Directory UMM :Data Elmu:jurnal:T:Tree Physiology:vol17.1997:
Tree Physiology 17, 115--123
© 1997 Heron Publishing----Victoria, Canada
Relationships between gas exchange adaptation of Sitka× interior
spruce genotypes and ribosomal DNA markers
SHIHE FAN, STEVEN C. GROSSNICKLE and BEN C. S. SUTTON
Forest Biotechnology Centre, B.C. Research, Inc., 3650 Wesbrook Mall, Vancouver, B.C. V3S 2L2, Canada
Received February 10, 1996
Summary Adaptive physiological changes were investigated in seven populations of Sitka (Picea sitchensis (Bong.)
Carr.) × interior spruce (P. glauca (Moench) Voss × P. engelmannii Parry ex Engelm.) spanning the Nass--Skeena transition
zone in British Columbia, Canada. Each population was represented by an Si rDNA index that was calculated from the
relative optical densities on a gel autoradiogram of five ribosomal DNA bands characteristic of Sitka spruce and interior
spruce. This index estimates the proportion of the genome
contributed by interior spruce. Physiological adaptations were
assessed by gas exchange parameters measured under both
well-watered and drought conditions. Under well-watered conditions, Sitka spruce populations had higher maximal photosynthesis at saturating light and ambient CO2, higher quantum
yield at the light compensation point, and higher dark respiration than interior spruce populations. Sitka spruce populations
also reached maximal photosynthesis at lower photosynthetically active radiation and higher CO2 concentrations, and had
higher stomatal densities that resulted in lower stomatal limitations to photosynthesis than interior spruce populations. In
contrast, interior spruce populations exhibited greater drought
tolerance than Sitka spruce populations. Their gas exchange
rates declined at a slower rate in response to drought. They
maintained higher gas exchange rates in response to moderate
to severe drought (predawn plant water potentials = −1.5 MPa),
and their photosynthetic rates recovered faster when they were
rewatered after exposure to drought. Comparison of the seven
populations indicated that physiological parameters were significantly related to the Si rDNA index. An increase in Si rDNA
index was associated with proportional changes in physiological measurements, suggesting that genetic interchange among
species with contrasting ecological adaptations can enhance
the environmental adaptation of natural populations.
Keywords: DNA analysis, drought, Picea engelmannii, Picea
glauca, Picea sitchensis.
Introduction
Introgressive hybridization occurs between Sitka spruce
(Picea sitchensis (Bong.) Carr.) and interior spruce (P. glauca
(Moench) Voss × P. engelmannii Parry ex. Engelm.) in the
Nass--Skeena transition zone in northwestern British Colum-
bia, Canada (Roche 1969, Daubenmire 1972). Sitka spruce
naturally occurs in the maritime climate on the Pacific coast of
North America (Burns and Honkala 1990). The parental species of interior spruce, Engelmann spruce (P. engelmannii) and
white spruce (P. glauca), have contrasting ecological adaptations. Engelmann spruce grows in cool, humid environments
at high elevations, whereas white spruce is found in dry, cool,
continental conditions across North America (Burns and
Honkala 1990). Reproductive difficulties sometimes occur
when two distinctive spruce species are crossed, but Sitka,
Engelmann, and white spruce are phylogenetically close and
hybridize easily (Wright 1955). It is important to understand
the consequences of introgression between these spruce species, which may help develop breeding and dispersal strategies.
Populations taken from the Sitka × interior spruce introgression zone are highly heterogeneous in both their nuclear and
plastid DNA (Sutton et al. 1991a, 1991b, 1994). Sitka spruce
has different ecological adaptations than interior spruce, therefore, the DNA exchange between these species has caused
corresponding alterations in physiological attributes. Changes
in drought and freezing tolerance of Sitka × interior spruce
populations correspond with their nuclear ribosomal DNA
(rDNA) patterns (Grossnickle et al. 1996). These results are
consistent with evolutionary studies, which suggest that introgression may produce new ecotypes both by transferring existing adaptive characteristics from one population to another and
by giving rise to new adaptations (Rieseberg and Wendel
1993).
Because of the scarcity of experimental evidence for adaptive variabilities resulting from DNA exchange among species
with different ecological adaptations (Grossnickle et al. 1996),
little is known about the significance of DNA changes on
morphological, physiological and phenological attributes of
trees (Mitton 1995). Individual forest tree species are usually
distributed over a large geographic range and their genotype
and phenotype vary with environmental variation across the
range (Abrams 1994). It is important, therefore, to establish a
relationship between genotypic (DNA) and phenotypic (i.e.,
physiological) attributes that is independent of environmental
variability.
Following the study of Grossnickle et al. (1996), we examined the adaptive significances of DNA exchange in seven
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FAN, GROSSNICKLE AND SUTTON
Sitka × interior spruce introgressive hybrid populations from
the Nass--Skeena transition zone. Specifically, gas exchange
characteristics were measured under both well-watered and
drought conditions, and their relationships with the rDNA
values of the populations examined. Results suggested that
DNA exchange between Sitka spruce and interior spruce has
broadened the ecological adaptations of spruce populations in
the Nass--Skeena transition zone.
Materials and methods
Plant materials
Seven Sitka (Picea sitchensis) × interior (P. glauca × P. engelmannii) spruce populations were sampled from the Nass-Skeena transition zone in northwestern B.C., Canada. Four
were wild seed collections, one was an operational seedlot
pooled from the Bulkley Valley seed orchard, and two were
seedlots from open pollinated families within the Bulkley
Valley seed orchard (each consisting of five families based on
the localities of the original parental trees). A brief summary
of the seed collection is given in Table 1 and a detailed description has been given by Sutton et al. (1994).
The history of seedling culture has been detailed by Grossnickle et al. (1996). In brief, seedlings were 1-year-old container-grown plants raised at the B.C. Ministry of Forests
Saanich nursery (Sidney, B.C., Canada; 48°28′ N and 124°10′ W).
Seedlings were transplanted in the second year to a farm field
site at the University of B.C. (Vancouver, B.C., 49°15′ N and
123°15′ W). Seedlings used in the experiment had been
planted in 11-liter pots to facilitate rapid and nondisruptive
removal for physiological testing. The pots were randomly
placed within the field site design and submerged in the soil so
that seedlings were exposed to normal site edaphic conditions.
Just before the third growing season, potted seedlings were
relocated to an outdoor compound at B.C. Research, Inc. (2 km
away from the farm field site). During the third year, seedlings
were watered every three days and fertilized every other week
with 20,8,20 (N,P,K) Plant Prod forestry seedling fertilizer
(Plant Products Co., Ltd., Brampton, ON, Canada) (equivalent
to 200 ppm N). Experiments were started in late June immediately after seedlings had set terminal buds.
DNA Analysis
Each value of Si rDNA index listed in Table 1 was calculated
from rDNA data taken on eight experimental seedlings within
a population. Details of the DNA analysis procedures have
been described previously (Sutton et al. 1994, Grossnickle et
al. 1996). In brief, rDNA was isolated from three buds from
each seedling. After purification, rDNA was digested with the
restriction enzyme, HindIII, followed by electrophoresis in a
0.8% agarose gel. Ribosomal DNA was transferred to Hybond
filters (Amersham, Oakville, ON, Canada) by Southern blotting, and hybridized with a α32P-dATP labeled probe, Eco 2.0
(yeast 18S RNA gene). Ribosomal DNA bands were visualized by autoradiography, and an Si rDNA index was then
calculated, as described previously, for each seedling according to the optical density of the five characteristic bands (Sutton et al. 1994, Grossnickle et al. 1996). The Si rDNA index
gives an overall estimation of interior spruce DNA contribution to the genome of each seedling.
Photosynthetic characterization of well-watered and
drought-treated seedlings
Photosynthetically active radiation (PAR) versus net photosynthesis (Pn) curves and intercellular CO2 (Ci ) versus Pn
curves were constructed from the seven test populations.
Measurements were taken on newly developed, unshaded,
lateral branches in the upper crown with an LI-6200 Portable
(closed-loop) Photosynthesis System and a 250-ml leaf cuvette
(Li-Cor, Inc., Lincoln, NE). Curves were constructed from six
seedlings for each population.
The PAR--Pn response curves were measured twice on the
same branch of each seedling under both well-watered (predawn plant water potentials (Ψpd) of about −0.2 MPa) and
moderate drought (Ψpd of −1.0 to −1.1 MPa) conditions. Measurements were taken in an environmentally controlled growth
room providing the following conditions: 22 °C air temperature, 40 ± 5% relative humidity, and 400 ± 10 µl l −1 ambient
CO2. Light was provided by two Sylvania 1000 W Metalarc®
metal halide lamps (Osram Sylvania Inc., Manchester, NH). To
obtain the PAR--Pn response curve, PAR was varied by changing the thickness of plastic sheets between the seedling and the
light. Air temperature in the leaf cuvette was held virtually
constant during measurements with a 360-mm external electrical fan. Measurements were started with dark-adapted seed-
Table 1. Summary of seed source information and the interior spruce rDNA index (mean ± SE; n = 8) for each test population.
