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Effects of Climate and Water Stress on Physiological Performance in California's Redwoods

by

Anthony Ray Ambrose

B.S. (Humboldt State University) 1992

A dissertation submitted in partial satisfaction of the

requirements for the degree of

Doctor of Philosophy

in

Integrative Biology

in the

GRADUATE DIVISION

of the

UNIVERSITY of CALIFORNIA, BERKELEY

Committee in charge:

Professor Todd E. Dawson, Chair Professor David D. Ackerly

Professor Dennis D. Baldocchi Professor Stephen C. Sillett

Fall 2009

UMI Number: 3410797

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Effects of Climate and Water Stress on

Physiological Performance in California's Redwoods

(2009)

by

Anthony Ray Ambrose

Abstract

Effects of Climate and Water Stress on

Physiological Performance in California's Redwoods

by

Anthony Ray Ambrose

Doctor of Philosophy in Integrative Biology

University of California, Berkeley

Professor Todd E. Dawson, Chair

The physiology of trees is strongly influenced by environmental conditions.

However, our understanding of how changes in tree size affect their response to the

environment remains limited. This dissertation examines the effects of climate and water

stress on physiological performance in different-sized coast redwood (Sequoia

sempervirens) and giant sequoia (Sequoiadendron giganteum) trees in both greenhouse

and field settings.

When exposed to soil water stress and high vapour pressure deficit (VPD), S.

giganteum saplings maintained higher leaf water potentials () and gas exchange rates

than S. sempervirens saplings. S. giganteum saplings also maintained higher leaf gas

exchange rates at a given leaf and VPD, but also exhibited greater stomatal sensitivity

to decreasing and increasing VPD. Variation in leaf soluble sugar carbon isotope

discrimination (A13C) and leaf water oxygen isotope enrichment (A180) in saplings was

related to differences in gas exchange rates and VPD. Taller trees have lower leaf fthan

1

shorter trees due to greater effects of gravity and friction on water transport. Variation in

A C and A O along vertical gradients in mature trees of different height was related to

changes in leaf W, leaf gas exchange capacity, leaf structure, and canopy microclimate.

Adjustments in treetop branch water transport capacity and cavitation vulnerability partially compensated for lower leaf Y'vn. taller trees. Despite these adjustments, increasing leaf mass per unit area (LMA) led to decreasing mass-based gas exchange rates with height. Species-level differences in LMA, wood density, and area-based gas

exchange rates constrained other structural and physiological responses to increased

height. Treetop transpiration and stomatal conductance also decreased with increasing

height in S. sempervirens, although shorter trees exhibited greater stomatal sensitivity to

increasing VPD than taller trees.

These results reveal that the two redwood species exhibit fundamental differences

in their physiological response to climate and water stress that reflects contrasting

environmental conditions in their native habitat. They also suggest that the physiological

response of redwoods to climate change will vary with tree size and age, and that the

combined analysis of A CandA O can be useful for examining climate and water stress

effects on physiological performance in redwoods.

Chair Date

I dedicate this work to my wife Christine Ambrose.

i

TABLE OF CONTENTS

Dedication i

Table of Contents ii

Acknowledgments iv

Chapter 1: Effects of Humidity and Water Potential on Leaf Gas Exchange and

Stable Carbon and Oxygen Isotopes in California Redwoods

ABSTRACT 2

INTRODUCTION 3

MATERIALS AND METHODS 10

RESULTS 20

DISCUSSION 26

REFERENCES 35

TABLES 48

FIGURES 50

Chapter 2: Effects of Height on Treetop Branch Hydraulics, Leaf Structure and Gas

Exchange in California Redwoods

ABSTRACT 58

INTRODUCTION 59

ii

MATERIALS AND METHODS 62

RESULTS 72

DISCUSSION 78

REFERENCES 89

TABLES 100

FIGURES 102

Chapter 3: Effects of Height on Treetop Transpiration and Stomatal Conductance

in Coast Redwood

ABSTRACT 111

INTRODUCTION 112

MATERIALS AND METHODS 117

RESULTS 127

DISCUSSION 132

REFERENCES 139

TABLES 149

FIGURES 153

i i i

ACKNOWLEDGMENTS

I would like to express my deepest gratitude to all those who contributed directly or

indirectly to the completion of this dissertation. This work would not have been possible

without the contributions of many people and institutions. First, I wish to thank my

advisor and committee chair, Todd Dawson, and committee member Stephen Sillett, for

outstanding support, assistance, friendship, and direct contributions to all aspects of the

work presented here. I greatly appreciate all of your help over the last several years and

look forward to many more years of fruitful collaboration. I offer many thanks to

committee members David Ackerly and Dennis Baldocchi for thoughtful comments,

ideas, and discussions which greatly improved the quality of this dissertation and

contributed to my overall education. I also thank my qualifying exam committee

members Wayne Sousa and Mary Power for stimulating discussions and feedback. I

greatly appreciate the direct and indirect contributions of George Koch and Robert Van

