Tree Physiology 36, 252–266
doi:10.1093/treephys/tpv141
Research paper
Limited variation found among Norway spruce half-sib families
in physiological response to drought and resistance to embolism
Daniel J. Chmura1,3, Marzenna Guzicka1, Katherine A. McCulloh2 and Roma Żytkowiak1
1Institute
of Dendrology, Polish Academy of Sciences, ul. Parkowa 5, 62-035 Kórnik, Poland; 2Department of Botany, University of Wisconsin-Madison, Madison, WI 53706, USA;
author ([email protected])
3Corresponding
Received June 3, 2015; accepted December 10, 2015; published online January 19, 2016; handling Editor Maurizio Mencuccini
Projections of future climates suggest that droughts (Ds) may become more frequent and severe in many regions. Genetic variation, especially within populations in traits related to D resistance, is poorly investigated in forest trees, but this knowledge is
necessary to better understand how forests will respond to water shortages. In this study, we investigated variability among
seven open-pollinated half-sib families of a single population and two population-level progenies of Norway spruce (Picea abies
(L.) H. Karst.) in their gas exchange response to imposed D and xylem vulnerability to embolism. During their third growing season, saplings were subjected to three treatments—control (C), D (for 19 weeks) and broken drought (BD, 54 days without watering starting in mid-July, then well-watered). In response to D, all families reduced their stomatal conductance (gs) and
light-saturated rates of photosynthesis (Amax) in a similar way. After rewatering, the xylem water potential (Ψ) recovered in the
BD treatment, but gs and Amax remained lower than in C. Needle starch concentration was altered in both D treatments compared
with C. Xylem of D-exposed trees was more vulnerable to embolism than in C. The minimum attained safety margin remained
positive for all families, indicating that no catastrophic hydraulic failure occurred in stem xylem during D. Significant family variation
was found for Ψ early in the D (midday Ψ between −1.2 and −1.8 MPa), and for needle damage, but not for sapling mortality.
Family variation found at the initial stages of D, and not afterward, suggests that all families responded similarly to greater
D intensity, exhibiting the species-specific response. Limited variation at the family level indicates that the response to D and the
traits we examined were conservative within the species. This may limit breeding opportunities for increased D resistance in
Norway spruce in light of expected climatic changes.
Keywords: cavitation, drought vulnerability, hydraulic conductivity, photosynthesis, stomatal conductance.
Introduction
Water availability is one of the most important factors limiting
tree growth, survival and species distribution. Numerous studies,
both in natural and controlled conditions, have found negative
impacts of drought (D) on aboveground growth and survival of
seedlings, saplings and mature trees (e.g., ­Fritts 1966, ­Vose and
­Swank 1994, ­Hanson et al. 2001, ­Dohrenbusch et al. 2002,
­Ogaya and ­Peñuelas 2007, ­Allen et al. 2010). Ongoing changes
in climate associated with increases in atmospheric concentrations of greenhouse gases will likely impose new stresses on
trees and forests. Projections of future climates suggest that
elevated temperatures will change hydrology and exacerbate
water shortages, even if the total amount of precipitation does
not change (­Christensen et al. 2007, ­EEA 2008). Thus, Ds may
become more frequent and more severe in future climates.
Therefore, D resistance will undoubtedly be an important trait for
maintaining healthy and productive forests in the coming
decades.
Norway spruce (Picea abies (L.) H. Karst.) is a significant component of many forest communities and an important commercial
tree species in Europe. However, during the last decades, spruce
stands throughout Europe experienced health deterioration and
© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Variation among Norway spruce half-sib families 253
increasing mortality (­Solberg 2004, ­
Šrámek et al. 2008,
­Hentschel et al. 2014). Although the exact causes of that mortality are unknown, a greater evaporative demand in warmer
atmosphere during the summer may be a contributing factor.
High xylem conductivity and stomatal conductance (gs), and
thus lower water-use efficiency, during D have been identified as
the risk factors for dieback, even in mature Norway spruce trees
(­Hentschel et al. 2014). Further, moisture stress weakens trees
and predisposes them to other stress agents, such as insects
and diseases (­Solberg 2004). Early tree life stages are the most
vulnerable to climatic influences, including water shortages.
Thus, it is important to identify mechanisms responsible for D
resistance in seedlings.
Trees have multiple physiological, anatomical, morphological
and life-history mechanisms to resist D. During the growing season, dehydration avoidance is typically more common in woody
plants than desiccation tolerance (­Chaves et al. 2003), although
a wide range exists in the water potential at which trees close
their stomata during D. Trees tend to avoid dehydration by minimizing water loss and maximizing water uptake, for instance by
effective stomatal regulation and/or deep rooting. With increasing evaporative demand, stomata close preventing excessive
dehydration, but also limiting CO2 diffusion into the leaf, and thus
photosynthesis (­Cornic 2000, ­Yordanov et al. 2000, ­Chaves
et al. 2002). However, stomatal closure during water shortage
is necessary not only to prevent excessive dehydration but also
D-induced xylem dysfunction and hydraulic failure due to embolism propagation (­Sperry and T
­ yree 1988, ­1990, ­Tyree and
­Ewers 1991). A common measure used to compare the vulnerability of xylem to D is the xylem water potential inducing 50%
loss of hydraulic conductivity (Ψ50). Among woody species,
median vulnerability to embolism increases in accordance with
the gradient of increasing rainfall (­Maherali et al. 2004). However, the Ψ50 does not reflect the inherent degree of risk associated with a given vulnerability to embolism. This threat is better
indicated by the hydraulic safety margin, which is commonly
defined as the midday water potential of a stem minus the Ψ50.
Across species, trees across the globe seem to operate at low
xylem safety margins, independent of rainfall (­Meinzer et al.
2009, ­Choat et al. 2012).
