Biology Research

B R I E F C O M M U N I C AT I O N

doi:10.1111/evo.12833

Increased susceptibility to fungal disease accompanies adaptation to drought in Brassica rapa Niamh B. O’Hara,1,2,3,4 Joshua S. Rest,2 and Steven J. Franks3

1Jacobs Technion-Cornell Institute, Cornell Tech, New York, New York 10011 2Department of Ecology and Evolution, Stony Brook University, Stony Brook, New York 11794 3Department of Biology, Fordham University, Bronx, New York 10458

4E-mail: [email protected]

Received November 20, 2014

Accepted November 23, 2015

Recent studies have demonstrated adaptive evolutionary responses to climate change, but little is known about how these re-

sponses may influence ecological interactions with other organisms, including natural enemies. We used a resurrection experiment

in the greenhouse to examine the effect of evolutionary responses to drought on the susceptibility of Brassica rapa plants to a

fungal pathogen, Alternaria brassicae. In agreement with previous studies in this population, we found an evolutionary shift to

earlier flowering postdrought, which was previously shown to be adaptive. Here, we report the novel finding that postdrought

descendant plants were also more susceptible to disease, indicating a rapid evolutionary shift to increased susceptibility. This was

accompanied by an evolutionary shift to increased specific leaf area (thinner leaves) following drought. We found that flowering

time and disease susceptibility displayed plastic responses to experimental drought treatments, but that this plasticity did not

match the direction of evolution, indicating that plastic and evolutionary responses to changes in climate can be opposed. The

observed evolutionary shift to increased disease susceptibility accompanying adaptation to drought provides evidence that even

if populations can rapidly adapt in response to climate change, evolution in other traits may have ecological effects that could

make species more vulnerable.

K E Y W O R D S : Alternaria brassicae, drought, flowering time, rapid evolution, resurrection approach.

Ongoing changes in climate, including warming and altered pre-

cipitation, have been increasing drought severity and frequency

over the past 50 years (IPCC 2014). These changes in climate are

having widespread effects in many populations, including rapid

evolutionary shifts in traits such as phenology (Parmesan and

Yohe 2003). Studies in the emerging field of eco-evolutionary

dynamics suggest that such rapid evolutionary responses may po-

tentially influence ecological interactions (Pelletier et al. 2009;

Dargent et al. 2013), but our understanding of the ecological ef-

fects of evolutionary responses to climate change remains limited.

Although adaptive evolutionary responses could potentially help

populations cope with climate change (Penuelas and Filella 2001;

Hoffmann and Sgro 2011; Franks et al. 2014), these benefits could

be lost if the evolutionary shifts also result in increased suscepti-

bility to predation or disease. This would be particularly likely if

there are trade-offs between responses to increased abiotic stress

and the ability to defend against natural enemies. Assessing the

ecological effects and potential costs of climate change adaptation

is critical for making predictions about the full effects of climate

change.

Here, we examine the evolution and plasticity of multiple

plant traits and the potential ecological effects of adaptation in a

unique system in which a rapid evolutionary response to a change

in climate has been demonstrated. Prior research found a rapid

adaptive evolutionary shift to earlier flowering in response to a

change in climate (drought) in southern California populations

of the annual plant Brassica rapa (Franks et al. 2007; Franks

and Weis 2008). In the current study, we investigate whether

2 4 1 C© 2015 The Author(s). Evolution C© 2015 The Society for the Study of Evolution. Evolution 70-1: 241–248