Pelt to many aspects of this research. I thank Jim Spickler, Cameron Williams, Marie

Antoine, Giacomo Renzullo and Hiroaki Ishii for assistance in the field and laboratory;

Dana Horton and David Barajas for assistance with sample processing; Jarmilla

Pittermann for assistance with hydraulic measurements; Stefania Mambelli and Paul

Brooks for assistance with stable isotope analyses; Christina Bentrup for soil moisture

data processing; Steve Burgess for advice and assistance with sapflow measurements;

Kim DeLong for assistance with greenhouse experiments; and Kevin Tu for advice and

assistance with modeling and many other aspects of this research. Chapter 1 of this

dissertation will be published as a separate paper with Kevin Tu, Stefania Mambelli, and

iv

Todd Dawson as co-authors. Chapter 2 of this dissertation has already been published as

a separate paper in the journal Plant, Cell and Environment (2009) with Stephen Sillett

and Todd Dawson as co-authors. Chapter 3 of this dissertation will be published as a

separate paper with Stephen Sillett, George Koch, Robert Van Pelt, Marie Antoine, and

Todd Dawson as co-authors. I greatly appreciate all of these co-authors outstanding

contributions to these papers. I thank all of my fellow members of the Dawson Lab for

providing a wonderful and stimulating environment for my graduate education and for all

kinds of assistance throughout the years, including Kevin Simonin, Emily Limm, Greg

Goldsmith, Adam Roddy, Michal Shuldman, Allison Kidder, Kali Lader, Ansgar

Kahman, Radika Baskar, Natasha Hausmann, Elizabeth Wenk, Jim Johnstone, Vanessa

Boukili, Rich Brenner, Lou Santiago, and Rafael Oliviera. I appreciate the support and

assistance of the staff of the Department of Integrative Biology at UC Berkeley, including

Mei Griebenow, Emily Howard, Connie Hsu, and Michael Schneider. Humboldt

Redwoods State Park, Kings Canyon National Park, and UC Berkeley Center for Forestry

kindly granted permission to conduct research at my study sites. I gratefully

acknowledge financial support from the Save-the-Redwoods League, Global Forest

Science, the National Science Foundation, and many small grants provided by the

Department of Integrative Biology. Finally, I give special thanks to my wife Christine

Ambrose for unlimited and unconditional support and assistance throughout my graduate

career.

v

Chapter 1

Effects of Humidity and Water Potential on Leaf Gas Exchange

and Stable Carbon and Oxygen Isotopes in California Redwoods

1

ABSTRACT

We examined how soil water deficit and humidity affect leaf water potential, gas

exchange, and stable C and O isotope ratios in coast redwood (Sequoia sempervirens) and

giant sequoia (Sequoiadendron giganteum) saplings grown under controlled conditions.

We also tested if the combined analysis of C and O isotopes can be used to infer leaf gas

exchange responses to environmental changes in mature S. sempervirens and S.

giganteum trees in the field. The effect of soil water deficit on sapling leaf water

potential (), stomatal conductance (gs), and net photosynthesis (An) was generally

greater at low humidity than at high humidity. Across all treatments, S. giganteum

saplings maintained higher leaf W, gs, and An and higher gs and An at a given and leaf-

to-air vapour pressure deficit (VPD) compared to S. sempervirens saplings. Leaf soluble

sugar carbon isotope discrimination (A13CLSS) was positively correlated with both^n and

gs but not^4/gs. Leaf water oxygen isotope enrichment (A18OL) was negatively correlated

with gs and positively correlated with VPD. A negative relationship between A CLSS and

A18OL was associated with greater sensitivity of gs compared to An across treatments. We

observed large vertical gradients in A13CLSS and A18OL in mature trees, which was

attributed to simultaneous changes in xylem W, microclimate, physiology, leaf

morphology, and canopy structure. Our results suggest that the vulnerability of S.

sempervirens to soil and atmospheric drought is greater than S. giganteum, and that

combined analysis of C and O isotopes to understand leaf gas exchange dynamics is

promising but requires careful consideration of physiological and microclimatic

heterogeneity within tall canopies.