Forests may be prone to injury and growth reductions associated with increasing temperatures and incidence of D, but the
extent of within-species variation in vulnerability to D is not well
documented (­Anderegg 2015). Although Norway spruce has
only low to moderate resistance to D when compared with other
tree species (­Niinemets and ­Valladares 2006, ­Modrzyński
2007), genetic variation among and within populations in traits
conferring D resistance is not well recognized. Individual trees
within a single population may vary in their response to D
(­Hentschel et al. 2014); however, the genetic basis of such
variation has not been investigated. Variation in resistance to
embolism has been found both among populations (­K avanagh
et al. 1999) and families (­Anekonda et al. 2002) in Pseudotsuga
menziesii (Mirb.) Franco and among populations in Populus
trichocarpa Torr. & A.Gray ex Hook. (­Sparks and ­Black 1999).
Yet, only limited variation among populations in Ψ50 was found
in two pine species (­Lamy et al. 2011, ­2014, ­Sáenz-Romero
et al. 2013) and the angiosperm Fagus sylvatica (L.)
(­Wortemann et al. 2011, ­Aranda et al. 2015). The extent of
population or family variation has also been found to vary by site
(­Corcuera et al. 2011, ­Lopez et al. 2013), thus suggesting high
phenotypic plasticity in some species. The large portion of variation in vulnerability to embolism may reside within rather than
among populations (­Wortemann et al. 2011, ­Lamy et al. 2014).
However, the generality of this pattern and the extent of withinpopulation variation are yet to be determined.
In this article, we present results on the response to imposed
D among seven open-pollinated half-sib families and two population-level progenies of Norway spruce. In 3-year-old saplings
exposed to D in the controlled environment study, we investigated gas exchange parameters—light-saturated rates of photosynthesis (Amax) and gs, and xylem vulnerability to embolism
assessed with a pressure sleeve method. We explored the questions of whether, and to what extent, the families within a single
population will show variable response to, and recovery from, D
in terms of gas exchange parameters, and what is the extent of
within-population variation in xylem vulnerability to embolism.
We expected saplings exposed to D treatment to produce more
embolism-resistant wood than the control (C) (­Beikircher and
­Mayr 2009, ­Fichot et al. 2010).
Materials and methods
Plant material, experimental conditions and layout
The study material consisted of seven open-pollinated maternal
half-sib families of Norway spruce, originating from the ‘restitution’ seed orchard of the ‘Kolonowskie’ provenance (­Chałupka
et al. 2008). In addition, two sets of open-pollinated seed, originating from two seed stands located in forest districts Koniecpol,
Poland (50°46′N, 19°45′E, altitude 240 m), and Český Krumlov,
Czech Republic (48°40′N, 14°20′E, altitude 800 m), were
included in the study. Mean annual air temperatures and precipitation sums are 7.5 °C and 623 mm, respectively, in Koniecpol,
and 7.7 °C and 826 mm in Český Krumlov (­Climate-Data.org).
Although the two latter seed sources likely consist of a mixture of
open-pollinated families, we hereafter interchangeably use the
terms ‘seed sources’ and ‘families’ for brevity. Most of the measurements reported in this article were done on seven half-sib
families, but needle damage was scored also on the two populationlevel seed sources.
Seedlings of all nine seed sources were raised in containers in
a nursery. One-year-old seedlings were replanted in spring 2012
into the 15-l pots filled with a mixture (v/v) of compost (50%),
peat (25%) and sand (25%). Each planting pot had a 2 cm
Tree Physiology Online at http://www.treephys.oxfordjournals.org
254 Chmura et al.
thick layer of gravel at the bottom to allow free drainage. The
­slow-release fertilizer (Osmocote® Exact 16–18 months, 15-3.58.3 NPK + 1.8 Mg + TE) was mixed into a growing medium at
the dose of 3.7 g l−1. No further fertilization was provided
throughout the study. During the second growing season (first
season in the experiment), seedlings were sprayed twice to control aphids and once to control fungi (chlorothalonil). The mean
tree height was 12.0 ± 4.5 cm (SD) at planting, and at the onset
of D, trees were on average 72.4 ± 12.1 cm tall.
The experiment was set under two rain-exclusion shelters
covered with polypropylene foil and a neutral shading net. The
photosynthetic photon flux density (PPFD) under the shelters at
seedling level was 47 ± 0.67% (SE) of full sunlight, measured
at seven occasions in both direct and diffuse sunlight. The shelters had open sides to minimize the effect on temperature. Air
temperatures under the shelters and outside were monitored
with the EL-USB-2 + dataloggers (Lascar Electronics Ltd,
­Salisbury, UK) between the end of April 2012 and the end of
February 2014. On average, air temperature was 0.31 ± 0.03 °C
(SE) higher under the shelters than outside. However, the largest warming effect up to 3.5 °C was observed during the freezing weather in winter and early spring, and even a cooling effect
was observed during the temperature maxima in summer.
The experiment was set up in a split-plot design with three
treatments as a whole-plot level and nine seed sources as a
subplot level in three replications. The three treatments were
control (C), D and broken drought (BD). Each seed source was
represented by 10 trees within the whole plot for a total of 810
trees in the experiment (9 families × 3 treatments × 3 replications × 10 trees). During the first year after transplanting, seedlings in all treatments were well-watered (typically by 1 l per
plant every 4–5 days during the growing season). In the third
growing season (second year after transplanting), D was applied
to the D and BD treatments starting at 11 July 2013 (Figure 1).
