Western North American Naturalist Western North American Naturalist Volume 66 Number 4 Article 1 12-8-2006 Testing hypothesized evolutionary shifts toward stress tolerance Testing hypothesized evolutionary shifts toward stress tolerance in hybrid in hybrid Helianthus Helianthus species species Larry C. Brouillette University of Georgia, Athens Maheteme Gebremedhin University of Georgia, Athens David M. Rosenthal University of Georgia, Athens and Portland State University, Portland, Oregon Lisa A. Donovan University of Georgia, Athens Follow this and additional works at: https://scholarsarchive.byu.edu/wnan Recommended Citation Recommended Citation Brouillette, Larry C.; Gebremedhin, Maheteme; Rosenthal, David M.; and Donovan, Lisa A. (2006) "Testing hypothesized evolutionary shifts toward stress tolerance in hybrid Helianthus species," Western North American Naturalist: Vol. 66 : No. 4 , Article 1. Available at: https://scholarsarchive.byu.edu/wnan/vol66/iss4/1 This Article is brought to you for free and open access by the Western North American Naturalist Publications at BYU ScholarsArchive. It has been accepted for inclusion in Western North American Naturalist by an authorized editor of BYU ScholarsArchive. For more information, please contact [email protected], [email protected].
Transcript
Western North American Naturalist Western North American Naturalist
in hybrid in hybrid HelianthusHelianthus species species
Larry C. Brouillette University of Georgia, Athens
Maheteme Gebremedhin University of Georgia, Athens
David M. Rosenthal University of Georgia, Athens and Portland State University, Portland, Oregon
Lisa A. Donovan University of Georgia, Athens
Follow this and additional works at: https://scholarsarchive.byu.edu/wnan
Recommended Citation Recommended Citation Brouillette, Larry C.; Gebremedhin, Maheteme; Rosenthal, David M.; and Donovan, Lisa A. (2006) "Testing hypothesized evolutionary shifts toward stress tolerance in hybrid Helianthus species," Western North American Naturalist: Vol. 66 : No. 4 , Article 1. Available at: https://scholarsarchive.byu.edu/wnan/vol66/iss4/1
This Article is brought to you for free and open access by the Western North American Naturalist Publications at BYU ScholarsArchive. It has been accepted for inclusion in Western North American Naturalist by an authorized editor of BYU ScholarsArchive. For more information, please contact [email protected], [email protected].
Evolution of tolerance to stressful environ-ments is a major area of ecological researchwith important applications to conservationbiology and crop breeding (Wang et al. 2003).Many studies report correlations between plantfunctional traits and environmental parame-ters, but fall short of demonstrating that traitsof interest were derived under a specific set ofenvironmental conditions (Grime 1977). Organ-isms with known evolutionary relationships,such as hybrid species and their ancestral par-ents, can be used to make stronger inferencesthat certain plant functional traits are adap-tive. In this study we use a hybrid system toexamine how environmental pressures directedthe evolution of tolerance and functional traitresponses to nutrient stress.
We focus on 4 species of sunflower thatoccur in the Great Basin. Due to variation inclimatic and edaphic conditions, the GreatBasin contains a patchwork of habitats span-ning broad ranges of water and nutrient levels
(Smith & Nowak 1990, Comstock and Ehle-ringer 1992). Helianthus annuus is a widelydistributed annual sunflower that occurs onmesic, clay-based soils in the western UnitedStates. Helianthus petiolaris has a more re-stricted distribution, occurring on relativelymore xeric, sandy soils (Heiser 1947). These 2species hybridized multiple times to produce3 ancient homoploid hybrid species (Riese-berg 1991). Hybridization produced individu-als with functional trait values outside therange of the parental species, potentially mak-ing hybrids suited for colonization of extremehabitats (Rieseberg et al. 2003, Rosenthal et al.2005b).