2

INTRODUCTION

Climate change models predict that increasing atmospheric greenhouse gas

concentrations will cause average annual temperatures in parts of the western United

States to increase 2.2 to 5.6 C by the end of the century (IPCC 2007; Karl, Mellilo &

Peterson 2009). As a result, ecosystems in the region will likely experience substantial

changes in precipitation and evaporation patterns, including a decline in springtime

precipitation, reduced snowpack, reduced fog frequency, and more frequent and severe

summer drought (Hayhoe et al. 2004; Lueng et al. 2004; Seager et al. 2007; Barnett et al.

2008; Bonfils et al. 2008; Pierce et al 2008; Johnstone & Dawson 2010). Reduced water

availability and increasing evaporative conditions will likely lead to greater levels of

water stress with severe consequences for the growth, productivity, and distribution of

trees and forests in the region. Understanding how key physiological processes

controlling water use and carbon gain may be affected by these changes is therefore

essential for the development of effective forest management strategies.

California's two redwood species, Sequoia sempervirens (D. Don) Endl (coast

redwood) and Sequoiadendron giganteum (Lindl.) Buchholz (giant sequoia), are the

tallest and largest trees on Earth, respectively, with individuals exceeding 115m height

and 55,000 m3 wood volume (Van Pelt 2001; Sillett unpublished data). However, both

species may be particularly susceptible to projected climate changes. Paleoecological

records show that redwood trees once had much wider geographical distributions than

they currently occupy (Miller 1988). Today, native S. sempervirens populations are

restricted to a narrow coastal fog belt in northern California and extreme southwestern

Oregon, while S. giganteum naturally occurs in approximately 67 isolated groves at mid-

3

elevations on the western slope of California's Sierra Nevada mountain range. These

contracted distributions appear to be the direct result of tree responses to past climatic

changes, especially those affecting soil water availability and the evaporative demand for

that water at the leaf level (Anderson & Smith 1994; Noss 2000). Both species also

appear to perform best with abundant soil and/or higher atmospheric moisture

(Stephenson 1996; Koch et al. 2004; Burgess & Dawson 2004; Simonin, Santiago &

Dawson 2009; Ewing et al. 2009). The range of S. sempervirens today confirms the

aforementioned as it is closely associated with the presence of frequent coastal fog, with

relatively mild temperatures and low vapour pressure deficits in most of the range

throughout the year (Dawson 1998). In contrast, S. giganteum grows in locations

experiencing a greater probability of atmospheric desiccation due to high temperatures

and saturation vapour pressure deficits during the summer. However, the probability in

the current climate of S. giganteum trees experiencing severe soil water deficits is low

because they are supplied with abundant soil water throughout the year that is replenished

by spring snowmelt (Rundel 1972). A significant decrease in soil water supply and/or a

marked increase in evaporative demand would likely lead to conditions that could cause

severe tree water deficits, potentially leading to lower photosynthesis and growth, treetop

die-back, reduced natural regeneration, increased mortality, and further range contraction.

In this study, we investigated the effects of humidity and water stress on leaf gas

exchange and C and O isotopes in S. sempervirens and S. giganteum trees in both

greenhouse and field settings. We sought to determine if the two redwood species

differed in their sensitivity to changes in important climatic drivers and evaluate whether

the simultaneous analysis of C and O isotopes can be used to infer leaf gas exchange

4

dynamics in redwoods under a range of environmental conditions. Because the effects of

seasonal and annual variation in climate are likely to vary with tree size and age, we

examined both saplings and mature redwood trees ranging from 28.9 to 112.9 m in

height. Our primary objectives were to: compare the physiological response of redwood saplings to changes in humidity and soil water availability in controlled greenhouse

conditions using gas exchange measurements and dual C and O isotope analyses; and test

the validity of using dual C and O stable isotope analyses to infer leaf physiological

performance along vertical climate and water potential gradients in mature redwood trees

under natural field conditions.