Before the onset of D, some seedling mortality (6%) occurred
in the experiment for unknown reasons, although most of those
seedlings died in winter 2012–13. Watering was completely
withheld from both D treatments, whereas the C treatment
remained well-watered. The D ended in the BD treatment after
54 days at 3 September 2013, when no recovery of xylem
water potential was observed from midday to predawn (Figure 1). The BD treatment was then watered to full saturation,
and then watering was maintained as in the C treatment. Drought
in the D treatment continued until 21 November 2013, which
was 133 days (19 weeks) after the onset of D. Watering in all
three treatments continued periodically during winter (200–
250 ml per plant), and was resumed the next spring.
Measurements and observations
Xylem water potential During the D experiment, xylem water
potential was measured regularly on seedlings of seven half-sib
families in all three treatments. Predawn (Ψpd) and midday (Ψmd)
Tree Physiology Volume 36, 2016
xylem water potential were measured on shoots with a pressure
chamber (Scholander bomb, PMS Instrument Co., Albany, OR,
USA). The shoots ∼5 cm long were gently wrapped in aluminum
foil and sealed in plastic bags several hours before measurements. A symmetrical pair of side shoots was taken for Ψpd and
Ψmd. At each of 23 sampling occasions between June and
­October 2013, 63 trees were measured for Ψ (7 families × 1
tree × 3 treatments × 3 replications).
Photosynthesis and gs During the D experiment, Amax and gs
were measured 11 times on seedlings from 7 half-sib families
with the intent to measure gas exchange regularly during the season, with more frequent measurements during recovery from D.
The Amax expressed on a leaf-area basis was measured with the
LI-COR LI-6400 portable photosynthesis system (LI-COR Biosciences, Lincoln, NE, USA). Due to logistical issues involving the
narrow pathways between plants that made it difficult to maneuver the LI-6400, the Amax was measured on detached shoots
typically within 5 min after cutting. Preliminary results obtained on
well-watered samples showed that during this time, no difference
was noted in Amax or gs between attached and detached shoots.
The PPFD (mean ± SE) was 1200 ± 0.03 µmol m−2 s−1 provided
with the red/blue LED light source, representing light-saturated
conditions. On the first two measurement dates, the PPFD was set
to 800 ± 0.08 µmol m−2 s−1. Photosynthesis was determined at a
reference CO2 concentration of 400 ± 0.02 µmol mol−1, ambient
humidity and measurement temperature (block temperature)
varying between 14 and 26 °C, depending on ambient temperature. Measurements were completed within 4 h around the solar
noon, and typically took 2.5–3 min to reach steady state. Needle
shading in the cuvette was avoided by plucking some needles that
would cause shading before measurement. Needles present in the
measurement chamber were collected, scanned and analyzed for
projected area with the WinRHIZO image analysis software
(­Version 2001a, Regent Instruments, Quebec City, QC, C
­ anada).
At seven measurement dates, at least 63 trees were measured
(one measurement per plant), and at the remaining four dates
(2 July, 16 July, 2 August and 10 September), 42 trees were
sampled with one replication omitted.
Carbohydrate dynamics During the D experiment, the dynamics of total nonstructural carbohydrates (TNC) in needles were
analyzed. For this purpose, needles from shoots measured for
gas exchange were used, except for two occasions (June and
October) when needles from shoots measured for midday water
potential were used. The TNC were determined with the method
by ­Hansen and M
­ øller (1975) and ­Haissig and D
­ ickson (1979).
Sugars were extracted from the oven-dried (65 °C to the constant weight) tissue powder in a methanol : chloroform : water
solution (12 : 5 : 3 by volume), and the residue was used for
starch determination. The extracted soluble sugars were determined colorimetrically with the athrone reagent at 625 nm within
Variation among Norway spruce half-sib families 255
Figure 1. Time course in 2013 of midday and predawn xylem water potential in Norway spruce saplings grown in three treatments: C—control,
D—drought and BD—broken drought. Shaded area indicates the D period in the BD treatment; in the D treatment, D continued until 21 November.
At each date, points show treatment means (N = 21 per treatment), and error bars show SEM. Asterisks indicate dates when family differences
were significant.
30 min. Starch in the insoluble material was gelled and c­ onverted
to glucose with amyloglucosidase. Glucose was measured with
glucose oxidase by mixing the sample with the peroxidase–­
glucose oxidase–o-dianisidine dihydrochloride reagent. Absorbance was measured at 450 nm after a 30-min incubation
period at 25 °C. Concentrations of soluble sugars were calculated from the regression equations based on the glucose standard solutions. The soluble sugars and starch were expressed as
percent tissue dry mass.
Xylem vulnerability to embolism Xylem vulnerability to embolism was measured on stem sections of saplings from the seven
half-sib families in the C and D treatments. Most of saplings for
the xylem hydraulics study were sampled during the 2014
growing season when they were recovering from D. However, 12
seedlings in the D treatment were sampled in fall of 2013. After
harvesting, ∼20-cm-long stem sections were immediately
wrapped with wet paper towel, sealed in plastic bags and transported to the laboratory (250 m). The samples were then recut
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256 Chmura et al.
with a razor blade under water, debarked and saturated under
vacuum, typically for >18 h, to remove embolisms. Subsequently,
the maximum xylem specific conductivity was measured (ks(max)),
and embolism propagation was induced in the sample with a
double-ended pressure sleeve (PMS Instrument Co.) used for
the air-injection method (­Cochard et al. 1992, ­2013, ­Sperry and
­Saliendra 1994). Each sample was run through a series of
increasing pressures in 1 MPa increments. At each pressure
step, the flow rate was measured using 20 mM KCl solution. We
used a variation of the tubing system described in ­Torres-Ruiz
et al. (2012) to measure flow rate on our samples. Instead of a
balance reservoir, we used a pipette of appropriate volume. The
flow rate was measured at two positions of the pressure head
(upstream reservoir). The zero-flow was estimated using the
two-point slope method (­Torres-Ruiz et al. 2012) and subtracted from the flow estimate to account for passive water
uptake. The whole sample was wrapped in parafilm during the
flow measurement to prevent desiccation. Specific xylem conductivity—ks (­Tyree and ­Ewers 1991)—was calculated using
Darcy’s law according to the formula given in ­Domec and ­Gartner
(2001).