B R I E F C O M M U N I C AT I O N

this adaptive evolutionary change may have ecological effects

by exploring plant susceptibility to a fungal pathogen. Altered

susceptibility is particularly likely because the early flowering

plants, which are able to escape drought, allocate resources to

rapid growth and development (Franks 2011), potentially leaving

fewer resources available for defense. We hypothesized that post-

drought descendant plants would show greater disease suscepti-

bility than predrought ancestral plants, with the drought causing

the evolution of increased susceptibility as a byproduct of selec-

tion for earlier flowering. We focused on defense against a fungal

pathogen, Alternaria brassicae, which was commonly observed

in our field sites with 21.9% (±21.1) to 35.2% (±32.7) of B. rapa tissue in quadrats sampled displaying symptoms (O’Hara

et al. 2016). This fungus is also known to have important effects

on both wild populations and agricultural varieties of crucifers

(Tewari and Conn 1993). We used a resurrection approach (Franks

et al. 2008), measuring pathogen response in ancestral predrought

(seeds field-collected in 1997) and descendant postdrought (seeds

field-collected in 2004) B. rapa populations grown under the same

conditions in the greenhouse. We conducted a full-factorial ex-

periment with the two plant populations (ancestral and descen-

dant), two levels of drought treatment (well watered and drought

stressed), and two levels of fungal inoculation (inoculated and

noninoculated control). We assessed phenotypes including flow-

ering time, disease susceptibility, and specific leaf area (SLA) to

determine how adaptive evolution in response to drought in B.

rapa affected disease susceptibility.

Methods STUDY SYSTEM

The plant-pathogen system we used is the foliar fungal pathogen

A. brassicae, which causes Alternaria black spot in its host B. rapa

L. (Brassicaceae, field mustard) (Conn et al. 1990). A. brassicae

is a necrotrophic fungus that causes damping off, leaf spots, de-

foliation, and reduced seed yield in B. rapa (Tewari 1991; Koike

et al. 2006). Brassicas have multiple lines of defense against Al-

ternaria fungi, including a waxy cuticle that forms a barrier to

invasion (Tewari and Skoropad 1976) and induced defenses upon

successful invasion, governed by multiple genes, including phy-

toalexins, which may impart partial, but not total resistance to the

disease in B. rapa (Nowicki et al. 2012). Because B. rapa is an

important crop species (bok choi, napa cabbage, oilseed, turnip,

polish canola), its response to this costly and destructive pathogen

has been extensively studied in agriculture (Rotem 1994; Meena

et al. 2010).

B. rapa PROPAGATION

Previous to this study, a large number of seeds (>10,000) were

collected from ripened seedpods (siliques) along a transect in a

natural population of B. rapa located on the University of Cal-

ifornia Irvine campus in May of 1997 (ancestors) and June of

2004 (descendants). The temporally distinct ancestral and de-

scendant populations are hereafter referred to as our populations.

Plants were grown for a generation (about 90 days) and crossed

within-population under greenhouse conditions to reduce mater-

nal effects (Franks et al. 2007). These F1 plants were crossed

within-population prior to this study to reduce storage effects and

the F2 (refreshed) generation was used in the current study. For all

crosses, at least 500 plants per population were crossed at random

once they started flowering, using a feather to transfer pollen, and

visiting each plant at least two times every 3 days.

A. brassicae CULTIVATION

B. rapa tissue infected with A. brassicae was collected from

Bodega Bay, California. This collection site is distant (702 km)

from our natural Brassica populations to avoid the potential issue

of coevolution affecting differential disease susceptibility in an-

cestral versus descendant plants. A. brassicae fungal spores were

isolated from the plant tissue and identified by the Oregon State

University Plant Clinic. Spore plugs were grown on carrot dex-

trose agar plates for one week followed by a week on carrot agar

plates under 12 hours of light and 12 of dark to encourage sporula-

tion. Fresh spores were collected the day of inoculation, strained

through gauze to remove hyphae, and adjusted to a concentra-

tion of 1 × 106 spores/ml in 0.05% Tween. All fungal work was conducted in sterile conditions, and was permitted under APHIS

license #P526P-11-00130.

EXPERIMENTAL DESIGN

Using B. rapa F2 seeds, we conducted a greenhouse experiment

(from February 18th to June 17th, 2012) growing 288 ances-

tral and 288 descendant plants from seed. Both populations were

subjected to a full-factorial combination of a pathogen treatment

(mock inoculated or inoculated with spores) and a drought treat-

ment (well watered or drought stressed).