We compare the parental Helianthus speciesto the 2 hybrid species, which occur on sandy,nutrient-poor soils in the Great Basin Desert.Helianthus anomalus is endemic to active sanddunes, which are very poor in fertility andorganic content (N ~0.01%, P ~0.4 mg ⋅ g–1,organic content ~0.01%; Rosenthal et al. 2005a,
TESTING HYPOTHESIZED EVOLUTIONARY SHIFTS TOWARD STRESS TOLERANCE IN HYBRID HELIANTHUS SPECIES
Larry C. Brouillette1,2,4, Maheteme Gebremedhin1, David M. Rosenthal1,3, and Lisa A. Donovan1
ABSTRACT.—We examined how plant traits related to growth and resource use have evolved during hybrid speciationand specialization into stressful habitats. Two desert sunflower species of homoploid hybrid origin are endemic to habi-tats with lower soil nutrient levels than those of their ancestral parent species. We hypothesized that the hybrid specieswould exhibit greater tolerance to low levels of soil nutrients than their parental species. The 2 hybrid species,Helianthus anomalus and H. deserticola, and their parental species, H. annuus and H. petiolaris, were compared forplant traits and growth through reproduction under 3 nutrient levels in a greenhouse study. An additional seedling studycompared species for maximum seedling relative growth rate under optimum conditions. The hybrid species did havegreater tolerance of nutrient limitation than the parental species, demonstrated by a resistance to change in stem heightand diameter growth across treatments. A similar trend was observed in total biomass at final harvest. This ability tomaintain growth may be partially explained by maintained investment in photosynthetic enzymes regardless of nutrienttreatment. Though the hybrid species were more tolerant of nutrient stress, differences in the hybrid response to nutri-ent stress compared to the parental species’ response were much smaller than expected from habitat comparisons.Helianthus anomalus has evolved a classic stress-tolerant phenotype, having long leaf lifespan, tough leaves, and slowerearly seedling relative growth rate. While both hybrid species have a conservative growth strategy, which confersgreater stress tolerance than the parental species possess, functional trait differences among the hybrids suggest that the2 species have experienced vastly different selective pressures.
1Department of Plant Biology, University of Georgia, Athens, GA 30602.22502 Miller Plant Sciences, Athens, GA 30602. 3Department of Biology, Portland State University, Portland, OR 97207.4E-mail: [email protected]
409
Ludwig et al. 2006). Helianthus deserticolaoccurs on stabilized sand dunes with higher fertility (N ~0.03%, P ~0.4 mg ⋅ g–1, organiccontent ~0.02%) than the H. anomalus habi-tat, but lower fertility than the nearby habitatsof both parental species (N ~0.6%, P ~0.6–0.7 mg ⋅ g–1, organic content ~0.04%–0.05%;Rosenthal et al. 2005a, Ludwig et al. 2006,L.A. Donovan unpublished data). Previousstudies have pointed toward nutrient availabil-ity as an important selective pressure in thehabitats of the hybrid species (Ludwig et al.2004, Rosenthal et al. 2005a). However, this isthe 1st study to examine differences in nutri-ent stress tolerance in the hybrid species rela-tive to the parental species. We determine howfunctional trait values in the hybrid specieshave shifted as the species became establishedin and adapted to resource-poor habitats inthe Great Basin Desert.
Because the hybrid species occur on nutri-ent-poor soil, we hypothesized that they wouldexhibit greater tolerance to soil nutrient limi-tations than the parental species. We definenutrient stress tolerance as the ability to resistchange in plant growth or biomass accumula-tion when nutrients vary from optimum tolimiting levels. We also expected pronouncedplant traits associated with low-resource habi-tats in the hybrid species, compared to theparental species. Suites of plant functional traitssuch as increased root mass ratios, lowered tis-sue turnover rates, and decreased transpira-tion rates have been proposed as adaptationsthat allow plants to inhabit resource-poorhabitats. These traits may come at a physiolog-ical cost, exhibited as lowered photosyntheticrates, decreased capacity for nutrient uptake,and/or reduction in maximum growth rate(Chapin et al. 1993). Variation in specific leafarea (SLA, leaf area per unit dry mass) mayalso be an important trait in stressful environ-ments. Traits such as leaf lifespan, leaf longev-ity, and maximum photosynthetic rate arestrongly correlated with SLA across plantfunctional groups (Reich et al. 1997).