Stable carbon and oxygen isotope theory

Stable carbon isotope ratios (813C) in plant material have been widely used as a

tool for examining plant water-carbon relations and physiological responses to the

environment (Adams & Grierson 2001; Dawson et al 2002). The heavy stable isotope

C is discriminated against during photosynthetic assimilation of CO2. Discrimination

results from fractionations that occur during CO2 diffusion through the stomata into the

leaf, as 13C02 diffuses more slowly than I2CC>2, and during the carboxylation reactions of

photosynthesis, as the primary carboxylating enzyme ribulose 1,5-bisphosphate (RuBP)

carboxylase/oxygenase (Rubisco) reacts preferentially with 12CC>2. The most frequently

used model for evaluating C isotope discrimination (A13C) relates A1 C to the ratio of

intercellular (c,) and atmospheric (ca) CO2 mole fractions as (Farquhar et al. 1982):

A13C = a + (b-a)^- (1)

5

where a is the fractionation caused by differential diffusion of CO2 and CO2 through

the stomata (4.4%o; O'Leary 1981), and b is the net fractionation associated with

enzymatic reactions, primarily Rubisco, during carboxylation (~27%o; Farquhar &

Richards 1984). The C[/ca ratio reflects the balance between the supply of CO2 via

stomata and the photosynthetic demand for CO2. According to Eqn 1, any factor that

lowers Cj/ca will cause a linear decline in A13C. Because cjca and consequently A13C

decrease as stomata close to conserve water, A C can be used as an indicator of the

severity of water deficit experienced by a plant (Dawson & Ehleringer 1993; Dawson et

al. 2002). Eqn 1 is a useful but simplified version of the full equation proposed by

Farquhar et al. (1982) and ignores other minor fractionations that occur during

photorespiration and dark respiration. It also ignores the effect of leaf mesophyll CO2

resistance, which reduces CO2 concentrations at the sites of carboxylation (cc), thereby

resulting in lower A13C (Siebt et al. 2008).

Although it is inferential, analysis of A C of plant material has an advantage over

cuvette-based gas exchange measurements because it provides a time-integrated, rather

than instantaneous, estimate of cjc&. The time period that A13C integrates varies with the

particular plant material analyzed. For example, A C in leaf or wood cellulose reflects

photosynthesis-weighted C[lc& over the course of a growing season (Dawson et al. 2002;

Gessler et al. 2009), while AI3C in leaf soluble sugars reflects photosynthesis-weighted

cj/Ca over the course of about 1 day (Brugnoli et al. 1988; Lauteri, Brugnoli & Spaccino

1993; Scartazza et al. 1998). Because cjca is influenced by both the supply of CO2

through the stomata and mesophyll as well as the demand for CO2 during assimilation,

1"%

the relative influence of each factor in determining A C cannot be easily identified. To

6

address this limitation, the stable oxygen isotope ratio (8 O) of plant material has been

increasingly used to separate the effects of changes in stomatal conductance (gs) from the

effects of changes in net photosynthetic assimilation (An) on 8 C and plant performance

(Saurer, Aellen & Siegwolf 1997; Barbour & Farquhar 2000; Scheidegger et al. 2000;

Cernusak et al. 2003; Cernusak, Farquhar & Pate 2005; Keitel et al. 2006; Brandes et al.

2007; Grams et al. 2007; Gessler et al. 2009). The basis for this approach stems from the

fact that 8180 is sensitive to changes in evaporative conditions, and indirectly to gs, but is

not sensitive to changes in A and the effects of photosynthetic demand for CO2 on Cj/ca.

Because stomatal closure typically occurs in response to increasing leaf-to-air vapour

1 ft

pressure deficit (VPD), an increase in 8 O is often linked to a decrease in gs.

Simultaneous analysis of the variation in both leaf 813C (or A13C) and 8180 (or A180) can

therefore provide information on the relative changes of gs and An in response to

changing environmental conditions.

Leaf water 180 (8 1 8 0L) is influenced by both the isotopic composition of source

water and evaporative enrichment in leaves during transpiration (Gonfiantini et al. 1965).

The steady-state enrichment of water at the sites of evaporation relative to source water

(A18Oe) can be modeled as (Craig & Gordon 1965; Dongmann et al. 1974; Farquhar &

Lloyd 1993): A18Oe=e++fc+(A18Ov-ekA (2)

where e+ is the equilibrium fractionation between liquid water and vapor at the air-water

interfaces, k is the kinetic fractionation that occurs during water vapor diffusion from the

leaf intercellular air space to the atmosphere, A18Ov is the isotopic enrichment of vapor in

the atmosphere compared with source water, and eje{ is the ratio of ambient to

intercellular vapour pressures. The equilibrium fractionation, e+, is calculated as