For cultivation, seeds were planted individually in separate 8

× 8 × 13 cm pots filled with Sunshine Mix #1 growth media (Sun Gro Horticulture, Vancouver, BC, Canada), with 1.4 g of slow re-

lease 14-14-14 Osmocote fertilizer and supplemented with Mir-

acle Gro All Purpose 20-20-20 fertilizer weekly during watering

(3.0 g/l) (Scotts, Marysville, OH, USA). To avoid room position

effects, plants were moved in blocks among randomized coordi-

nates in the greenhouse every 5 days. Blocks were small (about

eight plants) and included both populations. Inoculated plants

were kept separate from control plants to avoid cross-infection.

Light hours were gradually lengthened from 12 to 14 hours to

mimic the growing season. Because B. rapa is self-incompatible,

plants were hand pollinated between randomized pairs of plants

every three days, once they started flowering. All open flowers

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were pollinated. All plants were watered daily to saturation for

two weeks to allow establishment. After two weeks, we began the

drought and inoculation treatments.

Plants that received a drought treatment were watered to

saturation every 4 days. Plants that did not receive the drought

treatment continued to be watered to saturation daily. Soil mois-

ture was monitored using a Field Scout TDR 100 Soil Moisture

Meter (Spectrum Technologies). The moisture level in the soil of

drought treated plants was significantly lower than well-watered

plants (wet = 28.57% soil moisture (±0.30), dry = 21.55% soil moisture (±0.22), F = 351.7, p < 0.001). This drought treatment was designed to mimic field conditions based on field observa-

tions and precipitation records of the study population, which was

characterized by a wet period pre- and immediately postgermina-

tion followed by limited precipitation, rather than a sudden stop

in rain (Franks et al. 2007). Using this study design, plants were

kept alive but also experienced a drought treatment by infrequent

watering.

Plants were inoculated with A. brassicae by wounding 2-

week-old leaves (one leaf per plant and two wounds per leaf) with

a sterile pipette tip and placing 10 µl of a fresh spore solution on

each wound. Control plants were wounded and treated with 10 µl

of 0.05% Tween. We wounded the leaves to inoculate our plants

because A. brassicae enters leaves through wounds as well as

through stomata and by enzymatically degrading the cuticle and

cell wall and forming specialized penetration structures (Tsuneda

and Skoropad 1978). Immediately following inoculation, plants

were kept at 90% humidity for 3 days and then placed at ambient

humidity and either well watered or drought treated (as described

above). High humidity following inoculation is standard protocol

in plant pathology studies because it is known to encourage spore

germination. These conditions also mimic field conditions for

our study population that experience more moisture early in the

growing season and a high incidence of A. brassicae infection

(O’Hara et al. 2016).

TRAIT MEASUREMENTS

Host susceptibility was assessed in terms of disease severity, with

plants showing greater damage scored as more susceptible. The

disease severities of the leaves for a subset of 277 randomly se-

lected plants, including both noninoculated control and inoculated

plants, were scored 21 days postinoculation, using a visual index

(Fig. S1) that ranged from 1 to 10 based on the amount of chloro-

sis and necrosis (Buchwald and Green 1992). Generally, disease

severity scores were independently verified by two researchers

who were blinded to whether they were assessing ancestral or

descendant plants. Infected leaves displayed a highly significant

increase in disease severity (one-way ANOVA comparing inoc-

ulated vs. control plants: inoculated mean = 4.62 (±0.20), non-

inoculated mean = 3.64 (±0.16), F1,117 = 41.34, p < 0.001), demonstrating the efficacy of this treatment.

We quantitatively validated our visual index and the efficacy

of the inoculation with a detached leaf assay of 50 leaves. Prior to

inoculation, fully expanded leaves were detached from plants and

placed in petri dishes on filter paper premoistened with distilled

water and inoculated following the same procedure previously

described. Four days postinoculation, leaves were cleared, stained,

and visualized through a microscope. Leaves were cleared using

a 1:3 acetic acid to ethanol solution and shaken overnight at a

low speed, followed by a 1:5:1 acetic acid, ethanol, and glycerol

solution. After rinsing in water, leaves were boiled for 3 minutes

in a solution of 5% Parker black ink and distilled white vinegar,

and then destained using water that was acidified with a few

drops of vinegar, followed by a 5% vinegar wash (Vierheilig et al.