In this study we experimentally comparedthe growth and performance responses of 2hybrid Helianthus species to their ancestralparent species at different nutrient levels. Weexpected the hybrid species to exhibit greatertolerance to nutrient stress than their parentalspecies. We also examined a suite of traitsimportant in nutrient-poor habitats to iden-
tify putative mechanisms of nutrient stress tolerance.
METHODS
Seeds were collected in 2002 from a singlenatural population of each species and storedat 6°C until use. Seeds from H. annuus, H.anomalus, and H. deserticola were collected at the Little Sahara Recreational Area, JuabCounty, Utah, the only known place where all3 species presently co-occur. Seeds from H.petiolaris were collected from the populationused by Rieseberg et al. (2003) located 10miles south of Page along Hwy 89, CoconinoCounty, Arizona. For all germination experi-ments, seeds of all 4 species were cold strati-fied on moist filter paper for 4 weeks. Seedswere then removed from cold treatment,stored in the dark at room temperatureovernight, and transferred to fresh filter paperin petri dishes. Petri dishes were placed underfluorescent lights (~115 µmol ⋅ m–2s–1) with a12-hour photoperiod.
Nutrient Limitation Experiment
The nutrient limitation experiment was acomplete randomized block design, with 4species, 3 nutrient treatments, 3 blocks, and 3 replicates per block, totaling 108 plants.Seedlings with 0–2 true leaves were trans-planted into 25-cm pots in the University ofGeorgia greenhouses on 17–19 March 2004.Pots contained sand and baked clay in a 3:1mixture (Turface, Profile Products, BuffaloGrove, IL). The high, medium, and low nutri-ent treatments were 40 g, 4 g, and 2 g, respec-tively, of slow-release fertilizer with macro andmicronutrients (Osmocote Plus, Scotts Co.,Marysville, OH) applied on 23 March to theupper 2 cm of the soil. Nutrient levels werechosen based on a preliminary experiment inwhich the 40-g and 4-g treatment wereapplied to all 4 species and a 2-g treatmentwas applied to H. anomalus, which showed asignificant decrease in biomass over the smallchange in nutrient levels. Plants were watereddaily to maintain field capacity. Seven plantsdied from transplant shock and were excludedfrom all analyses.
We measured stem height 5 times duringthe experiment, starting on 23 March and con-tinuing at approximately 3-week intervals.
410 WESTERN NORTH AMERICAN NATURALIST [Volume 66
Measurements for the last 3 dates includedthe diameter of the stem at soil level. Wedetermined leaf lifetime by marking the mostrecently fully expanded mature leaf on 5 Mayand recording the date when 50% of the leafturned brown. On 27 May each plant wassampled for mature leaf traits by harvestingthe most recently expanded fully mature leafin early morning when leaves were fullyhydrated. Leaf area (LI-3100, LI-COR Inc.,Lincoln, NE), wet weight, and dry weightwere measured to estimate SLA and leaf suc-culence ( Jennings 1976). On 9 June a fruitripeness penetrometer was used to estimateleaf toughness on a mature leaf (McCormickFruit Tech., Yakima, WA). Variability of individ-ual measurements was large, so the mean of5–7 measurements was the estimate of leaftoughness for the plant.