The percentage loss of conductivity (PLC) was calculated at
each pressure step in relation to ks(max) for each tree. A vulnerability curve (VC) describing the relationship between PLC and
applied pressure was fitted to the data at the family within a
treatment level with a sigmoid equation (­Pammenter and ­Van
der W
­ illigen 1998):
PLC =
100
(1 + exp(a(Ψ − b)) (1)
where PLC is the percentage loss of conductivity at a given
water potential Ψ (negative of the applied positive pressure),
and a and b are fitted parameters. The 14 VCs were generated
(7 families × 2 treatments) based on 66 trees in total. Most of
the VCs were based on data from five or more trees, although
two VCs used data from four trees and one from three trees. The
nonlinear procedure in the JMP 9.0.0 statistical package (SAS
Institute Inc., Cary, NC, USA) was used to fit the VC parameters.
The parameter b is an estimate of the pressure at which 50%
loss of conductivity occurs (Ψ50) (­Domec and ­Gartner 2001).
We used formulas given in ­Domec and ­Gartner (2001) to estimate the air-entry point (Ψe), the full embolism point (Ψmax) and
the slope (s) of the linear part of the VC. We calculated xylem
hydraulic safety margin as a difference between minimum water
potential experienced in each treatment (Ψmin) and Ψ50 (­Meinzer
et al. 2009, ­Choat et al. 2012).
Needle damage and plant survival Starting at the end of
August 2013, needle discoloration was scored using a scoring
scale ranging from 1 (no visible signs of needle damage—­
needles bright green) to 5 (needles completely yellow—sapling
Tree Physiology Volume 36, 2016
dead). Before that time, no visible macroscopic signs of D damage were observed on needles. Needle discoloration was scored
12 times during the 2013 season (between 19 August and 14
November) when D was applied. All nine seed sources were
assessed for needle damage, and the trait was analyzed as the
percentage of trees within a plot scored at least at a given score
(i.e., ≥3, ≥4 and 5). An additional scoring campaign was done in
spring 2014; at the same time, seedling survival was assessed
by the examination of phloem color and condition at the base
of the stem. Phloem on live trees was green and fresh, and on
dead trees, it was brown and dry or decaying. Mortality rate in
spring 2014 did not take into account trees that died before the
onset of D.
Statistical analysis
Variation at each sampling date for Ψ, Amax, gs and starch content was analyzed with analysis of variance (ANOVA) using the
following model:
Yijk = µ + Ri + T j + RTij + Fk + FTjk + eijk
(2)
where Yijk is an individual observation, µ the overall mean, Ri the
random effect of ith replication (i = 1, …, 3), Tj the fixed effect
of jth treatment (j = 1, …, 3), RTij the random effect of treatment × replication interaction (the error term for R and T effects),
Fk the fixed effect of kth family (k = 1, …, 7, …, 9—depending
on the trait), FTjk the family × treatment interaction term and eijk
is the error term. For needle damage and mortality, where only
plot-level data were available, the FT term was omitted from the
model. In case of significant differences for a given effect, means
were compared with the Tukey–Kramer HSD test and with contrast analysis. Before the analysis, Ψ values were converted to
positive, and Ψ and gs data were ln-transformed in order to conform to the ANOVA requirements, but results are presented in
the original scale.
Xylem vulnerability parameters were available only as family
averages within two treatments. Therefore, the ANOVA model for
these traits included only the effects of family, treatment and
error. Treatment effects were compared with the Student’s t-test.
All the statistical analyses were performed in the JMP 9.0.0 statistical package (SAS Institute Inc.).
Results
Plant water status
Xylem water potential in the C treatment was maintained above
−0.5 MPa predawn and above −1.0 MPa midday; thus, seedlings
did not experience water stress in this treatment throughout the
experiment (Figure 1). In both D treatments, a significant reduction in Ψ was observed only after 5 days for midday values, and
9 days for predawn values. The Ψ values in the D and BD
­treatments did not differ significantly from each other until the
Variation among Norway spruce half-sib families 257
watering resumption in the BD treatment (Figure 1). When the
Ψpd reached a level of about −2.0 MPa, and no recovery
between Ψmd and Ψpd was observed, the BD treatment seedlings were rewatered to field capacity. After rewatering, the Ψ
values in the BD treatment increased and remained intermediate
between the values for the C and D treatments for 17 days predawn and 31 days midday, after which the Ψ values were the
same in the C and BD treatments (Figure 1).
All families responded similarly to the imposed treatments.
The family effect was significant only at two measurements
before the onset of D, and at three measurements between 6
and 16 August, when the Ψpd in D treatments dropped from
about −0.8 to −1.5 MPa and Ψmd dropped from −1.2 to
−1.8 MPa (Figure 1). Family response to treatments was variable at that period as indicated by the significant family × treatment interaction term on 9 August (see Table S1 available as
Supplementary Data at Tree Physiology Online). The family 138
that showed the least negative Ψ value before the onset of D
had among the most negative Ψ values during that discussed
period in August.
Photosynthesis and gs
Both Amax and gs did not differ significantly among treatments
before and directly after (5 days) imposing D (Figure 2). Both
traits were significantly reduced in D treatments compared with
the C at 3 weeks after imposing D (Figure 2, see Table S1
available as Supplementary Data at Tree Physiology Online).