Source
Seedlot
Seed zone1
Location
Elevation (m)
Si rDNA Index
1
2
3
4
5
6
7
1004
4075
4132
14657
6308
Five OP families
Five OP families
NST
BLK
SM
NST
Kitwanga, 55°20′ N; 128°05′ W
Morice West, 54°10′ N; 127°15′ W
Lower Nass, 54°58′ N; 129°38′ W
Meziadan Lake, 56°06′ N; 129°19′ W
Bulkley Valley, 53°30′ to 55°40′ N; 127° W
Morice Lake, 54°20′ N; 127°30′ W
North Babine, 55°35′ N; 126°40′ W
305
760
10
290
< 840
< 840
< 840
0.51 ± 0.06
0.92 ± 0.05
0.11 ± 0.03
0.36 ± 0.06
0.97 ± 0.09
0.81 ± 0.11
0.84 ± 0.03
1
NST = Nass--Skeena Transition, BLK = Bulkley Valley, SM = Spruce Maritime.
GAS EXCHANGE ADAPTATION AND RIBOSOMAL DNA MARKERS
lings. After a change in PAR, further measurements were not
made until after an interval of 15--20 min, which in a preliminary trial was found sufficient to allow photosynthesis to reach
a steady state.
The Ci --Pn curves were measured on well-watered seedlings
under the conditions described above except that PAR was 750
µmol m −2 s −1 and CO2 in the leaf cuvette was progressively
reduced from an initially high concentration (1100--1200 µl l−1)
by seedling photosynthesis (McDermitt et al. 1989). Before
each Ci --Pn measurement, the initial CO2 concentration was
sustained for 10 min by slowly pumping CO2 (compressed
CO2 mixed with air) into the LI-6200 system through an
external flow switch using a Li-Cor 6000-01 gas calibration
cylinder. This treatment acclimated stomata and photosynthesis to the high CO2 environment and stabilized stomatal conductance without significant variation during the 30- to 50-min
measuring period. After this acclimation treatment, CO2 in the
cuvette was drawn down by the seedling (McDermitt et al.
1989), and data logging was started when CO2 in the cuvette
was 900 to 1000 µl l −1. When CO2 in the cuvette reached the
CO2 compensation point for Pn, the remaining CO2 in the
system was drawn down further by both the online and an
external soda lime tube attached to the external flow switch.
This allowed the Ci--Pn curves to be extended to a CO2 concentration approaching zero. Both Pn and Ci were corrected for
system leakage following the manufacturer’s recommendations (Application No. 103, Li-Cor, Inc. 1991).
Gas exchange response to drought
After the measurements of PAR--Pn and Ci --Pn under well-watered conditions, seedlings were subjected to soil drought by
withholding water for approximately 35 to 40 days. The
drought was simulated under an outdoor rain shelter. The
evening before each measuring day, seedlings were moved to
the growth room and placed on a darkened growth table for
10 h. The next morning, Ψpd was measured with a pressure
chamber (Model 3005, Soil Moisture Equipment Corp. Santa
Barbara, CA) on a lateral branch from the upper crown of each
seedling before the lights were turned on. Two hours later, gas
exchange parameters (net photosynthesis (Pn), stomatal conductance to water vapor (gwv) and transpiration flux density
(TFD)) were measured on an upper crown lateral branch with
the LI-6200 system under conditions similar to those for Ci --Pn
measurements, except that CO2 concentration in the leaf cuvette was 390 ± 10 µl l −1. The same branch was measured
throughout the drought and rewatering period. When Pn decreased to just less than 0, the soil was fully rehydrated. Gas
exchange was measured again exactly 24 h after rehydration.
To compare the relative rates of gas exchange recovery after
rewatering, rates relative to the pre-drought values are presented. Total needle surface area of each lateral branch sample
enclosed in the leaf chamber for gas exchange measurements
was determined from a relationship with oven-dried (70 °C)
needle dry weight. Gas exchange measurements were then
recalculated accordingly.
117
Stomatal morphology
Stomatal sizes were measured on current-year needles of five
selected populations (Nos. 1--4, and 7 in Table 1, Nos. 5 and 6
were excluded to reduce sample sizes). Three needles from
each of the top, middle, and bottom positions on an uppercrown branch were randomly excised, cut in half, and the
mesophyll removed. Stomatal sizes (nine per needle) on the
needle epidermis were then determined under a light microscope. Stomatal density assessment (nine views per impression) was made on needle surface impressions made from
fingernail polish. Three impressions were made for each seedling.
Data analysis
Equation 1 was used to model changes in Pn, gwv, and TFD
with decrease in Ψpd:
Y = ae(b − cx),
(1)
where Y is either Pn, gwv or TFD; x is Ψpd; and a, b, and c are
model parameters. The last negative Pn data point of each
seedling was excluded from the data set used for modeling. To
compare the dehydration tolerance of populations, the values
of Ψpd at which Pn, gwv, or TFD were half their pre-drought
maxima were determined by means of Equation 1. The higher
the Ψpd value, the lower the dehydration tolerance. Equation 1
was also used to normalize Pn, gwv, and TFD to −1.5 MPa for
comparisons among populations.
Equation 2 defined PAR--Pn and Ci --Pn curves for each
seedling:
Y=a+
bx
,
c+x
(2)
where Y is Pn; x is either PAR or Ci ; a is dark respiration (Rd)
in PAR--Pn measurements or photorespiration in Ci --Pn measurements (because real Rd is generally inhibited at PAR above
500 µmol m −2 s −1 (Villar et al. 1994)); b is the potential
maximum Pn under saturating PAR or ambient CO2; and c is
the light utilization or CO2 affinity constant when Pn was half
maximal (Leopold and Kriedemann 1975). Equation 2 generally fitted the data well (r 2 from 0.97 to 1.00 for Ci --Pn curves
and 0.94 to 0.98 for PAR--Pn curves, respectively). Quantum
yield (φ), the initial slope at the light compensation point for
Pn, and carboxylation efficiency of ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco) (VRuBP), the initial slope at
the CO2 compensation point for Pn, were estimated from the
PAR--Pn and Ci --Pn curves, respectively (Farquhar et al. 1980).
Stomatal limitations (I) to Pn were estimated from Ci--Pn
curves by the differential method (Jones 1985):
I=
1/g wv
,
1/gwv + 1/gm
(3)
where gwv was the slope of the ‘‘supply function,’’ i.e., measured gwv; gm was the slope of the ‘‘demand function,’’ i.e., Ci--Pn
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FAN, GROSSNICKLE AND SUTTON
curve, at the operating point. The operating point was determined according to the ‘‘demand’’ and ‘‘supply’’ functions
fitted or measured for each individual seedling.
The relationship of the Si rDNA index to physiological
measurements in the seven populations was examined by the
Pearson product-moment correlation analysis, because the
Si rDNA index was designed to describe the genotype of a
population in a specific subzone in the Nass--Skeena transition
zone, and not individual experimental seedlings. This analysis
acknowledged the means and standard errors of both Si rDNA
indices and physiological measurements of each population.
This established a relationship between the Si rDNA index and
physiological parameters under both well-watered and drought
conditions. Population differences for specific physiological
parameters were evaluated by analysis of variance (with Systat
software, SYSTAT, Inc., Evanston, IL), except when effects of
DNA exchange were examined.
Results
Photosynthetic characterization of well-watered and
drought-treated seedlings
Sitka spruce populations had higher maximum Pn in saturating
light and under well-watered conditions (open symbols, Ψpd
approximately −0.2 MPa) and lower maximum Pn in moderate
drought (filled symbols, Ψpd of −1.0 to −1.1 MPa) (Figure 1a)
than interior spruce populations. Under well-watered and
moderate drought conditions, Sitka spruce populations
(Si rDNA index = 0.11) reached half maximal Pn at lower PAR
than interior spruce populations (Si rDNA index = 0.97), with
hybrids in between (Figure 1b). The light compensation point
for Pn was similar in both well-watered (52 to 54 µmol m −2 s −1)
and moderate drought (31 to 41 µmol m −2 s −1) conditions in all
Sitka, interior, and hybrid spruce populations.
Quantum yield (φ) of photosynthesis was higher (P > 0.04)
in Sitka spruce populations than in interior spruce populations,
and tended to decline as the Si rDNA index increased (Figure 1c). This trend of φ remained the same in both well-watered and drought-treated seedlings.
Under well-watered conditions, Sitka spruce populations
had higher potential maximal Pn at saturating CO2 concentrations than interior spruce populations (Figure 2a). Carbon
dioxide saturation of Pn also occurred at higher concentrations
in Sitka spruce populations than in interior spruce populations.
As the Si rDNA index increased, Ci at half maximal Pn occurred at lower CO2 concentrations (Figure 2b). However,
there was no difference in carboxylation efficiency of Rubisco
between all measured Sitka and interior spruce populations
(Figure 2c).
Under well-watered conditions, stomatal limitations to Pn
were slightly lower in Sitka spruce than in interior spruce
populations (Figure 3a). Population differences in stomatal
limitations were primarily a result of differences in stomatal
densities, which were higher in Sitka spruce populations than
in interior spruce populations (Figure 3b). Three to six rows of
stomata were common on the adaxial side of Sika spruce
needles, whereas two to three (two in most cases) rows of
Figure 1. (a) Net photosynthesis (Pn) versus photosynthetically active
radiation (PAR) in Sitka (Si rDNA index = 0.11, triangles) and interior
(Si rDNA index = 0.97, squares) spruce seedlings. (b) Photosynthetically active radiation at half maximal Pn in relation to Si rDNA index
in Sitka × interior spruce populations. (c) Photosynthetic quantum
yield at the light compensation point (φ) versus Si rDNA index in Sitka
× interior spruce populations. Open symbols indicate well-watered
conditions, filled symbols signify moderate (Ψpd at −1.0 to −1.1 MPa)
drought conditions.The symbol r is the Pearson product-moment correlation coefficient and ρ is the corresponding probability of significance.
stomata were typically observed on the adaxial side of interior
spruce needles. In contrast, stomatal size tended to be larger in
populations having higher Si rDNA indices (29.0 ± 0.9 µm for
interior spruce (Si rDNA index = 0.97)) than in populations
GAS EXCHANGE ADAPTATION AND RIBOSOMAL DNA MARKERS
119
Figure 3. (a) Stomatal limitations to Pn, and (b) stomatal densities on
needle surfaces versus Si rDNA index in Sitka × interior spruce
populations.