Gas exchange traits were measured on 5–6July for a subset of the plants (4 replicates, 4species, 2 treatments [high and low]) with aportable gas exchange system (LI-6400, LI-COR, Inc., Lincoln, NE). Inside the chamber,photosynthetically active radiation equalled2000 µmol ⋅ m–2s–1 and air temperature was~30°C. Measurements were taken at multipleexternal CO2 concentrations (ca) to determinerates of photosynthesis (A) over a range ofinternal leaf CO2 concentrations (ci). In order,measurements were taken at ca equal to 400,300, 200, 100, 50, and 400 ppm. At low ci, therelationship between photosynthetic rate andci may be approximated by a straight line, theslope of which is proportional to investment inrubisco. We estimated investment in rubiscousing the slope of a linear regression of A withci when ca was set to the values stated above.We averaged the 2 measurements at 400 ppmto estimate maximum photosynthesis (Amax).Date had no significant effect on measure-ments (P > 0.90) and was excluded from thefinal model. All measurements were correctedfor leaf area (LI-3100).
Gas exchange leaves were individually driedat 60°C, ground, and analyzed for N concen-tration (mg N ⋅ g–1; Carbo Erba NA 1500) andleaf carbon isotopic composition (leaf δ13C;Finnegan, continuous flow mass spectrometer,Bremen, Germany). Leaf δ13C provides anintegrated measure of ci over the lifetime ofthe leaf. Integrated ci is, in turn, a relative mea-sure of integrated water use efficiency (WUE),if leaf temperatures are similar (Farquhar et al.
1989, Ehleringer et al. 1992). A higher (lessnegative) leaf δ13C reflects greater WUE.Maximum photosynthetic rate, leaf N, andspecific leaf area were used to calculate photo-synthetic nitrogen-use efficiency (PNUE) fol-lowing Field and Mooney (1986).
All plants were harvested on 15–16 June.Biomass was partitioned into belowground, veg-etative, and reproductive components. Countswere taken of number of buds, flowers, andseed heads to estimate life history stage at har-vest. Biomass components were dried at 60°Cand weighed.
We modeled each variable as a function ofcategorical variables in a mixed model ANOVA(PROC MIXED, SAS Institute, Cary, NC):treatment, species, treatment × species inter-action, with block as a random effect. All bio-mass components were log-transformed to fitANOVA assumptions. Visual inspection of resid-ual plots revealed extreme outliers for leaf suc-culence (n = 2), specific leaf area (n = 2), totalbiomass (n = 1), and stem growth (n = 1).These observations were excluded from ouranalyses. Unadjusted values from the final 3measurements of stem height and stem diame-ter were analyzed in a repeated-measuresmixed model in PROC MIXED with an AR(1)covariance matrix. We used only the final 3measurements because they were available forboth growth measurements. Inclusion of stemheight for the first 2 dates did not change theconclusions of statistical tests. For traits mea-sured in all treatments, we separated the inter-action term into 6 components: linear andquadratic trends between and within the hybridand parental species (SAS CONTRAST state-ment). Partitioning the interaction term be-tween hybrid and parental species tested thehypothesis that parental species respond dif-ferently to nutrient stress than hybrid species.Further separating the interaction into “withinhybrids” and “within parentals” tested for spe-cies differences in response. For traits assessedonly in high and low treatments, only linearcomponents were examined. Evaluating thesea priori comparisons retained precision that islost in post hoc tests.
Seedling Relative Growth Rate Experiment
The relative growth rate study was a com-pletely randomized design with 4 species, 2harvests, and 12 replicates, giving a sample
2006] HELIANTHUS STRESS TOLERANCE 411
size of 96. On 17–19 March, seedlings weretransplanted into 6.5 cm × 25-cm pots (Deepots,Stuewe & Sons, Corvaillis, OR) containing thesame soil mixture described for the nutrientlimitation experiment. Seedlings were wateredto field capacity daily and received nonlimit-ing nutrient solution weekly (300 ppm PeteLite, J.R. Peters Laboratory, Allentown, PA).The plants were used to estimate maximumearly seedling relative growth rate (RGRmax)under optimum conditions for each of the 4species. Plants were harvested on 29 Marchand 12 April, dried at 60°C, and measured fortotal biomass. Log-transformed total biomasswas regressed against harvest date, species,and their interaction (SAS PROC GLM; Poorterand Lewis 1986). We used Fisher’s protectedleast significant difference tests to detect sig-nificant differences in the interaction term,indicating species with statistical differencesin RGRmax.