Afterward, the values did not differ between the two D treatments until rewatering in the BD treatment. Immediately
(1 day) after rewatering, the Amax and gs increased in the BD
treatment, but the values were intermediate between the D and
C treatments. The values further increased in the BD treatment,
but Amax remained lower than in the C until the last measurement (­Figure 2). The gs also remained lower in the BD than in
the C treatment throughout the rest of the experiment, but the
statistical significance of these differences varied by time
(­Figure 2). Family effect was not significant for Amax at any
time, and for gs, except for one measurement (Figure 2, see
Table S1 and Figure S1 available as Supplementary Data at
Tree Physiology Online).
Carbohydrate dynamics
Because only starch concentration, and not the glucose or total
soluble carbohydrate concentrations, exhibited variable dynamics among the treatments in the experiment, we present only
data on needle starch concentration. The family effect on needle
starch concentration was generally not significant, except for
three measurements (Figure 3, Figure S2 available as Supplementary Data at Tree Physiology Online). However, treatment
effect was significant, and in general no family × treatment interaction term was observed (see Table S1 available as Supplementary Data at Tree Physiology Online), meaning that all
families responded similarly to the imposed treatments. The
reduction of starch concentration from the initial peak to the low
level observed in all the treatments after water withholding was
not caused by D treatment. However, a month after water withholding, starch concentration was close to zero in both D treatments, whereas starch was accumulating in needles in the C
treatment (Figure 3). Rewatering in the BD treatment caused an
increase in needle starch concentration for at least 3 weeks after
rewatering, which coincided with the starch concentration falling
down in the C treatment (Figure 3). In the D treatment, starch
concentration remained at the level of ∼0.46% until the end of
the experiment.
Xylem vulnerability to embolism
Saplings in the D treatment had significantly less negative values
of Ψ50 and Ψe than those in the C treatment (Table 1, Figure 4).
The ks(max) and the slope of the linear part of the VC were significantly greater in the C treatment than in the D treatment (see
Figure S3 available as Supplementary Data at Tree Physiology
Online). The xylem safety margin was much narrower in the D
than in the C treatment (Table 1). The two treatments did not
differ in Ψmax.
The family effect was not significant for any parameter of VCs,
except safety margin, although within each treatment there was
some variation among families in examined parameters
(Table 1). The Ψe varied among families in the D treatment from
−1.9 to −3.0 MPa (Table 1), and the time when Ψe was permanently reached varied from 4 weeks after water withholding in
family 125 to 8.5 weeks in families 86 and 121 (Figure 5).
Midday xylem water potential in the D treatment did not reach
Ψmax in any family, and the Ψmin − Ψ50 hydraulic safety margins
were positive (Table 1), although Ψ50 values were approached
at some occasions during D in most families (see 95% confidence interval in Figure 5). Neither predawn nor midday xylem
water potential reached Ψe in the C treatment (not shown). Significant variation among families was found for safety margin
(Table 1), but also a significant family × treatment interaction
term, implying that safety margin varied among families but was
dependent on the treatment.
Needle damage and plant survival
During the D experiment, the most variation in needle damage
was found for the percentage of trees that scored ≥3 (Figure 6).
The family and treatment effects were significant (P ≤ 0.0099
and P ≤ 0.0268, respectively), except for the treatment effect at
2 out of 12 scoring campaigns (19 August and 29 August). In
general, the two D treatments had higher percentage of trees
with visible needle damage than the C treatment. There was
some recovery in the BD treatment observable 6 days after rewatering on 9 September (P = 0.0263 for the contrast between
5 September and 9 September in BD). Between 23 September
and 25 October, the percentage of trees with ­damage score ≥3
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258 Chmura et al.
Figure 2. Time course of light-saturated photosynthesis (Amax) in (a) and gs in (b) in Norway spruce saplings grown in three treatments: C—control,
D—drought and BD—broken drought. Shaded area indicates the D period in the BD treatment; in the D treatment, D continued until 21 November. At
each date, points show treatment means (N varied by date from 14 to 21 per treatment), and error bars show SEM. Different lower case letters at each
date indicate treatment means that were significantly different in the Tukey–Kramer test at α = 0.05 or contrast analysis at P ≤ 0.05 (only shown at
dates when treatment differences were significant). An asterisk indicates the date when family differences were significant.
was significantly lower in the BD than in the D treatment (Figure 6). Family differences were also significant; at the last
scoring campaign in November 2013, the percentage varied
among the families from 23 to 100% in the D treatment, and
from 10 to 66% in the BD treatment. The ranking of families
differed between these treatments, but the four families with
most damage (126, 122, 138 and 125) were at the top of
the damage ranking in the D and BD treatments throughout the
study.
Tree Physiology Volume 36, 2016
During the D experiment in 2013, no significant variation was
found for the percentage of trees scored ≥4, which was <4.5%
in the D and <3.5% in the BD treatment. This means that
although the percentage of trees at the score 3 increased successively throughout the experiment (Figure 6), few of those
trees advanced to a higher needle damage class. Only eight
trees (1%) were scored dead based on needle damage
(score 5) at the last scoring campaign in 2013 (six in the BD
treatment, and one each in the D and C treatments).
Variation among Norway spruce half-sib families 259
Figure 3. Time course of starch content in current-year needles of Norway spruce saplings grown in three treatments: C—control, D—drought and BD—
broken drought. Shaded area indicates the D period in the BD treatment; in the D treatment, D continued until 21 November. At each date, points show
treatment means (n = 21 per treatment, no data shown for the C treatment on 21 June because not all families were represented), and error bars show
SEM. Different lower case letters at each date indicate treatment means that were significantly different in the Tukey–Kramer test at α = 0.05 or contrast
analysis at P ≤ 0.05 (only shown at dates when treatment differences were significant). Asterisks indicate dates when family differences were significant.