Figure 2. (a) Net photosynthesis (Pn) versus intercellular CO2 concentration (Ci) in Sitka (Si rDNA index = 0.11, triangles) and interior
(Si rDNA index = 0.97, squares) spruce seedlings. (b) Intercellular
CO2 concentrations (Ci) at half maximal Pn, and (c) Rubisco carboxylation efficiency at the light compensation point (VRuBP) under wellwatered conditions versus Si rDNA index in Sitka × interior spruce
populations.
having lower Si rDNA indices (26.3 ± 0.8 µm for Sitka spruce
(Si rDNA index = 0.11)), although they were not statistically
different.
Gas exchange response to drought
Under well-watered conditions, Sitka spruce populations had
higher Pn, gwv, TFD, and Rd, than interior spruce populations,
with gas exchange parameters decreasing as the Si rDNA index
increased (Figure 4). However, when seedlings were subjected
to drought, Pn, gwv and TFD (normalized at Ψpd of −1.5 MPa)
were all lower in Sitka spruce populations having a lower
Si rDNA index (Figures 4a--4c). The exception, under drought
conditions, was Rd (measured at Ψpd of −1.0 −1.1 MPa), which
was similar for all Sitka × interior spruce populations (Figure 4d).
Lower gas exchange rates in drought-treated Sitka spruce
populations than in interior spruce populations could be ascribed to an intolerance to dehydration (Figure 5). Although
initially higher under well-watered conditions, Pn, gwv, and
TFD all dropped sharply in Sitka spruce populations as seedling Ψpd decreased. In contrast, these parameters declined at a
slower rate in drought-treated interior spruce populations. Because of these differences in sensitivity to dehydration, Pn, gwv,
and TFD fell to half of their predrought maximum at lower Ψpd
in populations with a higher Si rDNA index (Figure 6).
Twenty-four hours after drought-treated seedlings had been
watered, Pn, gwv, and TFD recovered partially in all populations, but the degree of recovery varied (Figure 7). Populations
with a higher Si rDNA index generally had a more rapid
recovery of Pn, gwv, and TFD after drought, measured by
recovery to predrought values, than populations with a lower
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FAN, GROSSNICKLE AND SUTTON
Figure 5. Changes in net photosynthesis (Pn) (a), stomatal conductance to water vapor (gwv) (b), and transpiration flux density (TFD) (c)
in relation to decreases in predawn water potentials (Ψpd) for Sitka
(Si rDNA index of 0.11, n) and interior (Si rDNA index of 0.97, u)
spruce seedlings.
Figure 4. Net photosynthesis (Pn) (a), stomatal conductance to water
vapor (gwv) (b), transpiration flux density (TFD) (c), and dark respiration rates (Rd) (d) versus Si rDNA index in Sitka × interior spruce
populations maintained under well-watered conditions (open symbols)
or subjected to moderate drought (Ψpd at −1.5 MPa) (filled symbols),
except dark respiration which was measured at −1.0 to −1.1 MPa.
Si rDNA index. Of the gas exchange processes, recovery from
drought was highest for TFD (45 to 65%), followed by Pn (25
to 45%), and gwv (18 to 35%).
Discussion
Populations with a higher Si rDNA index exhibited lower
maximum Pn under both saturating PAR and CO2 and well-watered conditions than those with a lower Si rDNA index. When
other environmental factors are not limiting, rates of CO2
diffusion to, and CO2 fixation at, the photosynthetic sites in
chloroplasts can limit photosynthesis. Carbon dioxide diffusion rates can be affected by stomatal and mesophyll conductance. Fixation of CO2 can be limited by the carboxylation
efficiency of Rubisco at low Ci , the regeneration of ribulose
1,5-bisphosphate at intermediate Ci , and the recycling of inor-
GAS EXCHANGE ADAPTATION AND RIBOSOMAL DNA MARKERS
Figure 6. Predawn plant water potentials (Ψpd) at which net photosynthesis (Pn) (a), stomatal conductance to water vapor (gwv) (b), and
transpiration flux density (TFD) (c) were at half of maximal values
versus Si rDNA index in Sitka × interior spruce populations. The
higher the Ψpd values (less negative) the lower a population’s dehydration tolerance.
ganic phosphate at high Ci concentrations (Farquhar et al.
1980, Sharkey 1985). Rubisco carboxylation efficiency, CO2
and light compensation points for Pn were all similar among
the seven populations, and corresponded to reported values for
Sitka spruce (Ludlow and Jarvis 1971, Leverenz and Jarvis
1979) and Engelmann spruce (DeLucia 1986). Carboxylation
efficiency could not, therefore, explain the differences in maximum Pn between Sitka and interior spruce populations.
Unlike carboxylation efficiency, gwv differed among the
populations as a result of differences in stomatal densities on
needle surfaces. Higher stomatal densities resulted in lower
stomatal limitations to Pn, and could partially explain higher
121
Figure 7. The recovery of net photosynthesis (Pn) (a), stomatal conductance to water vapor (gwv) (b), and transpiration flux density (TFD)
(c) of drought-treated seedlings in relation to Si rDNA index in Sitka
× interior spruce populations. After-drought measurements were taken
exactly 24 hours after rewatering and are expressed as the percentage
of the corresponding pre-drought value. Lines were drawn through the
data to provide a qualitative indication of the trend in recovery of gas
exchange versus Si rDNA index.
Pn in Sitka spruce populations than in populations having a
higher Si rDNA index. The importance of CO2 diffusion rate
to Pn was further suggested by the higher maximum Pn in
CO2-saturated than in light-saturated conditions in the Sitka ×
interior populations. Similar findings were found when comparing a coastal with a coastal × interior ponderosa pine (Pinus
ponderosa Dougl. ex Laws) population (Monson and Grant
1989). The estimated stomatal limitations for all Sitka × interior populations were comparable to reports for black spruce
(Picea mariana (Mill.) B.S.P.) (Stewart et al. 1995) and loblolly pine (Pinus taeda L.) (Teskey et al. 1986). Stomatal
anatomy seemed to be more important than carboxylation
efficiency for describing maximum Pn in the Sitka × interior
populations.
In contrast to well-watered conditions, interior spruce populations had greater dehydration tolerance of gas exchange
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FAN, GROSSNICKLE AND SUTTON
processes than Sitka spruce populations, measured by the Ψpd
at which Pn, gwv and TFD are at half their maximal values, and
percent recovery of gas exchange processes after drought.
Because stomatal limitations to Pn increase under drought
conditions in spruce species (cf Stewart et al. 1995), higher gwv
in interior than in Sitka spruce populations would explain their
higher Pn. Black spruce populations that have greater dehydration tolerance are able to maintain higher gwv and Pn under
drought, and grow faster on dry sites than populations with
more limited dehydration tolerance (Tan et al. 1992, Tan and
Blake 1993). Trees occurring naturally on dry sites are generally more dehydration tolerant (Kuhns et al. 1993) and better
able to maintain high gas exchange rates under drought conditions than trees of the same species occupying more mesic sites
(Larsen 1981, Seiler and Johnson 1988). Our data are consistent with previous findings of greater drought tolerance in
interior spruce than in Sitka spruce (Grossnickle et al. 1996).
Because of its unique geographic location, the Nass--Skeena
transition zone has a heterogeneous, transitional climate with
both maritime and continental characteristics (Roche and Haddock 1987, Sutton et al. 1994). Environmental heterogeneity
tends to sustain genetic polymorphism (Hedrick 1986), and
trees with high degrees of heterozygosity are thought to be
more tolerant to environmental stresses (Müller-Starck 1985,
Stutz and Mitton 1988, Mitton et al. 1989). Gene transfer
between ecologically differing species as the result of introgressive hybridization can result in improved environmental
adaptation (Oliver et al. 1995) as appears to be true in the case
of Sitka and interior spruces. Incorporation of interior spruce
genes into the Sitka spruce genome has produced hybrids that
have both retained the high photosynthetic capability of Sitka
spruce under nondrought conditions, and acquired the greater
drought and freezing tolerance of interior spruce (Grossnickle
et al. 1996). These diverse physiological features allow Sitka
× interior spruce hybrids to occupy environments over a range
of transitional habitats, and compete for sites that are not well
utilized by either pure Sitka or interior spruce. Thus, environmental variability and selection pressure during evolution may
explain the existence of hybrid populations between Sitka and
interior spruce in the Nass--Skeena transition zone.