RESULTS
Nutrient Limitation Experiment
Plant performance was assessed as stemgrowth (in height and diameter) and total bio-mass at harvest. All 4 species showed substan-tial reduction in growth and biomass produc-tion in response to lowered nutrient levels(Figs. 1, 2, Table 1). Hybrid species had a lessdrastic reduction in stem height and diametergrowth than the parental species when we com-pared the low nutrient treatment to the highnutrient treatment. The effect was seen as asignificant difference in linear response ofhybrid and parental species (Table 1). This wasparalleled by a similar, but not significant, pat-tern in total biomass (Fig. 1, Table 1; F = 0.14).Individuals of the hybrid species under lownutrient treatment produced approximately85% less biomass than those under high nutri-ent treatment. By comparison, parental speciesshowed a 90% reduction. We used percentageof inflorescences at the seed-head stage toassess life history status. These data suggestdifferences in life history stage between speciesand treatments at harvest, with H. anomalus inthe high treatment having the lowest percent-age of inflorescences at the seed-head stage(Table 2).
There was a highly significant effect oftreatment on leaf percent nitrogen, indicating
that foliar nitrogen concentration paralleledsoil nutrient levels. Hybrid species tended tohave a smaller difference between treatmentsthan parental species (Table 3). The oppositewas true of leaf δ13C, with parental speciesoverall having less of a change in WUE as a re-sponse to treatment than hybrid species (Table3; F = 2.93, P ≤ 0.10). There was also a differ-ence between hybrid and parental species’ re-sponses to nutrient stress for leaf succulence;hybrid species tended to maintain higher leafsucculence under low nutrients (Table 2; F =3.93, P ≤ 0.10). Hybrid species maintainedhigher investment in rubisco than parentalspecies under low nutrient conditions, as seenin the significant difference in linear responseof the initial slope of the A/ci curve (Fig. 3,Table 4; F = 4.36, P ≤ 0.05). Even though weexamined Amax and leaf-level PNUE, only mar-ginally significant differences in the linearresponse of hybrid and parental species weredetected for Amax (Table 3; F = 3.31, P ≤ 0.10).
In addition to the anticipated differencesbetween the hybrid and parental species, the2 hybrid species responded differently tonutrient treatment. Helianthus anomalus pro-duced leaves that were tougher than those ofthe other species (Fig. 3, Table 4; F = 94.82, P ≤ 0.0001). Additionally, H. anomalus producedlonger-lived leaves in response to the lowered
412 WESTERN NORTH AMERICAN NATURALIST [Volume 66
Total biomass at reproductive maturity
Species
H. annuus H. anomalus H. deserticola H. petiolarisT
otal
bio
mas
s (g
)0
40
80
120
160
High NutrientsMed. NutrientsLow Nutrients
Fig. 1. Mean total biomass measurements and 95% con-fidence intervals for 4 Helianthus species grown in a green-house at 3 nutrient levels (n = 101).
nutrients, assessed as a difference in linear nu-trient stress response between H. anomalus andH. deserticola (Fig. 3, Table 4; F = 9.31, P ≤0.01). We also observed a marginally signifi-cant quadratic response of H. anomalus rootmass ratio to the treatment levels, suggesting alower threshold for stress-induced root alloca-tion (Table 2; F = 3.43, P ≤ 0.10).
