for the PACMAD yields (Sage & Zhu, 2011; von Caemmerer et al., 2012). lineages (Edwards & Still, 2008; Edwards & Donoghue, Hibberd et al. (2008) stated that ‘although generating 2013). C4 rice is extremely ambitious, the polyphyletic evolu- However, adaptation to open, arid conditions does tion of C4 photosynthesis provides cause for optimism’. not itself appear to be sufficient for a group to develop The evolutionary lability of both C4 photosynthesis and salt tolerance. For example, the subfamily Danthonioi- salt tolerance in grasses, and the apparent link between deae contains nearly 300 species of tussock and pampas the two, might be considered encouraging, even if engi- grasses, found mainly in the Southern Hemisphere neering C4 photosynthesis or salt tolerance in commer- (Linder et al., 2010). Many species in this subfamily are cially viable crops may be more difficult than some found in open and relatively arid habitats (Bouchenak- have hoped (e.g. Flowers, 2004; Flowers & Flowers, Khelladi et al., 2010; Edwards & Smith, 2010), but the 2005; Zhu et al., 2010). subfamily shows a paucity of halophytes. Across the angiosperms, there are other examples of arid-adapted Acknowledgments groups that have rarely evolved salt tolerance, such as the Proteaceae and Cactaceae (Flowers et al., 2010). We are grateful to Colin Osborne and co-authors for Conversely, not all halophytes occur in open, arid envi- providing us with data on photosynthetic pathways, ronments, for example plants adapted to coastal salt Erika Edwards and co-authors for providing the phylog- marshes and mangrove forests. However, the potential eny, and to Rana Munns, Tim Flowers, Haris Saslis- for reduced transpiration rates in C4 plants may be an Lagoudakis, Camile Moray, Marilyn Ball, Susanne von advantage in salt-affected habitats even where water is Caemmerer and Bas Bruning for helpful feedback on not limited, as it may limit the physiological stress of the manuscript. osmotic adjustment. It is interesting to note that some C4 plants require References small amounts of Na+ for growth (Brownell & Cross- land, 1972), and so do not thrive in the absence of Na+ Aronson, J.A. 1989. HALOPH: A Data Base of Salt Tolerant Plants (Subbarao et al., 2003). Some C4 plants can use sodium of the World. Office of Arid Land Studies, University of ions as osmoticum to allow rapid grown under saline Arizona, Tucson, Arizona. conditions (Kronzucker et al., 2013). Sodium ions can Bennett, T.H., Flowers, T.J. & Bromham, L. 2013. Repeated also play a role in the concentration of CO in C evolution of salt-tolerance in grasses. Biol. Lett. 9: 20130029. 2 4 Bouchenak-Khelladi, Y., Verboom, G.A., Savolainen, V. & physiology through Na+-coupled pyruvate transport in Hodkinson, T.R. 2010. Biogeography of the grasses (Poa- chloroplasts (Furumoto et al., 2011). However, the + ceae): a phylogenetic approach to reveal evolutionary his- requirement for Na for growth is not universal in C4 tory in geographical space and geological time. Biol. J. Linn. plants, and some C4 grasses, including maize and sugar- Soc. 162: 543–557. cane, show no growth benefits from presence of Brownell, P. & Crossland, C. 1972. The requirement for sodium (Subbarao et al., 2003). sodium as a micronutrient by species having the C4 dicar- Further studies are required to tease apart the inter- boxylic photosynthetic pathway. Plant Physiol. 49: 794– 797. correlation of aridity, salinity, C4 photosynthesis and salt tolerance. It would also be interesting to see von Caemmerer, S., Quick, W.P. & Furbank, R.T. 2012. The whether C photosynthesis is more commonly associ- development of C4 rice: current progress and future chal- 4 lenges. Science 336: 1671–1672. ated with the evolution of particular strategies of salt Christin, P.A., Salamin, N., Kellogg, E.A., Vicentini, A. & Bes- tolerance. For example, salt-tolerant grasses may rely nard, G. 2009. Integrating phylogeny into studies of C4 vari- more heavily on salt exclusion than dicotyledonous ation in the Grasses. Plant Physiol. 149: 82. halophytes (Glenn et al., 1999), and it could be that C4 Christin, P.-A., Osborne, C.P., Chatelet, D.S., Columbus, J.T., photosynthesis is particularly beneficial to this strategy Besnard, G., Hodkinson, T.R. et al. 2013. Anatomical ena- of tolerance and less beneficial to other strategies such blers and the evolution of C4 photosynthesis in grasses. Proc. as salt accumulation. This could be tested by finding Natl. Acad. Sci. 110: 1381–1386. the correlation between specific salt tolerance traits and Dajic, Z. 2006. Salt stress. In: Physiology and Molecular Biology of the C pathway, both within the grass family, as well Stress Tolerance in Plants (K.V. Madhava Rao, A.S. Raghaven- 4 – as in other families. dra, K. Janardhan Reddy, eds), pp. 41 99. Springer, Dor- drecht, the Netherlands. The significant correlation between C and salt toler- 4 Des Marais, D.L. & Juenger, T.E. 2010. Pleiotropy, plasticity, ance in naturally occurring grass species suggests that and the evolution of plant abiotic stress tolerance. Ann. N. Y. C4 photosynthesis may provide advantages to the Acad. Sci. 1206:56–79. development of plant varieties that can grow in salt- Eallonardo, A.S., Leopold, D.J., Fridley, J.D. & Stella, J.C. affected areas. It is interesting to contrast the evolution- 2013. Salinity tolerance and the decoupling of resource axis ary and agricultural development of salt tolerance and plant traits. J. Veg. Sci. 24: 365–374.

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