In this and previous work (Grossnickle et al. 1996), seedlings of all seven populations were treated under identical
environmental conditions. Differences in physiological attributes (phenotypic variation) between populations were related
to their corresponding Si rDNA index (genotypic variation)
values. This is consistent with Cheverud’s (1988) argument
that phenotypic analysis can sometimes substitute for genetic
analysis. However, genetic estimates provide a more direct
method of defining population differences (Willis et al. 1991).
Nevertheless, caution is required in using physiological
(phenotypic) measurements for screening populations with
unknown genetic background. Apart from the genetic dependency, physiology of tree species is strongly influenced by
environmental conditions. Different physiological processes
may respond in various ways to environmental conditions. For
instance, Pn, gwv, and TFD responses in Sitka and interior
spruce populations changed under different watering regimes,
whereas these populations had similar PAR for maximum Pn,
φ and Rd as soil water availability varied. Elucidation of the
interactions among physiological processes and environmental conditions will lead to an understanding of the adaptive
significances of interspecific DNA exchange and the use of
physiological tests. Testing under both limiting and nonlimiting environmental conditions may provide more reliable
information for the understanding of the evolutionary consequences of species introgression.
In conclusion, introgressive hybridization between Sitka
spruce and interior spruce in British Columbia’s Nass--Skeena
transition zone has resulted in a wide array of genotypes.
Gradual mixing of interior and Sitka spruce populations has
caused a proportional reduction in high gas exchange capability under well-watered conditions that is associated with Sitka
spruce populations. On the other hand, greater gas exchange
capability under drought conditions is related to greater dehydration tolerance in interior spruce than in Sitka spruce populations. The wide range of physiological responses found in
Sitka × interior spruce populations could explain the persistence of a spruce hybrid swarm in the heterogeneous habitats of
the Nass--Skeena transition zone.
Acknowledgments
Funding for this research was provided by the Science Council of
British Columbia (Contract No. 175 (T-5)), the British Columbia
Ministry of Forests (Contract No. 18605-20/SXSS) grants to Steven
C. Grossnickle and Ben C.S. Sutton, and a Natural Science and
Engineering Research Council of Canada industrial postdoctoral fellowship to Shihe Fan.
References
Abrams, M.D. 1994. Genotypic and phenotypic variation as stress
adaptations in temperate tree species: a review of several case
studies. Tree Physiol. 14:833--442.
Burns, R.M. and B.H. Honkala. 1990. Silvics of North America.
Volume 1, Conifers. USDA For. Serv., Agric. Handbook No. 654.
Washington, DC, pp 187--226, 260--267.
Cheverud, J.M. 1988. A comparison of genetic and phenotypic correlations. Evolution 42:958--968.
Daubenmire, R. 1972. Taxonomic and ecological relationships between Picea glauca and Picea sitchensis and their ecological interpretation. Can. J. Bot. 46:787--798.
DeLucia, E.H. 1986. Effect of low temperature on net photosynthesis,
stomatal conductance and carbohydrate concentration in Engelmann spruce (Picea engelmannii Parry ex Engelm.) seedlings. Tree
Physiol. 2:143--154.
Farquhar, G.D., S. von Caemmerer and J.A. Berry. 1980. A biochemical model of photosynthetic (CO2) assimilation in leaves of C3
species. Planta 149:78--90.
Grossnickle, S.C., B.C.S. Sutton, R.S. Folk and J.R. Gawley. 1996.
Relationship between nuclear DNA markers and physiological parameters for Sitka × interior spruce populations. Tree Physiol.
16:547--556.
Hedrick, P.W. 1986. Genetic polymorphism in heterogeneous environments: a decade later. Annu. Rev. Ecol. System. 17:535--566.
Jones, H.G. 1985. Partitioning stomatal and nonstomatal limitations to
photosynthesis. Plant, Cell Environ. 8:95--104.
GAS EXCHANGE ADAPTATION AND RIBOSOMAL DNA MARKERS
Kuhns, M.R., W.W. Stroup and G.M. Gebre. 1993. Dehydration tolerance of five bur oak (Quercus macrocarpa) seed sources from
Texas, Nebraska, Minnesota, and New York. Can. J. For. Res.
23:387--393.
Larsen, J.B. 1981. Geographic variation in winter drought resistance
of Douglas-fir (Pseudotsuga menziesii Mirb. Franco). Silvae Genet.
30:4--5.
Leopold, A.C. and P.E. Kriedemann. 1975. Plant growth and development. McGraw-Hill Book Co., Toronto, pp 102--103.
Leverenz, J.W. and P.G. Jarvis. 1979. Photosynthesis in Sitka spruce.
VIII. The effects of light flux density and direction on the rate of net
photosynthesis and the stomatal conductance of needles. J. Appl.
Ecol. 16:919--932.
Ludlow, M.M. and P.G. Jarvis. 1971. Photosynthesis in Sitka spruce
(Picea sitchensis (Bong.) Carr.). J. Appl. Ecol. 8:925--953.
Mitton, J.B. 1995. Genetics and the physiological ecology of conifers.
In Ecophysiology of Coniferous Forests. Eds. W.K. Smith and T.M.
Hinckley. Academic Press, New York, pp 1--36.
Mitton, J.B., H.P. Stutz, W.S. Schuster and K.L. Shea. 1989. Genotypic differentiation at PGM in Engelmann spruce from wet and dry
sites. Silvae Genet. 38:217--221.
Mcdermitt, D.K., J.M. Norman, J.T. Davis, T.M. Ball, T.J. Arkebauer,
J.M. Welles and S.R. Roemer. 1989. CO 2 response curves can be
measured with a field-portable closed-loop photosynthesis system.
Ann. Sci. For. 46 (suppl.):416s--420s.
Monson, R.K. and M.C. Grant. 1989. Experimental studies of ponderosa pine. III. Differences in photosynthesis, stomatal conductance,
and water-use efficiency between two genetic lines. Am. J. Bot.
76:1041--1047.
Müller-Starck, G. 1985. Genetic differences between ‘tolerant’ and
‘sensitive’ beeches (Fagus sylvatica L.) in an environmentally
stressed adult forest stand. Silvae Genet. 34:241--246.
Oliver, M.J., D.L. Ferguson and J.J. Burke. 1995. Interspecific gene
transfer: implications for broadening temperature characteristics of
plant metabolic processes. Plant Physiol. 107:429--434.
Rieseberg, L.H. and J.F. Wendel. 1993. Introgression and its consequences in plants. In Hybrid Zones and Evolutionary Processes. Ed.
R.G. Harrison. Oxford University Press, New York, pp 70--109.
Roche, L. 1969. A genecological study of the genus Picea in British
Columbia. New Phytol. 68:505--554.
Roche, L. and P.G. Haddock. 1987. Sitka spruce (Picea sitchensis) in
North America with special reference to its role in British forestry.
Proc. Royal Soc., Edinburgh, 93B:1--12.
123
Seiler, J.R. and J.D. Johnson. 1988. Physiological and morphological
responses of three half-sib families of loblolly pine to water-stress
conditioning. For. Sci. 34:487--495.
Sharkey, T.D. 1985. Photosynthesis in intact leaves of C 3 plants:
physics, physiology and rate limitations. Bot. Rev. 51:53--105.
Stewart, J.D., A. Zine El Abidine and P.Y. Bernier. 1995. Stomatal and
mesophyll limitations of photosynthesis in black spruce seedlings
during multiple cycles of drought. Tree Physiol. 15:57--64.
Stutz, H.P. and J.B. Mitton. 1988. Variation in Engelmann spruce
associated with variation in soil moisture. Arct. Alp. Res. 20:461-465.
Sutton, B.C.S., D.J. Flanagan and Y.A. El-Kassaby. 1991a. A simple
and rapid method for estimating representation of species in spruce
seedlots using chloroplast DNA restriction fragment. Silvae Genet.
38:119--123.
Sutton, B.C.S., D.J. Flanagan, J.R. Gawley, C.H. Newton, D.T. Lester
and Y.A. El-Kassaby. 1991b. Inheritance of chloroplast and mitochondrial DNA in Picea and composition of hybrids from introgression zones. Theor. Appl. Genet. 82:242--248.
Sutton, B.C.S., S.C. Pritchard, J.R. Gawley, C.H. Newton and G.K.
Kiss. 1994. Analysis of Sitka spruce-interior spruce introgression in
British Columbia using cytoplasmic and nuclear DNA probes. Can.
J. For. Res. 24:278--285.
Tan, W. and T.J. Blake. 1993. Drought tolerance, abscisic acid and
electrolyte leakage in faster- and slower-growing black spruce
(Picea mariana) progenies. Physiol. Plant. 89:817--823.
Tan, W., T.J. Blake and T.J.B. Boyle. 1992. Drought tolerance in fasterand slower-growing black spruce (Picea mariana) progenies: I.
Stomatal and gas exchange responses to osmotic stress. Physiol.
Plant. 85:639--644.
Teskey, R.O., J.A. Fites, L.J. Samuelson and B.C. Bongarten. 1986.
Stomatal and nonstomatal limitations to net photosynthesis in Pinus
taeda L. under different environmental conditions. Tree Physiol.
2:131--142.
Villar, R., A.A. Hel and J. Merino. 1994. Comparison of methods to
estimate dark respiration in the light in leaves of two woody species.
Plant Physiol. 105:167--72.
Willis, J.H., J.A. Coyne and M. Kirkpatrick. 1991. Can one predict the
evolution of quantitative characters without genetics? Evolution
45:441--444.