Under low resources, H. deserticola had amean maximum photosynthetic rate compara-ble to high nutrient plants, but the response ofthe 2 hybrid species was not significantly dif-ferent (Table 3; F = 1). Additionally, the linearresponse of specific leaf area to the nutrientlevels in H. deserticola indicates that it pro-duces thicker leaves in response to nutrient
2006] HELIANTHUS STRESS TOLERANCE 413
H. a
nom
alus
40
80
120
160
Pla
nt S
tem
Dia
met
er (
cm)
0
5
10
15
H. d
eser
ticol
a
Pla
nt H
eigh
t (cm
)
40
80
120
160
H. p
etio
laris
Date
40
80
120
160
High NutrientsMedium NutrientsLow Nutrients
May 5 May 25 June 16
H. a
nnuu
s
40
80
120
160
0
5
10
15
May 5 May 25 June 16
Date
0
5
10
15
0
5
10
15
Plant height and stem diameter growth
Fig. 2. Stem height and diameter for 4 Helianthus species grown under 3 nutrient levels in a greenhouse (n = 101).Plotted values are arithmetic means at each date for each species–treatment combination. Least squared estimates of themeans are given for each of 3 dates used in the repeated measures analysis. Measurements were repeated over all indi-vidual plants in the experiment at each time increment. Error bars represent 95% confidence intervals.
stress, unlike the other study species (Fig. 3,Table 4; F = 5.90, P ≤ 0.05).
Seedling Relative Growth Rate Experiment
Helianthus anomalus had a significantlyslower early seedling RGRmax than H. deserti-cola and H. petiolaris. We estimated seedlingRGRmax for H. anomalus to be lower than thatof H. annuus, but the difference was not sig-nificant (Fig. 4).
DISCUSSION
We conclude that the hybrid species aremore tolerant of nutrient stress than theirparental species. We observed significant dif-ferences in stem height and diameter growththat suggested higher stress tolerance in thehybrid species. A similar trend was seen intotal biomass, but differences were not signifi-cant. We found evidence that hybrid specieshave higher tolerance of nutrient limitation,but differences were much subtler than ex-pected. The final harvest date arbitrarily trun-cated the life cycles of the species, and thefailure to detect expected differences in bio-mass could be due to early cutoff of H. anom-alus, which tends to persist in the field until1st frost in many populations. A better test ofthe effects of nutrient limitation on fitnesswould have been a direct measure of seed set.Pollinators are excluded from the greenhouse,so seed weight and number are impossible to
obtain without frequent crossing throughoutthe experiment.
Foliar nitrogen concentration was less re-sponsive to treatment in hybrid species com-pared to parental species. This seems to beimportant in the field because selection analy-ses show a strong association between foliarnitrogen content and fitness in the H. anom-alus habitat (Ludwig et al. 2004). High levelsof foliar nitrogen may account for the greaterinvestment in rubisco and marginally higherability to maintain Amax found in hybrid speciesunder low nutrient conditions. The resistanceto changing investment in photosynthesis de-spite lower nitrogen availability is one possibleexplanation for increased nutrient stress toler-ance of the hybrid species. Under low nutri-ent conditions, hybrid species also tended tohave higher water-use efficiency and leaf suc-culence, which are putative drought resistancetraits. Water does not appear to be a majorlimiting factor for H. anomalus in its home hab-itat, so drought-resistance traits in H. anomalusmay result from genetic correlations amongstress-resistance traits (Chapin et al. 1993).
Helianthus anomalus produced tougher,longer-lived leaves than the other species andshowed a reduced early seedling RGRmax.While RGRmax was not significantly lower thanboth parentals in this study, a subsequent ex-periment showed that H. anomalus had a sig-nificantly lower RGRmax than H. annuus (datanot shown). Helianthus anomalus also tended
414 WESTERN NORTH AMERICAN NATURALIST [Volume 66
TABLE 1. Analysis of plant growth variables and total biomass. Degrees of freedom and F-values for the appropriatemixed model analysis of our 3 measures of tolerance: height growth, stem diameter growth, and total biomass at harvest(n = 101). Significant F-values are denoted as follows: + (P ≤ 0.10), * (P ≤ 0.05), ** (P ≤ 0.01), and *** (P ≤ 0.0001).
Height growth Stem diameter growth Total biomass____________________ _____________________ ________________Source df F df F df F
416 WESTERN NORTH AMERICAN NATURALIST [Volume 66TA
BL
E3.