1998). The number of spores invading leaf tissue was counted at

100× magnification. Infected, stained leaves had an average of 9.5 (±8.7) spores per wound, while uninfected plants were free of symptoms and spores. We also found that spore counts were

correlated with the disease severity scores (Pearson correlation: r

= 0.784, p = < 0.001). Plants were monitored daily and the date of flowering was

recorded for all 527 plants that germinated. The experiment was

conducted until all plants senesced. Specific leaf area (SLA), the

ratio of the light capturing surface area of a leaf per unit of dry

leaf mass (Milla and Reich 2007), was also measured. SLA is of-

ten altered in response to stress and is informative about resource

allocation (Cornelissen et al. 2003). To calculate SLA, the newest

fully expanded leaf was collected from a randomly selected subset

of 280 plants 58 days postplanting, scanned, desiccated with silica

beads, and then weighed. Leaf area in scanned images was mea-

sured using ImageJ (Schneider et al. 2012). SLA was calculated

by dividing the area of each leaf by its dry weight.

DATA ANALYSIS

To determine if evolutionary shifts in plant traits (flowering time,

disease susceptibility, and SLA) occurred, we compared ancestral

and descendant plants, following the resurrection protocol (Franks

et al. 2008). We tested for differences in trait means under all

treatments using a three-way ANOVA, with population (ancestor

or descendant), pathogen treatment (fungal or mock inoculated),

drought treatment (well watered or drought stressed) and their in-

teractions as fixed effects. A two-way ANOVA was used to test for

an effect of population and drought treatment on disease suscep-

tibility since only inoculated plants display disease susceptibility.

We then tested two hypotheses using one-way ANOVAs: (1) traits

(flowering time, disease susceptibility, and SLA) evolved in this

population, which we tested by comparing trait means of ancestors

to descendants for each trait within inoculation and drought treat-

ments, and (2) traits displayed plasticity in response to a drought

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treatment in the greenhouse, which we tested by comparing trait

means of wet to dry treated plants within inoculation treatment and

temporal populations. One-way ANOVAs were used because they

are direct tests of these a priori hypotheses. Trait values (flowering

time, disease susceptibility, and SLA) were dependent variables,

with each treatment tested (population, inoculation, and drought

treatment) modeled as fixed effects in specific analyses. For all

models of disease susceptibility, only data collected for inoculated

plants were used. All analyses were conducted on transformed

data (Table S1) using R 3.0.1 stats package (R Core Team 2013).

Results EVOLUTION OF FLOWERING TIME AND DISEASE

SUSCEPTIBILITY

We found evidence for the rapid evolution of earlier flowering,

with population significantly affecting flowering time (three-way

ANOVA; Table 1). We also found the same pattern of descen-

dants flowering earlier than ancestors in each treatment, but this

shift was only significant under the well-watered/noninoculated

condition (ANOVA: F1, 131 = 5.278, p = 0.023; Fig. 1A). We found evidence for an evolutionary shift to greater

pathogen susceptibility (two-way ANOVA; Table 1). When we

analyzed each treatment separately, we also found that descen-

dants were more susceptible to the pathogen than the ancestors

under both well watered/inoculated (ANOVA: F1, 67 = 16.25, p < 0.001; Fig. 1B) and drought treated/inoculated (ANOVA:

F1, 65 = 5.302, p = 0.025; Fig. 1B) conditions. Thus the evolution- ary shift to earlier flowering was accompanied by an evolutionary

increase in disease susceptibility.

We found evidence for the rapid evolution of increased

SLA (three-way ANOVA; Table 1). We also found the same

pattern of descendants having greater SLA than ancestors in

each treatment, although this was only significant under the well

watered/noninoculated condition (ANOVA: F1, 69 = 4.160, p = 0.045; Fig. 1C).