Table 1. Values of Ψ50, Ψmax and Ψe, slope of the VC, Ψmin, xylem hydraulic safety margins and ks(max) for seven half-sib families of Norway spruce
growing in the drought (D) and control (C) treatments. Analysis of variance P values for the main effects are given at the bottom of a table. Standard
errors (in parentheses) were available only for Ψ50, Ψmin, safety margin and ks(max) because other variables were calculated from the parameters of
Eq. (1) fitted at the level of family within treatment. Bold values show statistically significant effects.
Treatment
Family Ψ50 (MPa)
D
D
D
D
D
D
D
86
98
121
122
125
126
138
Mean
C
C
C
C
C
C
C
86
98
121
122
125
126
138
Mean
ANOVA P values
Family
Treatment
Family × treatment
Replication
Replication × treatment
−4.58 (0.14)
−4.25 (0.18)
−4.56 (0.15)
−3.99 (0.17)
−4.14 (0.33)
−4.55 (0.14)
−3.85 (0.11)
−4.27 (0.11)
−4.97 (0.14)
−5.01 (0.17)
−4.43 (0.10)
−4.95 (0.07)
−4.81 (0.11)
−4.50 (0.13)
−4.32 (0.14)
−4.71 (0.11)
Ψmax (MPa) Ψe (MPa) Slope of the
Ψmin (MPa)
VC (% MPa−1)
Safety margin
ks(max) (kg m−1
Ψmin − Ψ50 (MPa) MPa−1 s−1) × 10−4
−6.15
−6.09
−6.57
−5.86
−6.37
−6.26
−5.43
−6.11
−6.39
−6.89
−5.57
−5.76
−6.00
−5.89
−5.41
−5.99
1.77 (0.33)
1.32 (0.29)
1.72 (0.22)
0.58 (0.05)
1.01 (0.26)
1.22 (0.19)
1.17 (0.21)
2.36 (0.37)
2.50 (0.89)
3.10 (0.36)
2.98 (0.49)
2.54 (0.75)
2.83 (0.90)
2.54 (0.46)
1.25 (0.11)
3.93 (0.09)
4.02 (0.02)
3.40 (0.05)
3.84 (0.03)
3.71 (0.02)
3.48 (0.08)
3.39 (0.06)
2.68 (0.21)
3.80 (0.75)
4.66 (0.45)
4.30 (0.96)
3.73 (0.78)
4.05 (0.54)
4.18 (0.75)
4.25 (0.60)
3.68 (0.06)
4.12 (0.26)
0.0073
0.0010
0.0028
0.2427
0.4847
0.9669
0.0002
–
–
–
0.4735
0.0298
–
–
–
0.3105
0.5971
–
–
–
−3.00
−2.41
−2.54
−2.12
−1.90
−2.84
−2.26
−2.44
−3.54
−3.12
−3.28
−4.13
−3.62
−3.10
−3.23
−3.43
0.8571
0.0063
–
–
–
31.7
27.2
24.8
26.7
22.3
29.2
31.6
27.7
35.1
26.5
43.6
61.4
42.1
35.9
45.9
41.5
0.5890
0.0225
−2.81 (0.33)
−2.93 (0.29)
−2.84 (0.22)
−3.41 (0.05)
−3.13 (0.26)
−3.33 (0.19)
−2.68 (0.21)
−3.02 (0.09)
−1.04 (0.09)
−0.99 (0.02)
−1.03 (0.05)
−1.11 (0.03)
−1.10 (0.02)
−1.02 (0.08)
−0.93 (0.06)
−1.03 (0.02)
0.1253
0.0015
0.4231
0.2427
0.4847
Tree Physiology Online at http://www.treephys.oxfordjournals.org
260 Chmura et al.
Figure 4. Percentage loss of xylem conductivity vs negative pressure in the control (C) and drought (D) treatments in seven families of Norway spruce.
Each point represents a family mean within a treatment (n = from 3 to 6). Lines show model fit according to Eq. (1) for each treatment.
At the scoring campaign in spring 2014 after the D, the percentage of trees with damaged needles increased, compared
with the end of the previous year (Figure 6). Treatment effect
was significant (P ≤ 0.0155) for the percentage of trees scored
≥4 and at score 5, but not for trees scored ≥3. Significantly more
trees were damaged in the D treatment (62% for score ≥4, and
55% for score 5) than in the other two treatments (23 and 2%
for score ≥4, and 7 and 0.5% for score 5 in the BD and C treatments, respectively). No significant variation among families was
found for needle damage in spring 2014.
Similar to needle damage, seedling mortality in spring 2014
varied significantly among treatments (P = 0.0044), but not
among families (P = 0.1303). Between the onset of D (June
2013) and the scoring of survival in spring 2014, 62% of saplings died in the D treatment, whereas only 8% died in the BD
treatment and 0.5% in the C treatment. Although the family
effect was not significant, mortality varied among families from
36 to 85% in the D treatment.
Discussion
Despite its potential importance for modeling vegetation
responses and forest management under projected climate
change, knowledge of within-species variation in response to
D and in traits associated with D resistance in forest trees is
still limited (­Anderegg 2015). In this controlled environment
study, we investigated the response of gas exchange
Tree Physiology Volume 36, 2016
­ hysiology to imposed D, stem xylem vulnerability, needle
p
damage and survival in saplings of seven open-pollinated halfsib families and two population-level progenies of Norway
spruce. We were specifically interested in exploring the extent
of within-population variation in examined traits in this important European tree species. The results of our study contribute
new knowledge to growing evidence of the patterns of withinspecies variation in vulnerability to embolism in coniferous
trees.
Most of the variation in our experiment was associated with
the applied treatments—C, D and BD. However, we observed
significant differences among families in xylem water potential
(Ψ) before the onset of D, which likely reflected the inherent
physiology of the examined families. After the onset of D, it was
only during the early period of D when the stress level (Ψmd
between −1.2 and −1.8 MPa) allowed family differences to be
resolved. After that, the stress was too much to detect variation
among families. These observations indicate that the mechanisms regulating xylem water potential in response to D were
conservative and species specific, and indicate that within-­
population variation in this species may manifest only at mild D.