Mea
ns o
f pho
tosy
nthe
tic t
raits
and
dat
a an
alys
es. L
east
squ
ared
est
imat
es o
f mea
n tr
ait
valu
es w
ith 9
5% c
onfid
ence
inte
rval
bou
nds
(in p
aren
thes
es) f
or p
lant
fun
ctio
nal
trai
ts m
easu
red
only
at h
igh
and
low
nut
rien
t lev
els
(n =
32)
. F-v
alue
s an
d de
gree
s of
free
dom
from
the
mix
ed m
odel
ana
lysi
s ar
e re
port
ed b
elow
the
trai
t val
ues.
Sig
nific
ant F
-val
ues
are
deno
ted
with
sym
bols
: + (P
≤0.
10),
* (P
≤0.
05),
** (P
≤0.
01),
and
***
(P≤
0.00
01).
Trea
tmen
tA
max
PNU
EL
eaf %
nitr
ogen
Lea
f δ13
CSp
ecie
s (µ
mol
CO
2⋅m
–2s–
1 )
(µm
ol C
O2
⋅[m
ol N
]–1 s
–1)
(%)
(ppt
)
HIG
H
Hel
iant
hus
annu
us36
.4(2
8.6,
44.
3)25
2.8
(184
.2, 3
21.5
)3.
9(3
.43,
4.3
7)–3
0.4
(–30
.86,
–29
.93)
Hel
iant
hus
anom
alus
33.8
(26.
0, 4
1.7)
272.
1(2
08.8
, 335
.5)
3.32
(2.8
2, 3
.82)
–31.
89(–
32.3
8, –
31.3
9)H
elia
nthu
s de
sert
icol
a33
.4(2
5.6,
41.
2)25
3(1
92.3
, 313
.6)
3.85
(3.3
1, 4
.39)
–31.
75(–
32.2
9, –
31.2
2)H
elia
nthu
s pe
tiola
ris
39.5
(31.
6, 4
7.3)
340.
5(2
71.2
, 409
.8)
4.08
(3.5
0, 4
.67)
–31.
19(–
31.7
8, –
30.6
1)
LO
W Hel
iant
hus
annu
us25
.1(1
7.3,
32.
9)30
5.5
(242
.0, 3
68.9
)1.
58(1
.08,
2.0
8)–2
9.66
(–30
.16,
–29
.17)
Hel
iant
hus
anom
alus
26.9
(19.
1. 3
5.0)
349.
4(2
86.1
, 412
.8)
1.82
(1.3
5, 2
.29)
–31.
04(–
31.5
1, –
30.5
8)H
elia
nthu
s de
sert
icol
a34
(26.
1, 4
1.8)
313.
7(2
53.0
, 374
.5)
2.39
(1.8
5, 2
.92)
–30.
68(–
31.2
1, –
30.1
4)H
elia
nthu
s pe
tiola
ris
25.1
(17.
2, 3
2.9)
319
(257
.0, 3
81.1
)2.
09(1
.59,
2.5
9)–3
1.23
(–31
.73,
–30
.73)
Sour
cedf
Fdf
Fdf
Fdf
F
Spec
ies
3, 2
20.
323,
22
1.62
3, 2
22.
48+
3, 2
214
.43*
**Tr
eatm
ent
1, 2
29.
10**
1, 2
25.
28*
1, 2
211
2.36
***
1, 2
213
.41*
**Sp
ecie
s ×
Trea
tmen
t3,
22
1.49
1, 2
21.
283,
22
1.54
3, 2
21.
64L
inea
r pa
rent
al ×
hybr
id1,
22
3.31
+1,
22
2.17
1, 2
23.
88+
1, 2
22.
93+
Lin
ear
annu
us×
petio
lari
s1,
22
0.16
1, 2
21.
671,
22
0.45
1, 2
22.
29L
inea
r an
omal
us×
dese
rtic
ola
1, 2
21
1, 2
20.