PLASTIC RESPONSES TO DROUGHT

We found a plastic response in flowering time to water avail-

ability, based on a significant effect of the drought treatment

(three-way ANOVA; Table 1). However, the pattern among the

different treatment groups was not consistent. Flowering time was

significantly earlier for well watered versus drought stressed an-

cestral/inoculated plants (ANOVA: F1, 130 = 5.360, p = 0.022) and descendant/inoculated plants (ANOVA: F1, 133 = 6.761, p = 0.010), but later, although nonsignificantly, for well-watered versus drought stressed ancestral/noninoculated plants, while

descendant/noninoculated plants were unaltered by watering

(Fig. 1A).

Disease susceptibility showed a plastic response to the

drought treatment (two-way ANOVA; Table 1). We analyzed each

treatment separately and found that disease susceptibility was not

affected by watering for ancestral/inoculated plants, but that de-

scendant/inoculated plants were significantly more susceptible to

disease under well-watered conditions (ANOVA: F1, 64 = 8.60, p = 0.005; Fig. 1B).

There was evidence for plasticity of SLA in response to water

availability based on a significant watering treatment effect (three-

way ANOVA; Table 1). However, one-way ANOVAs within each

treatment group showed no significant plastic response to wa-

tering. Instead we saw a consistent but nonsignificant pattern of

lower SLA in the wet treatment for all groups (Fig. 1C).

Discussion In this study, we found that a natural population of B. rapa that

evolved to flower earlier also evolved increased susceptibility to

disease and greater SLA (thinner leaves) within seven generations

during the course of a natural drought (Fig. 1; Table 1). This work

adds to a growing body of evidence that rapid adaptive evolution

can occur in natural populations and contribute to climate change

responses (Penuelas and Filella 2001; Hoffmann and Sgro 2011;

Franks et al. 2014). These findings also support the idea that

contemporary evolution can play an important role in shaping

ecological interactions, which is the central premise of the recent

field of eco-evolutionary dynamics (Pelletier et al. 2009). As with

prior work in this system (Franks et al. 2007) and other recent

studies (Nevo et al. 2012; Sultan et al. 2013; Bustos-Segura et al.

2014), the resurrection approach (Franks et al. 2008) allowed

direct assessment of evolutionary change.

The evolutionary shift to earlier flowering we found is con-

sistent with previous studies using these predrought ancestral and

postdrought descendant populations (Franks et al. 2007; Franks

and Weis 2008; Franks 2011). While the shift to earlier flowering

was seen previously, the evolution of greater pathogen suscepti-

bility shown in this study is a novel finding. Variation in disease

resistance in natural plant populations has been mapped at a spatial

scale looking at local adaptation, but to our knowledge, previously

had not been followed over time (Thrall and Burdon 2003; Laine

et al. 2011; Pautasso et al. 2012). In contrast to the evolution of

early flowering following drought, which is adaptive and allows

plants to increase fitness by escaping drought conditions (Franks

et al. 2007), the reason for the evolution of increased susceptibil-

ity to pathogenic disease following drought is less clear. Although

not tested directly in this study, one possibility is that selection

for earlier flowering resulted in plants with thinner leaves and

lower defenses that were more susceptible to disease (due to a

trade-off between growth and defense). Our finding of an evo-

lutionary increase in SLA resulting in thinner leaves following

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Table 1. Evolution and plastic responses to treatments.

Df Mean square F value p value

Response variable—flowering time Population 1 0.080 11.907 <0.001 Inoculation 1 0.026 3.892 0.049 Watering 1 0.030 4.490 0.035 Pop × Inoc 1 0.001 0.106 0.745 Pop × Watering 1 0.001 0.156 0.693 Inoc × Watering 1 0.059 8.823 0.003 Pop × Inoc × Watering 1 0.002 0.299 0.585 Error 519 0.007

Response variable—disease susceptibility Population 1 0.772 21.778 <0.001 Watering 1 0.425 11.993 0.001 Pop × Watering 1 0.056 1.575 0.212 Error 132 0.036