However, we found that the time when Ψe was reached varied by
31 days among families. If more frequent, but not particularly
severe Ds occur in the future, that variation may contribute to
selective advantage of some families over others.
The gas exchange parameters were reduced in both D
treatments, and the response was consistent among all the
­
Variation among Norway spruce half-sib families 261
Figure 5. The time course of midday xylem water potential (Ψmd) in seven half-sib families of Norway spruce growing in the D treatment in relation to
the Ψe and Ψ50 values for each family indicated by horizontal dashed lines. At each date, points show mean ± 95% confidence interval (n = 3). The
vertical broken line indicates date of water withholding. Note the difference in Y-axis scale for family 98. The distance between the lowest point and
Ψ50 line, shows the minimum safety margin attained during D.
Tree Physiology Online at http://www.treephys.oxfordjournals.org
262 Chmura et al.
Figure 6. Percentage of trees with needle damage score ≥3 in three treatments: C—control, D—drought and BD—broken drought during the D
experiment (2013) and in the spring after D experiment (2014). Bars show treatment means (N = 27) and whiskers show SEM.
e­ xamined families of Norway spruce. Stomatal closure, and thus
stomatal limitation of photosynthesis, is the first response to slow
water stress in plants (­Chaves et al. 2002). It is also a mechanism
preventing D-induced xylem cavitation (­Sperry 2000, ­Maherali
et al. 2006). In saplings of Norway spruce and Picea mariana
(Mill.) Britton, Sterns & Poggenb. subjected to a D of the magnitude equivalent to that in our experiment (in terms of Ψ experienced), a similar level of reduction in photosynthesis and gs was
found (­Ditmarová et al. 2010, ­Balducci et al. 2013). In our study,
we observed a sudden, but reversible decrease in Amax, and especially in gs, in all treatments on 6 September (Figure 2, see also
Figure S1 available as Supplementary Data at Tree Physiology
Online), which is hard to interpret with the available data. One possible cause might be the return to relatively high maximum air
temperatures after a period of lower maximum air temperatures
(see Figure S4 available as Supplementary Data at Tree Physiology
Online). However, all families exhibited this pattern and the amongfamily variation in gas exchange was mostly not significant. In
P. menziesii, differences in δ13C, and thus water-use efficiency,
resulted from variable stomatal behavior between two populations,
but no variation was found among families within populations
(­Aitken et al. 1995). Similarly, differences in δ13C, and thus gs,
have been observed between populations in Pinus ponderosa
Douglas ex P.Lawson & C.Lawson, suggesting that local adaptation
can evolve within a species (­Kerr et al. 2015). In Picea glauca
(Moench) Voss, no variation was found among families in CO2
assimilation and gs in response to D (­Bigras 2005). These observations together with our results suggest that gas exchange
parameters, although sensitive to D, may be indicative of the
Tree Physiology Volume 36, 2016
response typical for the s­ pecies or p
­ opulation, but are rather poor
indicators of within-population variation in D resistance.
Needle starch concentration in the C treatment exhibited the
natural dynamics for current-year needles in Norway spruce
associated with needle morphogenesis and a sink/source transition (­Hampp et al. 1994, ­Egger and ­Hampp 1996). The final
seasonal decline in needle starch indicates export of carbohydrates from needles, which become a source of photosynthates for other tissues (­Egger and H
­ ampp 1996) and may
play a role in building frost resistance (­Morin et al. 2007,
­Charrier and ­Ameglio 2011, ­Charrier et al. 2013). However,
that last peak of starch concentration was absent from the D
treatment, and was delayed and much higher in needles of the
BD compared with the C treatment, indicating that after rewatering, trees in the BD treatment overcompensated for the
starch deprivation during D. The dynamics of leaf starch in the
D and BD treatments, directly reflecting reduction and recovery
of Amax and gs, indicates that gs was a major limitation of photosynthesis during D. However, the slow recovery of photosynthesis and gs after rewatering in the BD treatment indicate that
some limitation due to embolism likely also took place. Xylem
water potentials in the D and BD treatments were very close to
each other up to the point of rewatering in the BD treatment (3
September), when Ψ in saplings of all families in the D treatment was close to or below their Ψe point (see Figure 5).
These observations suggest that some embolism likely
occurred also in the BD treatment.
Variation in xylem vulnerability is associated with D resistance. Water potentials in stem xylem inducing 50% loss of
Variation among Norway spruce half-sib families 263
c­ onductivity have been considered lethal in conifers (­Brodribb
and C
­ ochard 2009, ­Brodribb et al. 2010), although recovery
has been observed over seasonal time periods in a wide range
of conifer species, including Norway spruce (­Mayr et al. 2002,
­2003b, ­2006, ­McCulloh et al. 2011). However, Ψ50 is physiologically relevant because at that point, xylem water potentials
operate along the steepest part of VC causing a severe risk of
catastrophic hydraulic failure (­Meinzer et al. 2009). The Ψ50
values found in our study were very similar to those reported for
stemwood of similarly sized Norway spruce (­Mayr et al. 2003a,
­Rosner et al. 2007) and P. mariana (­Balducci et al. 2015), and
were within the range of Ψ50 reported for Pinaceae
(­Martínez-Vilalta et al. 2004, ­Bouche et al. 2014). The Ψe and
ks(max) values were also close to those reported for 3-year-old
Norway spruce (­Rosner et al. 2007). In our study, we found no
statistically significant differences among examined families in
Ψ50 and other parameters of xylem vulnerability (except for
safety margin), but significant differences between the D and C
treatments. ­Rosner et al. (2007) found no variation in Ψ50
among eight clones of Norway spruce, although clonal differences in stemwood Ψ50 were found in P. menziesii (­Dalla-Salda
et al. 2009, ­2011). All these observations indicate that xylem
vulnerability may show little within-population variation in N
­ orway
spruce.