Wright, J.H. 1955. Species crossability in spruce in relation to distribution and taxonomy. For. Sci. 1:319--49.
© 1997 Heron Publishing----Victoria, Canada
Relationships between gas exchange adaptation of Sitka× interior
spruce genotypes and ribosomal DNA markers
SHIHE FAN, STEVEN C. GROSSNICKLE and BEN C. S. SUTTON
Forest Biotechnology Centre, B.C. Research, Inc., 3650 Wesbrook Mall, Vancouver, B.C. V3S 2L2, Canada
Received February 10, 1996
Summary Adaptive physiological changes were investigated in seven populations of Sitka (Picea sitchensis (Bong.)
Carr.) × interior spruce (P. glauca (Moench) Voss × P. engelmannii Parry ex Engelm.) spanning the Nass--Skeena transition
zone in British Columbia, Canada. Each population was represented by an Si rDNA index that was calculated from the
relative optical densities on a gel autoradiogram of five ribosomal DNA bands characteristic of Sitka spruce and interior
spruce. This index estimates the proportion of the genome
contributed by interior spruce. Physiological adaptations were
assessed by gas exchange parameters measured under both
well-watered and drought conditions. Under well-watered conditions, Sitka spruce populations had higher maximal photosynthesis at saturating light and ambient CO2, higher quantum
yield at the light compensation point, and higher dark respiration than interior spruce populations. Sitka spruce populations
also reached maximal photosynthesis at lower photosynthetically active radiation and higher CO2 concentrations, and had
higher stomatal densities that resulted in lower stomatal limitations to photosynthesis than interior spruce populations. In
contrast, interior spruce populations exhibited greater drought
tolerance than Sitka spruce populations. Their gas exchange
rates declined at a slower rate in response to drought. They
maintained higher gas exchange rates in response to moderate
to severe drought (predawn plant water potentials = −1.5 MPa),
and their photosynthetic rates recovered faster when they were
rewatered after exposure to drought. Comparison of the seven
populations indicated that physiological parameters were significantly related to the Si rDNA index. An increase in Si rDNA
index was associated with proportional changes in physiological measurements, suggesting that genetic interchange among
species with contrasting ecological adaptations can enhance
the environmental adaptation of natural populations.
Keywords: DNA analysis, drought, Picea engelmannii, Picea
glauca, Picea sitchensis.
Introduction
Introgressive hybridization occurs between Sitka spruce
(Picea sitchensis (Bong.) Carr.) and interior spruce (P. glauca
(Moench) Voss × P. engelmannii Parry ex. Engelm.) in the
Nass--Skeena transition zone in northwestern British Colum-
bia, Canada (Roche 1969, Daubenmire 1972). Sitka spruce
naturally occurs in the maritime climate on the Pacific coast of
North America (Burns and Honkala 1990). The parental species of interior spruce, Engelmann spruce (P. engelmannii) and
white spruce (P. glauca), have contrasting ecological adaptations. Engelmann spruce grows in cool, humid environments
at high elevations, whereas white spruce is found in dry, cool,
continental conditions across North America (Burns and
Honkala 1990). Reproductive difficulties sometimes occur
when two distinctive spruce species are crossed, but Sitka,
Engelmann, and white spruce are phylogenetically close and
hybridize easily (Wright 1955). It is important to understand
the consequences of introgression between these spruce species, which may help develop breeding and dispersal strategies.
Populations taken from the Sitka × interior spruce introgression zone are highly heterogeneous in both their nuclear and
plastid DNA (Sutton et al. 1991a, 1991b, 1994). Sitka spruce
has different ecological adaptations than interior spruce, therefore, the DNA exchange between these species has caused
corresponding alterations in physiological attributes. Changes
in drought and freezing tolerance of Sitka × interior spruce
populations correspond with their nuclear ribosomal DNA
(rDNA) patterns (Grossnickle et al. 1996). These results are
consistent with evolutionary studies, which suggest that introgression may produce new ecotypes both by transferring existing adaptive characteristics from one population to another and
by giving rise to new adaptations (Rieseberg and Wendel
1993).
Because of the scarcity of experimental evidence for adaptive variabilities resulting from DNA exchange among species
with different ecological adaptations (Grossnickle et al. 1996),
little is known about the significance of DNA changes on
morphological, physiological and phenological attributes of
trees (Mitton 1995). Individual forest tree species are usually
distributed over a large geographic range and their genotype
and phenotype vary with environmental variation across the
range (Abrams 1994). It is important, therefore, to establish a
relationship between genotypic (DNA) and phenotypic (i.e.,
physiological) attributes that is independent of environmental
variability.
Following the study of Grossnickle et al. (1996), we examined the adaptive significances of DNA exchange in seven
116
FAN, GROSSNICKLE AND SUTTON
Sitka × interior spruce introgressive hybrid populations from
the Nass--Skeena transition zone. Specifically, gas exchange
characteristics were measured under both well-watered and
drought conditions, and their relationships with the rDNA
values of the populations examined. Results suggested that
DNA exchange between Sitka spruce and interior spruce has
broadened the ecological adaptations of spruce populations in
the Nass--Skeena transition zone.
Materials and methods
Plant materials
Seven Sitka (Picea sitchensis) × interior (P. glauca × P. engelmannii) spruce populations were sampled from the Nass-Skeena transition zone in northwestern B.C., Canada. Four
were wild seed collections, one was an operational seedlot
pooled from the Bulkley Valley seed orchard, and two were
seedlots from open pollinated families within the Bulkley
Valley seed orchard (each consisting of five families based on
the localities of the original parental trees). A brief summary
of the seed collection is given in Table 1 and a detailed description has been given by Sutton et al. (1994).
The history of seedling culture has been detailed by Grossnickle et al. (1996). In brief, seedlings were 1-year-old container-grown plants raised at the B.C. Ministry of Forests
Saanich nursery (Sidney, B.C., Canada; 48°28′ N and 124°10′ W).
Seedlings were transplanted in the second year to a farm field
site at the University of B.C. (Vancouver, B.C., 49°15′ N and
123°15′ W). Seedlings used in the experiment had been
planted in 11-liter pots to facilitate rapid and nondisruptive
removal for physiological testing. The pots were randomly
placed within the field site design and submerged in the soil so
that seedlings were exposed to normal site edaphic conditions.
Just before the third growing season, potted seedlings were
relocated to an outdoor compound at B.C. Research, Inc. (2 km
away from the farm field site). During the third year, seedlings
were watered every three days and fertilized every other week
with 20,8,20 (N,P,K) Plant Prod forestry seedling fertilizer
(Plant Products Co., Ltd., Brampton, ON, Canada) (equivalent
to 200 ppm N). Experiments were started in late June immediately after seedlings had set terminal buds.
DNA Analysis
Each value of Si rDNA index listed in Table 1 was calculated
from rDNA data taken on eight experimental seedlings within
a population. Details of the DNA analysis procedures have
been described previously (Sutton et al. 1994, Grossnickle et
al. 1996). In brief, rDNA was isolated from three buds from
each seedling. After purification, rDNA was digested with the
restriction enzyme, HindIII, followed by electrophoresis in a
0.8% agarose gel. Ribosomal DNA was transferred to Hybond
filters (Amersham, Oakville, ON, Canada) by Southern blotting, and hybridized with a α32P-dATP labeled probe, Eco 2.0
(yeast 18S RNA gene). Ribosomal DNA bands were visualized by autoradiography, and an Si rDNA index was then
calculated, as described previously, for each seedling according to the optical density of the five characteristic bands (Sutton et al. 1994, Grossnickle et al. 1996). The Si rDNA index
gives an overall estimation of interior spruce DNA contribution to the genome of each seedling.
Photosynthetic characterization of well-watered and
drought-treated seedlings
Photosynthetically active radiation (PAR) versus net photosynthesis (Pn) curves and intercellular CO2 (Ci ) versus Pn
curves were constructed from the seven test populations.
Measurements were taken on newly developed, unshaded,
lateral branches in the upper crown with an LI-6200 Portable
(closed-loop) Photosynthesis System and a 250-ml leaf cuvette
(Li-Cor, Inc., Lincoln, NE). Curves were constructed from six
seedlings for each population.
The PAR--Pn response curves were measured twice on the
same branch of each seedling under both well-watered (predawn plant water potentials (Ψpd) of about −0.2 MPa) and
moderate drought (Ψpd of −1.0 to −1.1 MPa) conditions. Measurements were taken in an environmentally controlled growth
room providing the following conditions: 22 °C air temperature, 40 ± 5% relative humidity, and 400 ± 10 µl l −1 ambient
CO2. Light was provided by two Sylvania 1000 W Metalarc®
metal halide lamps (Osram Sylvania Inc., Manchester, NH). To
obtain the PAR--Pn response curve, PAR was varied by changing the thickness of plastic sheets between the seedling and the
light. Air temperature in the leaf cuvette was held virtually
constant during measurements with a 360-mm external electrical fan. Measurements were started with dark-adapted seed-
Table 1. Summary of seed source information and the interior spruce rDNA index (mean ± SE; n = 8) for each test population.