Response variable-SLA Population 1 37.52 5.994 0.015 Inoculation 1 192.94 30.824 <0.001 Watering 1 62.68 10.013 0.002 Pop × Inoc 1 4.36 0.696 0.405 Pop × Watering 1 0.02 0.004 0.952 Inoc × Watering 1 0.24 0.039 0.844 Pop × Inoc × Watering 1 0.28 0.044 0.834 Error 272 6.26

Shown are the results of three-way and two-way ANOVAs with population (ancestors or descendants), inoculation (inoculated or noninoculated, but

dropped when modeling disease susceptibility since only inoculated plants are included), watering (well watered or drought stressed) and their interactions

as fixed-effects in analyses conducted on transformed response variables: flowering time, disease susceptibility, and SLA. Significant effects of population

indicate evolution, while significant effects of the inoculation and watering treatments indicate plasticity. Parameters significantly different from zero

(p < 0.05) are bolded.

drought is consistent with this scenario. However, it is also pos-

sible that selection did act directly to increase susceptibility, or

that selection acted on other correlated traits. It is also possi-

ble that the increase in susceptibility was due to bottleneck and

drift events, although the population size remained qualitatively

large during the drought, suggesting that this was not a major fac-

tor. Future studies that examine the genetic basis of these traits,

or that directly manipulate the traits, would be useful in testing

among these alternatives and determining the mechanism of the

evolutionary shift to increased susceptibility and its relationship

to change in flowering time.

Our experimental design allowed us to investigate both evo-

lution in response to a natural drought and plasticity in response

to an experimentally induced drought. Plasticity and evolution in

many systems are predicted to be in the same direction (Pigli-

ucci et al. 2006; Wund et al. 2008), but studies have also found

that this relationship varies between highly adaptive, moderately

adaptive and maladaptive plasticity (Ghalambor et al. 2007; Gha-

lambor et al. 2015). We found that some traits were plastic and

some were not, and we also found that plastic responses in general

(for flowering time and disease susceptibility but not SLA) did

not match evolutionary responses. Flowering time evolved over

seven generations of drought to become earlier, but plants flow-

ered later as a plastic response to experimentally induced drought

(Fig. 1; Table 1). Similarly, experimentally induced drought re-

sulted in a plastic reduction in disease susceptibility, while the

evolutionary response over seven generations was increased sus-

ceptibility (Fig. 1; Table 1). The contrast in these two patterns

suggests that plasticity is not likely to have played a role in the

evolutionary increase in susceptibility observed in descendant

plants and that, in fact, the rapid evolutionary changes we ob-

served occurred despite plasticity generally acting in opposition

to the direction of evolution. For disease susceptibility, it is also

possible that the well-watered treatment may directly favor the

success of the pathogen (Agrios 2005). In contrast, evolution and

plasticity were in the same direction for SLA (Fig. 1; Table 1).

Although the reasons for this were not directly tested in this study,

we hypothesize that drought causes thinner leaves due to resource

limitation, resulting in a plastic increase in SLA under drought

conditions, and earlier flowering results in thinner leaved due to

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Figure 1. . Evolutionary changes in a natural B. rapa population between predrought ancestors (dark bars) and postdrought descendants

(light bars). We found a significant evolutionary shift to (A) earlier flowering, (B) greater disease susceptibility, and (C) increased specific

leaf area (SLA). There was also evidence for plasticity in these traits, with the direction of the response opposite to the evolutionary

response (A–B) except for SLA (C). The plants were grown under well-watered (wet) or drought stressed (dry) treatments and were

inoculated (fungus) or mock inoculated (none) with fungal spores. A three-way ANOVA (two-way for disease susceptibility) was conducted

on transformed data (Table S1) with population, inoculation, and watering treatments as fixed-effects, followed by specific pairwise

comparisons within treatments conducted using one-way ANOVAs. Untransformed means are shown with standard errors. Statistics are

provided in Table 1 (two- and three-way ANOVAs) and the text (one-way ANOVAs). Sample sizes are shown above each bar.

a trade-off between flowering time and growth, with selection for

earlier flowering causing an evolutionary increase in SLA. In any

case, the results of this study indicate that plastic and evolutionary

responses are not necessarily congruent, and that rapid evolution

can occur despite plasticity acting in the opposite direction.