111,
22
01,
22
0.2
TAB
LE
4. R
esul
ts o
f mix
ed m
odel
AN
OV
A e
xam
inin
g se
lect
ed p
lant
func
tiona
l tra
its. M
eans
and
sta
ndar
d er
rors
are
plo
tted
in F
ig. 2
. Sig
nific
ant F
-val
ues
are
deno
ted
by s
ymbo
ls a
sfo
llow
s: +
(P≤
0.10
), *
(P≤
0.05
), **
(P≤
0.01
), an
d **
* (P
≤0.
0001
). T
he to
tal n
umbe
r of
indi
vidu
als
assa
yed
was
101
, exc
ept f
or A
/cifo
r w
hich
32
plan
ts w
ere
subs
ampl
ed.
Lea
f life
time
Lea
f tou
ghne
ssSp
ecifi
c le
af a
rea
Initi
al s
lope
ofA
/cicu
rve
____
____
____
____
___
____
____
____
____
____
____
____
____
____
___
____
____
____
____
____
_So
urce
dfF
dfF
dfF
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Spec
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3, 8
78.
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86
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3, 8
624
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87
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2.64
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14.6
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211
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3.23
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1.42
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71.
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0.01
1, 8
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70.
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29
to increase investment in roots in the interme-diate nutrient treatment. These observationssuggest that H. anomalus has a classic stress-tolerant phenotype. Under this paradigm, longleaf lifespan decreases demand and higherroot allocation increases uptake of the limitingresource. These traits probably come at a cost ofreduced seedling RGRmax (Chapin et al. 1993).
Our data suggest that H. deserticola has adifferent strategy than H. anomalus. Unlike inH. anomalus habitat, water becomes scarce inH. deserticola habitat early in the growing sea-son (Rosenthal et al. 2005a). Water and nutri-ents likely co-limit growth in H. deserticola
habitat, imposing selective pressures differentfrom those in H. anomalus habitat (Ludwig etal. 2004). Under low nutrients, H. deserticolaproduced leaves with low SLA, a trait corre-lated to putative adaptations to multiple abi-otic stresses (Riech et al. 1997). It is also inter-esting that some photosynthetic traits wereunresponsive to treatment in hybrids becausewe have observed unexpectedly high photo-synthetic rates in H. deserticola growing in itsnutrient-poor home habitat (D.M. Rosenthalunpublished data).
Though we initially expected more obviousdifferences, we demonstrate that H. anomalus
2006] HELIANTHUS STRESS TOLERANCE 417
Fig. 3. Model least squared estimates of the means of plant functional traits: (A) leaf lifespan, (B) leaf toughness, (C)specific leaf area, and (D) initial slope of A/ci curve. Traits were measured on 4 Helianthus species grown in a green-house under 3 nutrient levels. The total number of individuals assayed was 101, except for A/ci where 32 plants weresubsampled. For gas exchange measurements, such as initial slope of A/ci curve, we subsampled individuals from highand low nutrient levels. Error bars represent 95% confidence intervals.
and H. deserticola are more tolerant of nutri-ent stress than their parental species. Addi-tionally, we report shifts in the means of H.anomalus traits predicted by correlationalstudies of plant adaptations to abiotic stress.Helianthus anomalus also has a reduced seed-ling RGRmax, which is likely a physiologicalcost of stress resistance. Helianthus deserticolaappears to acclimate to stress by shifting spe-cific leaf area. Hybrid species maintain greaterinvestment in photosynthetic enzymes despitesevere nutrient limitation. While the 2 speciesexhibit similar growth responses to nutrientstress, H. anomalus and H. deserticola appearto have evolved different suites of traits toadapt to distinct low resource habitats.
ACKNOWLEDGMENTS
We thank A. Howard, C. Gormally, and 2anonymous reviewers for comments on earlierdrafts. Research was supported by NSF grantIBN-0131078 to L.A. Donovan and a UGAPlant Biology Department small grant to L.C.Brouillette.
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