Contrary to our expectation, trees exposed to D did not produce more embolism-resistant wood compared with those in the
C treatment. However, droughted trees had a less steep VC than
in the C. Based on our findings, it is not clear whether the difference in vulnerability between the D and C treatments was due to
differences that occurred in the wood that was formed during or
after the D. Preliminary results of wood anatomy measurements
indicated that trees in the D treatment produced no or only small
amount of latewood compared with C (M. Guzicka, unpublished
data), which may have contributed to differences we observed
in xylem vulnerability between treatments (­Domec and ­Gartner
2002). Studies on xylem anatomy are warranted to help interpret the patterns of variation we found in xylem hydraulics.
There is an extremely small chance that the seedlings that were
randomly assigned to the two treatments had wood that exhibited different vulnerability to embolism before the D was
imposed, but that is highly unlikely. Another possibility is that the
treatment imposed ‘cavitation fatigue’ on the droughted seedlings (­Hacke et al. 2001). Cavitation fatigue is observed in some
species when woody tissue that has been exposed to D stress
exhibits a more vulnerable response to subsequent Ds, and has
been observed in Norway spruce before (­Rosner and ­K awamoto
2010). It could be that some damage incurred during the D, for
example, stretching of the margo of the pit membrane during
the propagation of a bubble from one tracheid to the next, made
the tissue more vulnerable to D stress of the imposed air
­pressures when the VCs were measured. Our findings show that
D-exposed saplings of this generally D-vulnerable species may
have an increased chance of survival during D through a mechanism that delays their xylem hydraulic failure. However, because
in the D treatment the measured xylem water potentials reached
only Ψe, and not Ψ50, it is difficult to assess the field relevance
of the slope of VC between these two points. Positive values of
the Ψmin − Ψ50 safety margins suggest that no catastrophic
hydraulic failure occurred in stem wood. Thus, a greater mortality of saplings in the D than in C treatment apparently was not
caused by excessive hydraulic failure of stem xylem. Typically,
xylem in roots is more vulnerable to embolism than in stems
(­Alder et al. 1996, ­Sperry and I­keda 1997, ­K avanagh et al.
1999). If root xylem was embolized in droughted trees, it could
cause increased mortality observed in the spring following the
D. At the end of 2013, all trees were rewatered, and thus, recovery should be observed as long as xylem remained at least partially functional.
Visual scoring of needle damage was not a good indicator of
D response. This method of assessment seemed attractive
despite its subjectivity, because the observations were repeatable (checked for the observer). Although we found significant
family variation in needle damage score ≥3 at most observation campaigns, it apparently did not reflect well the condition
of plants during D. After overwintering, the needle damage
increased to the level where differences among treatments
dominated over family variation. Yet, even then the visual scoring had little value as determined by the discrepancy in assessment of mortality between needle scoring and the examination
of phloem. Those observations highlight that macroscopic
assessment of D damage in Norway spruce based on the
appearance of needles is problematic and may give a false
impression of resistance to D. Thus, the measurements of
xylem water potential are indispensable in order to assess the
physiological condition of spruce saplings. This is unfortunate
for breeding programs where fast screening of many genotypes is necessary.
The limited set of families may contribute to the lack of variation we observed at the family level in our study. Assessment of
more genotypes, perhaps of contrasting phenotypes, would help
to generalize our findings about the patterns of variation in
xylem hydraulics within this species. However, the number of
families was restricted partly because of the time efficiency of
the methods used in the study. Patterns of genetic variation in
resistance to embolism using broader sets of populations and/or
families have been reported only recently with the advent of new
methods allowing relatively high-throughput assessment of
xylem vulnerability (­Cochard 2002). With the use of those novel
methods, no variation among populations in xylem hydraulic vulnerability was reported in both conifers (­Lamy et al. 2011,
­2 014, ­
S áenz-Romero et al. 2013) and angiosperms
(­Wortemann et al. 2011), but significant within-population variation was found in F. sylvatica (­Wortemann et al. 2011). The
results of our study indicate that rather small within-population
Tree Physiology Online at http://www.treephys.oxfordjournals.org
264 Chmura et al.
variation should be expected for vulnerability to embolism in
Norway spruce.
In conclusion, we found only limited variation among families
within a single population in gas exchange parameters response
to D and in xylem vulnerability to embolism. Families showed
variable percentages of needle damage initially, but we did not
find significant variation in sapling mortality, despite quite a
wide range of that variation. We also found family variation in Ψ
only at the initial stages of D, but not afterward, suggesting that
all families responded in a similar way to a greater D intensity,
thus exhibiting the species-specific response. However, the
variation we found in the time when Ψe was attained among
families may possibly be the mechanism underpinning variation
in D survival among trees in populations consisting of many
families. In summary, the observed responses to D and their
underlying mechanisms seem general for the species, with little
variation among families—this pattern of variation may limit
breeding opportunities for increased D resistance in tree
improvement programs of Norway spruce in light of expected
changes in climate.
Supplementary data
Supplementary data for this article are available at Tree Physiology
Online.
Acknowledgments
We thank the editor and three anonymous reviewers for their
constructive comments that helped to improve an earlier version
of the manuscript. We also thank Henryka Przybył, Roman
Rożkowski and Damian Michałowicz for field assistance, and
Maria Hładyszewska for help with measurements of xylem
hydraulics.
Conflict of interest
None declared.
Funding
This work was supported by National Science Centre, Poland (N
N309 713340) and National Science Foundation (IOS1146751 to K.A.M.).
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