Source
Seedlot
Seed zone1
Location
Elevation (m)
Si rDNA Index
1
2
3
4
5
6
7
1004
4075
4132
14657
6308
Five OP families
Five OP families
NST
BLK
SM
NST
Kitwanga, 55°20′ N; 128°05′ W
Morice West, 54°10′ N; 127°15′ W
Lower Nass, 54°58′ N; 129°38′ W
Meziadan Lake, 56°06′ N; 129°19′ W
Bulkley Valley, 53°30′ to 55°40′ N; 127° W
Morice Lake, 54°20′ N; 127°30′ W
North Babine, 55°35′ N; 126°40′ W
305
760
10
290
< 840
< 840
< 840
0.51 ± 0.06
0.92 ± 0.05
0.11 ± 0.03
0.36 ± 0.06
0.97 ± 0.09
0.81 ± 0.11
0.84 ± 0.03
1
NST = Nass--Skeena Transition, BLK = Bulkley Valley, SM = Spruce Maritime.
GAS EXCHANGE ADAPTATION AND RIBOSOMAL DNA MARKERS
lings. After a change in PAR, further measurements were not
made until after an interval of 15--20 min, which in a preliminary trial was found sufficient to allow photosynthesis to reach
a steady state.
The Ci --Pn curves were measured on well-watered seedlings
under the conditions described above except that PAR was 750
µmol m −2 s −1 and CO2 in the leaf cuvette was progressively
reduced from an initially high concentration (1100--1200 µl l−1)
by seedling photosynthesis (McDermitt et al. 1989). Before
each Ci --Pn measurement, the initial CO2 concentration was
sustained for 10 min by slowly pumping CO2 (compressed
CO2 mixed with air) into the LI-6200 system through an
external flow switch using a Li-Cor 6000-01 gas calibration
cylinder. This treatment acclimated stomata and photosynthesis to the high CO2 environment and stabilized stomatal conductance without significant variation during the 30- to 50-min
measuring period. After this acclimation treatment, CO2 in the
cuvette was drawn down by the seedling (McDermitt et al.
1989), and data logging was started when CO2 in the cuvette
was 900 to 1000 µl l −1. When CO2 in the cuvette reached the
CO2 compensation point for Pn, the remaining CO2 in the
system was drawn down further by both the online and an
external soda lime tube attached to the external flow switch.
This allowed the Ci--Pn curves to be extended to a CO2 concentration approaching zero. Both Pn and Ci were corrected for
system leakage following the manufacturer’s recommendations (Application No. 103, Li-Cor, Inc. 1991).
Gas exchange response to drought
After the measurements of PAR--Pn and Ci --Pn under well-watered conditions, seedlings were subjected to soil drought by
withholding water for approximately 35 to 40 days. The
drought was simulated under an outdoor rain shelter. The
evening before each measuring day, seedlings were moved to
the growth room and placed on a darkened growth table for
10 h. The next morning, Ψpd was measured with a pressure
chamber (Model 3005, Soil Moisture Equipment Corp. Santa
Barbara, CA) on a lateral branch from the upper crown of each
seedling before the lights were turned on. Two hours later, gas
exchange parameters (net photosynthesis (Pn), stomatal conductance to water vapor (gwv) and transpiration flux density
(TFD)) were measured on an upper crown lateral branch with
the LI-6200 system under conditions similar to those for Ci --Pn
measurements, except that CO2 concentration in the leaf cuvette was 390 ± 10 µl l −1. The same branch was measured
throughout the drought and rewatering period. When Pn decreased to just less than 0, the soil was fully rehydrated. Gas
exchange was measured again exactly 24 h after rehydration.
To compare the relative rates of gas exchange recovery after
rewatering, rates relative to the pre-drought values are presented. Total needle surface area of each lateral branch sample
enclosed in the leaf chamber for gas exchange measurements
was determined from a relationship with oven-dried (70 °C)
needle dry weight. Gas exchange measurements were then
recalculated accordingly.
117
Stomatal morphology
Stomatal sizes were measured on current-year needles of five
selected populations (Nos. 1--4, and 7 in Table 1, Nos. 5 and 6
were excluded to reduce sample sizes). Three needles from
each of the top, middle, and bottom positions on an uppercrown branch were randomly excised, cut in half, and the
mesophyll removed. Stomatal sizes (nine per needle) on the
needle epidermis were then determined under a light microscope. Stomatal density assessment (nine views per impression) was made on needle surface impressions made from
fingernail polish. Three impressions were made for each seedling.
Data analysis
Equation 1 was used to model changes in Pn, gwv, and TFD
with decrease in Ψpd:
Y = ae(b − cx),
(1)
where Y is either Pn, gwv or TFD; x is Ψpd; and a, b, and c are
model parameters. The last negative Pn data point of each
seedling was excluded from the data set used for modeling. To
compare the dehydration tolerance of populations, the values
of Ψpd at which Pn, gwv, or TFD were half their pre-drought
maxima were determined by means of Equation 1. The higher
the Ψpd value, the lower the dehydration tolerance. Equation 1
was also used to normalize Pn, gwv, and TFD to −1.5 MPa for
comparisons among populations.
Equation 2 defined PAR--Pn and Ci --Pn curves for each
seedling:
Y=a+
bx
,
c+x
(2)
where Y is Pn; x is either PAR or Ci ; a is dark respiration (Rd)
in PAR--Pn measurements or photorespiration in Ci --Pn measurements (because real Rd is generally inhibited at PAR above
500 µmol m −2 s −1 (Villar et al. 1994)); b is the potential
maximum Pn under saturating PAR or ambient CO2; and c is
the light utilization or CO2 affinity constant when Pn was half
maximal (Leopold and Kriedemann 1975). Equation 2 generally fitted the data well (r 2 from 0.97 to 1.00 for Ci --Pn curves
and 0.94 to 0.98 for PAR--Pn curves, respectively). Quantum
yield (φ), the initial slope at the light compensation point for
Pn, and carboxylation efficiency of ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco) (VRuBP), the initial slope at
the CO2 compensation point for Pn, were estimated from the
PAR--Pn and Ci --Pn curves, respectively (Farquhar et al. 1980).
Stomatal limitations (I) to Pn were estimated from Ci--Pn
curves by the differential method (Jones 1985):
I=
1/g wv
,
1/gwv + 1/gm
(3)
where gwv was the slope of the ‘‘supply function,’’ i.e., measured gwv; gm was the slope of the ‘‘demand function,’’ i.e., Ci--Pn
118
FAN, GROSSNICKLE AND SUTTON
curve, at the operating point. The operating point was determined according to the ‘‘demand’’ and ‘‘supply’’ functions
fitted or measured for each individual seedling.
The relationship of the Si rDNA index to physiological
measurements in the seven populations was examined by the
Pearson product-moment correlation analysis, because the
Si rDNA index was designed to describe the genotype of a
population in a specific subzone in the Nass--Skeena transition
zone, and not individual experimental seedlings. This analysis
acknowledged the means and standard errors of both Si rDNA
indices and physiological measurements of each population.
This established a relationship between the Si rDNA index and
physiological parameters under both well-watered and drought
conditions. Population differences for specific physiological
parameters were evaluated by analysis of variance (with Systat
software, SYSTAT, Inc., Evanston, IL), except when effects of
DNA exchange were examined.
Results
Photosynthetic characterization of well-watered and
drought-treated seedlings
Sitka spruce populations had higher maximum Pn in saturating
light and under well-watered conditions (open symbols, Ψpd
approximately −0.2 MPa) and lower maximum Pn in moderate
drought (filled symbols, Ψpd of −1.0 to −1.1 MPa) (Figure 1a)
than interior spruce populations. Under well-watered and
moderate drought conditions, Sitka spruce populations
(Si rDNA index = 0.11) reached half maximal Pn at lower PAR
than interior spruce populations (Si rDNA index = 0.97), with
hybrids in between (Figure 1b). The light compensation point
for Pn was similar in both well-watered (52 to 54 µmol m −2 s −1)
and moderate drought (31 to 41 µmol m −2 s −1) conditions in all
Sitka, interior, and hybrid spruce populations.
Quantum yield (φ) of photosynthesis was higher (P > 0.04)
in Sitka spruce populations than in interior spruce populations,
and tended to decline as the Si rDNA index increased (Figure 1c). This trend of φ remained the same in both well-watered and drought-treated seedlings.
Under well-watered conditions, Sitka spruce populations
had higher potential maximal Pn at saturating CO2 concentrations than interior spruce populations (Figure 2a). Carbon
dioxide saturation of Pn also occurred at higher concentrations
in Sitka spruce populations than in interior spruce populations.
As the Si rDNA index increased, Ci at half maximal Pn occurred at lower CO2 concentrations (Figure 2b). However,
there was no difference in carboxylation efficiency of Rubisco
between all measured Sitka and interior spruce populations
(Figure 2c).
Under well-watered conditions, stomatal limitations to Pn
were slightly lower in Sitka spruce than in interior spruce
populations (Figure 3a). Population differences in stomatal
limitations were primarily a result of differences in stomatal
densities, which were higher in Sitka spruce populations than
in interior spruce populations (Figure 3b). Three to six rows of
stomata were common on the adaxial side of Sika spruce
needles, whereas two to three (two in most cases) rows of
Figure 1. (a) Net photosynthesis (Pn) versus photosynthetically active
radiation (PAR) in Sitka (Si rDNA index = 0.11, triangles) and interior
(Si rDNA index = 0.97, squares) spruce seedlings. (b) Photosynthetically active radiation at half maximal Pn in relation to Si rDNA index
in Sitka × interior spruce populations. (c) Photosynthetic quantum
yield at the light compensation point (φ) versus Si rDNA index in Sitka
× interior spruce populations. Open symbols indicate well-watered
conditions, filled symbols signify moderate (Ψpd at −1.0 to −1.1 MPa)
drought conditions.The symbol r is the Pearson product-moment correlation coefficient and ρ is the corresponding probability of significance.
stomata were typically observed on the adaxial side of interior
spruce needles. In contrast, stomatal size tended to be larger in
populations having higher Si rDNA indices (29.0 ± 0.9 µm for
interior spruce (Si rDNA index = 0.97)) than in populations
GAS EXCHANGE ADAPTATION AND RIBOSOMAL DNA MARKERS
119
Figure 3. (a) Stomatal limitations to Pn, and (b) stomatal densities on
needle surfaces versus Si rDNA index in Sitka × interior spruce
populations.