An important caveat here, as with any greenhouse study,

is that the effects on all traits of our experimental drought in

the greenhouse could be different from natural drought in the

field, and field-based estimates of these traits may produce differ-

ent results. We attempted to approximate field conditions in the

greenhouse as much as possible by watering early in the season

to mimic the wet spring and then drought treating by infrequent

watering, as experienced in the field in the dry summers.

While plants can show some rapid evolutionary and plas-

tic responses to climate change, there are also constraints that

may substantially limit the ability of many plant populations to

sufficiently cope with the ongoing rapid rate of changes in cli-

mate (Franks et al. 2014). One type of constraint to evolution

that could hinder responses to climate change is negative genetic

correlation, which can be the result of trade-offs (Conner and

Hartl 2004, Etterson and Shaw 2001). One important trade-off in

plants involves defense against natural enemies such as herbivores

and pathogens, and growth or reproduction (Simms and Rausher

1987). This trade-off between growth and defense has been well

studied and underlies much of plant defense theory (Mole 1994).

Plants are under selection for increased growth and competitive

ability at the expense of defense allocation in introduced ranges

where specialist natural enemies are absent (Blossey and Notzold

1995). In this case, the adaptation conferring greater competitive

ability results in an ecological cost of decreased defense, which

is only paid if natural enemies are present. Though rarely consid-

ered, ecological costs to contemporary adaptive evolution could

also hinder the ability of populations to respond to climate change.

The evolutionary shifts to earlier flowering, increased SLA, and

greater susceptibility to disease we found in our study are con-

sistent with such a trade-off, particularly if the shift to earlier

flowering caused plants to produce thinner leaves that left them

more vulnerable to disease. Although our study was not designed

to test for such a trade-off directly, the fact that such trade-offs in

growth and defense appear to be common indicate that climate-

induced selection on one trait could cause correlated evolutionary

responses in other ecologically important traits.

Earlier flowering is a ubiquitous response to both drought

and the earlier start to growing seasons seen globally as the cli-

mate warms (Penuelas and Filella 2001; Parmesan and Yohe 2003;

Miller-Rushing and Primack 2008; Munguia-Rosas et al. 2011).

Although this general shift to earlier flowering could be taken

as evidence that plant populations can adapt to climate change,

there could be negative consequences and costs to such adapta-

tions. Specifically, our study shows that a shift to earlier flowering

in response to a climatic change in a natural plant population is

accompanied by increased susceptibility to fungal disease. Such

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ecological consequences of adaptation could impose constraints

on the long-term ability of populations to persist as climatic con-

ditions continue to change.

ACKNOWLEDGMENTS We thank A.E. Weis for seed collection, J. Gurevitch for help with ex- perimental design, D. Futuyma, J. J. Weber, A.L. Mila, J.R. True, and J. Gurevitch for feedback on this manuscript, E. Lowry and M. O’Neal for counsel and supplies, the Stony Brook greenhouse for support, E.P. Lauture, J.J. Lee, F. Chowdhury, H. Tahir, W. Amidi-Abraham, D.T. Cas- saro, A. Gill, and M. Flenniken for greenhouse help. Financial support included an American Fellowship from AAUW to N. O’Hara, a grant (DEB-1142784) from the National Science Foundation to S. Franks, and a grant (R01GM108904) from NIGMS-NIH to J.S.R. The authors have no conflict of interest to declare.

DATA ARCHIVING Dryad: http://dx.doi.org/10.5061/dryad.h3p05.

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Associate Editor: A . Angert Handling Editor: J . Conner

Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website:

Figure S1: The visual index that was used to measure disease severity of B. rapa plants infected with A. brassicae 21 days post inoculation. Table S1: Fixed factors and response variables with transformations conducted.

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