Figure 2. (a) Net photosynthesis (Pn) versus intercellular CO2 concentration (Ci) in Sitka (Si rDNA index = 0.11, triangles) and interior
(Si rDNA index = 0.97, squares) spruce seedlings. (b) Intercellular
CO2 concentrations (Ci) at half maximal Pn, and (c) Rubisco carboxylation efficiency at the light compensation point (VRuBP) under wellwatered conditions versus Si rDNA index in Sitka × interior spruce
populations.
having lower Si rDNA indices (26.3 ± 0.8 µm for Sitka spruce
(Si rDNA index = 0.11)), although they were not statistically
different.
Gas exchange response to drought
Under well-watered conditions, Sitka spruce populations had
higher Pn, gwv, TFD, and Rd, than interior spruce populations,
with gas exchange parameters decreasing as the Si rDNA index
increased (Figure 4). However, when seedlings were subjected
to drought, Pn, gwv and TFD (normalized at Ψpd of −1.5 MPa)
were all lower in Sitka spruce populations having a lower
Si rDNA index (Figures 4a--4c). The exception, under drought
conditions, was Rd (measured at Ψpd of −1.0 −1.1 MPa), which
was similar for all Sitka × interior spruce populations (Figure 4d).
Lower gas exchange rates in drought-treated Sitka spruce
populations than in interior spruce populations could be ascribed to an intolerance to dehydration (Figure 5). Although
initially higher under well-watered conditions, Pn, gwv, and
TFD all dropped sharply in Sitka spruce populations as seedling Ψpd decreased. In contrast, these parameters declined at a
slower rate in drought-treated interior spruce populations. Because of these differences in sensitivity to dehydration, Pn, gwv,
and TFD fell to half of their predrought maximum at lower Ψpd
in populations with a higher Si rDNA index (Figure 6).
Twenty-four hours after drought-treated seedlings had been
watered, Pn, gwv, and TFD recovered partially in all populations, but the degree of recovery varied (Figure 7). Populations
with a higher Si rDNA index generally had a more rapid
recovery of Pn, gwv, and TFD after drought, measured by
recovery to predrought values, than populations with a lower
120
FAN, GROSSNICKLE AND SUTTON
Figure 5. Changes in net photosynthesis (Pn) (a), stomatal conductance to water vapor (gwv) (b), and transpiration flux density (TFD) (c)
in relation to decreases in predawn water potentials (Ψpd) for Sitka
(Si rDNA index of 0.11, n) and interior (Si rDNA index of 0.97, u)
spruce seedlings.
Figure 4. Net photosynthesis (Pn) (a), stomatal conductance to water
vapor (gwv) (b), transpiration flux density (TFD) (c), and dark respiration rates (Rd) (d) versus Si rDNA index in Sitka × interior spruce
populations maintained under well-watered conditions (open symbols)
or subjected to moderate drought (Ψpd at −1.5 MPa) (filled symbols),
except dark respiration which was measured at −1.0 to −1.1 MPa.
Si rDNA index. Of the gas exchange processes, recovery from
drought was highest for TFD (45 to 65%), followed by Pn (25
to 45%), and gwv (18 to 35%).
Discussion
Populations with a higher Si rDNA index exhibited lower
maximum Pn under both saturating PAR and CO2 and well-watered conditions than those with a lower Si rDNA index. When
other environmental factors are not limiting, rates of CO2
diffusion to, and CO2 fixation at, the photosynthetic sites in
chloroplasts can limit photosynthesis. Carbon dioxide diffusion rates can be affected by stomatal and mesophyll conductance. Fixation of CO2 can be limited by the carboxylation
efficiency of Rubisco at low Ci , the regeneration of ribulose
1,5-bisphosphate at intermediate Ci , and the recycling of inor-
GAS EXCHANGE ADAPTATION AND RIBOSOMAL DNA MARKERS
Figure 6. Predawn plant water potentials (Ψpd) at which net photosynthesis (Pn) (a), stomatal conductance to water vapor (gwv) (b), and
transpiration flux density (TFD) (c) were at half of maximal values
versus Si rDNA index in Sitka × interior spruce populations. The
higher the Ψpd values (less negative) the lower a population’s dehydration tolerance.
ganic phosphate at high Ci concentrations (Farquhar et al.
1980, Sharkey 1985). Rubisco carboxylation efficiency, CO2
and light compensation points for Pn were all similar among
the seven populations, and corresponded to reported values for
Sitka spruce (Ludlow and Jarvis 1971, Leverenz and Jarvis
1979) and Engelmann spruce (DeLucia 1986). Carboxylation
efficiency could not, therefore, explain the differences in maximum Pn between Sitka and interior spruce populations.
Unlike carboxylation efficiency, gwv differed among the
populations as a result of differences in stomatal densities on
needle surfaces. Higher stomatal densities resulted in lower
stomatal limitations to Pn, and could partially explain higher
121
Figure 7. The recovery of net photosynthesis (Pn) (a), stomatal conductance to water vapor (gwv) (b), and transpiration flux density (TFD)
(c) of drought-treated seedlings in relation to Si rDNA index in Sitka
× interior spruce populations. After-drought measurements were taken
exactly 24 hours after rewatering and are expressed as the percentage
of the corresponding pre-drought value. Lines were drawn through the
data to provide a qualitative indication of the trend in recovery of gas
exchange versus Si rDNA index.
Pn in Sitka spruce populations than in populations having a
higher Si rDNA index. The importance of CO2 diffusion rate
to Pn was further suggested by the higher maximum Pn in
CO2-saturated than in light-saturated conditions in the Sitka ×
interior populations. Similar findings were found when comparing a coastal with a coastal × interior ponderosa pine (Pinus
ponderosa Dougl. ex Laws) population (Monson and Grant
1989). The estimated stomatal limitations for all Sitka × interior populations were comparable to reports for black spruce
(Picea mariana (Mill.) B.S.P.) (Stewart et al. 1995) and loblolly pine (Pinus taeda L.) (Teskey et al. 1986). Stomatal
anatomy seemed to be more important than carboxylation
efficiency for describing maximum Pn in the Sitka × interior
populations.
In contrast to well-watered conditions, interior spruce populations had greater dehydration tolerance of gas exchange
122
FAN, GROSSNICKLE AND SUTTON
processes than Sitka spruce populations, measured by the Ψpd
at which Pn, gwv and TFD are at half their maximal values, and
percent recovery of gas exchange processes after drought.
Because stomatal limitations to Pn increase under drought
conditions in spruce species (cf Stewart et al. 1995), higher gwv
in interior than in Sitka spruce populations would explain their
higher Pn. Black spruce populations that have greater dehydration tolerance are able to maintain higher gwv and Pn under
drought, and grow faster on dry sites than populations with
more limited dehydration tolerance (Tan et al. 1992, Tan and
Blake 1993). Trees occurring naturally on dry sites are generally more dehydration tolerant (Kuhns et al. 1993) and better
able to maintain high gas exchange rates under drought conditions than trees of the same species occupying more mesic sites
(Larsen 1981, Seiler and Johnson 1988). Our data are consistent with previous findings of greater drought tolerance in
interior spruce than in Sitka spruce (Grossnickle et al. 1996).
Because of its unique geographic location, the Nass--Skeena
transition zone has a heterogeneous, transitional climate with
both maritime and continental characteristics (Roche and Haddock 1987, Sutton et al. 1994). Environmental heterogeneity
tends to sustain genetic polymorphism (Hedrick 1986), and
trees with high degrees of heterozygosity are thought to be
more tolerant to environmental stresses (Müller-Starck 1985,
Stutz and Mitton 1988, Mitton et al. 1989). Gene transfer
between ecologically differing species as the result of introgressive hybridization can result in improved environmental
adaptation (Oliver et al. 1995) as appears to be true in the case
of Sitka and interior spruces. Incorporation of interior spruce
genes into the Sitka spruce genome has produced hybrids that
have both retained the high photosynthetic capability of Sitka
spruce under nondrought conditions, and acquired the greater
drought and freezing tolerance of interior spruce (Grossnickle
et al. 1996). These diverse physiological features allow Sitka
× interior spruce hybrids to occupy environments over a range
of transitional habitats, and compete for sites that are not well
utilized by either pure Sitka or interior spruce. Thus, environmental variability and selection pressure during evolution may
explain the existence of hybrid populations between Sitka and
interior spruce in the Nass--Skeena transition zone.
In this and previous work (Grossnickle et al. 1996), seedlings of all seven populations were treated under identical
environmental conditions. Differences in physiological attributes (phenotypic variation) between populations were related
to their corresponding Si rDNA index (genotypic variation)
values. This is consistent with Cheverud’s (1988) argument
that phenotypic analysis can sometimes substitute for genetic
analysis. However, genetic estimates provide a more direct
method of defining population differences (Willis et al. 1991).
Nevertheless, caution is required in using physiological
(phenotypic) measurements for screening populations with
unknown genetic background. Apart from the genetic dependency, physiology of tree species is strongly influenced by
environmental conditions. Different physiological processes
may respond in various ways to environmental conditions. For
instance, Pn, gwv, and TFD responses in Sitka and interior
spruce populations changed under different watering regimes,
whereas these populations had similar PAR for maximum Pn,
φ and Rd as soil water availability varied. Elucidation of the
interactions among physiological processes and environmental conditions will lead to an understanding of the adaptive
significances of interspecific DNA exchange and the use of
physiological tests. Testing under both limiting and nonlimiting environmental conditions may provide more reliable
information for the understanding of the evolutionary consequences of species introgression.
In conclusion, introgressive hybridization between Sitka
spruce and interior spruce in British Columbia’s Nass--Skeena
transition zone has resulted in a wide array of genotypes.
Gradual mixing of interior and Sitka spruce populations has
caused a proportional reduction in high gas exchange capability under well-watered conditions that is associated with Sitka
spruce populations. On the other hand, greater gas exchange
capability under drought conditions is related to greater dehydration tolerance in interior spruce than in Sitka spruce populations. The wide range of physiological responses found in
Sitka × interior spruce populations could explain the persistence of a spruce hybrid swarm in the heterogeneous habitats of
the Nass--Skeena transition zone.
Acknowledgments
Funding for this research was provided by the Science Council of
British Columbia (Contract No. 175 (T-5)), the British Columbia
Ministry of Forests (Contract No. 18605-20/SXSS) grants to Steven
C. Grossnickle and Ben C.S. Sutton, and a Natural Science and
Engineering Research Council of Canada industrial postdoctoral fellowship to Shihe Fan.
References
Abrams, M.D. 1994. Genotypic and phenotypic variation as stress
adaptations in temperate tree species: a review of several case
studies. Tree Physiol. 14:833--442.
Burns, R.M. and B.H. Honkala. 1990. Silvics of North America.
Volume 1, Conifers. USDA For. Serv., Agric. Handbook No. 654.
Washington, DC, pp 187--226, 260--267.
Cheverud, J.M. 1988. A comparison of genetic and phenotypic correlations. Evolution 42:958--968.
Daubenmire, R. 1972. Taxonomic and ecological relationships between Picea glauca and Picea sitchensis and their ecological interpretation. Can. J. Bot. 46:787--798.
DeLucia, E.H. 1986. Effect of low temperature on net photosynthesis,
stomatal conductance and carbohydrate concentration in Engelmann spruce (Picea engelmannii Parry ex Engelm.) seedlings. Tree
Physiol. 2:143--154.
Farquhar, G.D., S. von Caemmerer and J.A. Berry. 1980. A biochemical model of photosynthetic (CO2) assimilation in leaves of C3
species. Planta 149:78--90.
Grossnickle, S.C., B.C.S. Sutton, R.S. Folk and J.R. Gawley. 1996.
Relationship between nuclear DNA markers and physiological parameters for Sitka × interior spruce populations. Tree Physiol.
16:547--556.
Hedrick, P.W. 1986. Genetic polymorphism in heterogeneous environments: a decade later. Annu. Rev. Ecol. System. 17:535--566.
Jones, H.G. 1985. Partitioning stomatal and nonstomatal limitations to
photosynthesis. Plant, Cell Environ. 8:95--104.
GAS EXCHANGE ADAPTATION AND RIBOSOMAL DNA MARKERS
Kuhns, M.R., W.W. Stroup and G.M. Gebre. 1993. Dehydration tolerance of five bur oak (Quercus macrocarpa) seed sources from
Texas, Nebraska, Minnesota, and New York. Can. J. For. Res.
23:387--393.
Larsen, J.B. 1981. Geographic variation in winter drought resistance
of Douglas-fir (Pseudotsuga menziesii Mirb. Franco). Silvae Genet.
30:4--5.
Leopold, A.C. and P.E. Kriedemann. 1975. Plant growth and development. McGraw-Hill Book Co., Toronto, pp 102--103.
Leverenz, J.W. and P.G. Jarvis. 1979. Photosynthesis in Sitka spruce.
VIII. The effects of light flux density and direction on the rate of net
photosynthesis and the stomatal conductance of needles. J. Appl.
Ecol. 16:919--932.
Ludlow, M.M. and P.G. Jarvis. 1971. Photosynthesis in Sitka spruce
(Picea sitchensis (Bong.) Carr.). J. Appl. Ecol. 8:925--953.
Mitton, J.B. 1995. Genetics and the physiological ecology of conifers.
In Ecophysiology of Coniferous Forests. Eds. W.K. Smith and T.M.
Hinckley. Academic Press, New York, pp 1--36.
Mitton, J.B., H.P. Stutz, W.S. Schuster and K.L. Shea. 1989. Genotypic differentiation at PGM in Engelmann spruce from wet and dry
sites. Silvae Genet. 38:217--221.
Mcdermitt, D.K., J.M. Norman, J.T. Davis, T.M. Ball, T.J. Arkebauer,
J.M. Welles and S.R. Roemer. 1989. CO 2 response curves can be
measured with a field-portable closed-loop photosynthesis system.
Ann. Sci. For. 46 (suppl.):416s--420s.
Monson, R.K. and M.C. Grant. 1989. Experimental studies of ponderosa pine. III. Differences in photosynthesis, stomatal conductance,
and water-use efficiency between two genetic lines. Am. J. Bot.
76:1041--1047.
Müller-Starck, G. 1985. Genetic differences between ‘tolerant’ and
‘sensitive’ beeches (Fagus sylvatica L.) in an environmentally
stressed adult forest stand. Silvae Genet. 34:241--246.
Oliver, M.J., D.L. Ferguson and J.J. Burke. 1995. Interspecific gene
transfer: implications for broadening temperature characteristics of
plant metabolic processes. Plant Physiol. 107:429--434.
Rieseberg, L.H. and J.F. Wendel. 1993. Introgression and its consequences in plants. In Hybrid Zones and Evolutionary Processes. Ed.
R.G. Harrison. Oxford University Press, New York, pp 70--109.
Roche, L. 1969. A genecological study of the genus Picea in British
Columbia. New Phytol. 68:505--554.
Roche, L. and P.G. Haddock. 1987. Sitka spruce (Picea sitchensis) in
North America with special reference to its role in British forestry.
Proc. Royal Soc., Edinburgh, 93B:1--12.
123
Seiler, J.R. and J.D. Johnson. 1988. Physiological and morphological
responses of three half-sib families of loblolly pine to water-stress
conditioning. For. Sci. 34:487--495.
Sharkey, T.D. 1985. Photosynthesis in intact leaves of C 3 plants:
physics, physiology and rate limitations. Bot. Rev. 51:53--105.
Stewart, J.D., A. Zine El Abidine and P.Y. Bernier. 1995. Stomatal and
mesophyll limitations of photosynthesis in black spruce seedlings
during multiple cycles of drought. Tree Physiol. 15:57--64.
Stutz, H.P. and J.B. Mitton. 1988. Variation in Engelmann spruce
associated with variation in soil moisture. Arct. Alp. Res. 20:461-465.
Sutton, B.C.S., D.J. Flanagan and Y.A. El-Kassaby. 1991a. A simple
and rapid method for estimating representation of species in spruce
seedlots using chloroplast DNA restriction fragment. Silvae Genet.
38:119--123.
Sutton, B.C.S., D.J. Flanagan, J.R. Gawley, C.H. Newton, D.T. Lester
and Y.A. El-Kassaby. 1991b. Inheritance of chloroplast and mitochondrial DNA in Picea and composition of hybrids from introgression zones. Theor. Appl. Genet. 82:242--248.
Sutton, B.C.S., S.C. Pritchard, J.R. Gawley, C.H. Newton and G.K.
Kiss. 1994. Analysis of Sitka spruce-interior spruce introgression in
British Columbia using cytoplasmic and nuclear DNA probes. Can.
J. For. Res. 24:278--285.
Tan, W. and T.J. Blake. 1993. Drought tolerance, abscisic acid and
electrolyte leakage in faster- and slower-growing black spruce
(Picea mariana) progenies. Physiol. Plant. 89:817--823.
Tan, W., T.J. Blake and T.J.B. Boyle. 1992. Drought tolerance in fasterand slower-growing black spruce (Picea mariana) progenies: I.
Stomatal and gas exchange responses to osmotic stress. Physiol.
Plant. 85:639--644.
Teskey, R.O., J.A. Fites, L.J. Samuelson and B.C. Bongarten. 1986.
Stomatal and nonstomatal limitations to net photosynthesis in Pinus
taeda L. under different environmental conditions. Tree Physiol.
2:131--142.
Villar, R., A.A. Hel and J. Merino. 1994. Comparison of methods to
estimate dark respiration in the light in leaves of two woody species.
Plant Physiol. 105:167--72.
Willis, J.H., J.A. Coyne and M. Kirkpatrick. 1991. Can one predict the
evolution of quantitative characters without genetics? Evolution
45:441--444.
Wright, J.H. 1955. Species crossability in spruce in relation to distribution and taxonomy. For. Sci. 1:319--49.