(Bottinga& Craig 1969):

s+ = 2.644 -3.206 f10>\

V ^ 7 + 1.534

rl0e\ , T2 , V - M J

(3)

where T\ is leaf temperature (K). The kinetic fractionation, e^, is calculated as (Farquhar

et al. 1989; Cappa et al. 2003):

^ + 2 1 5 . ( 4 )

r,+rb

where rs and n> are the stomatal and boundary layer resistances to water vapour diffusion

(m2 s mol"1), and 32 and 21 are associated fractionation factors. The mean A180 of bulk

leaf water (A18OL) is expected to be less than at the sites of evaporation, with the

discrepancy between the two increasing as transpiration increases (Walker et al. 1989;

Flanagan, Comstock & Ehleringer 1991). This discrepancy occurs because the diffusion

of enriched water from the sites of evaporation back towards the leaf water pool is

opposed by the convective flow of unenriched source water from the xylem to the sites of

evaporation, described as the Peclet effect (Farquhar & Gan 2003). Under steady-state

conditions, A18OL can be calculated as (Farquhar & Lloyd 1993; Farquhar & Gan 2003):

A 0 L = A " Q , ( 1 - O ( 5 )

where $ is the dimensionless Peclet number describing the ratio of convection to

diffusion of enrichment as:

o-W (6)

0 1

where E is transpiration rate (mol m" s"), L is an effective path length (m), C is the molar

concentration of water (55.5 x 103 mol m"3), and D is the diffusivity of H2180 in water

(2.66 x 10"9 m2 s"1). With increasing E, water within the leaf mesophyll becomes

increasingly dominated by the convective flux of unenriched water to the sites of

evaporation, thereby reducing A18OL. Because E is closely correlated with gs at similar

VPD, A180L can potentially serve as an integrated measure of the stomatal supply of CO2

to leaves independent of the effects of photosynthetic demand for CO2 (Barbour &

Farquhar 2000).

We analyzed carbon isotope discrimination in leaf soluble sugars (A1JCLSS)

because it provides a short-term measure of integrated leaf gas exchange that we

hypothesized would closely correspond with instantaneous cuvette-based gas exchange

measurements. Similarly, we analyzed leaf water oxygen isotope enrichment (A OL)

rather than organic matter A180 because we were interested in evaluating short-term

responses to humidity and water stress on a comparable timescale as provided by

instantaneous gas exchange measurements and A CLSS- We did not analyze A O in leaf

soluble sugars because no reliable method for this currently exists. However, leaf sugars

are expected to be in close isotopic equilibrium with the water in which they were

formed, after taking into account an equilibrium fractionation of approximately +27%o

(Sternberg, De Niro & Savidge 1986; Barbour et al. 2000; Cernusak et al. 2003; Barbour

2007).

9

MATERIALS AND METHODS

Greenhouse experiment

Plant material and treatments

In fall 2006 we obtained 48 S. sempervirens and 48 S. giganteum saplings

approximately 3 years old and 1-2 m tall from a local nursery. The S. sempervirens

saplings were clones of the 'Aptos Blue' variety and the S. giganteum saplings were

grown from seed obtained from Calaveras County, California. Saplings were repotted in

60 L pots, watered daily, and supplied with nutrient solution (10-5-4) once every month.

No evidence of root binding was observed throughout the study. The saplings were

randomly divided into two equal groups, and the effects of humidity and soil water

availability on leaf water potential, gas exchange, and stable C and O isotope ratios were

examined during two sampling periods in May and June 2007. Within each sampling

period, 6 saplings of each species were randomly assigned to one of 4 treatment groups:

1) well-watered and high humidity, 2) soil water withheld and high humidity, 3) well-

watered and low humidity, and 4) soil water withheld and low humidity. Saplings of

each species were randomly assigned to one of two greenhouses with differing relative

humidity. Half of the saplings of each species in each greenhouse were provided with

saturating water daily to serve as controls, and the other half had water withheld for 13-

18 d. High and low humidity were maintained using greenhouse climate controls (Argus

Control Systems Ltd., White Rock, British Columbia, Canada), supplemented by

humidifiers and dehumidifiers. Relative humidity (RH) in the two greenhouses was 54.6

16.1 and 78.5 18.6 % during the first sampling period and 49.6 15.1 and 76.7 20.9

% during the second sampling period [mean 1 standard deviation (SD)]. Air

10

temperature (Ta) in the two greenhouses was 24.6 7.2 and 23.0 6.3 C during the first

sampling period and 24.6 7.4 and 25.1 5.8 C during the second sampling period

(mean 1 SD). These ambient conditions corresponded to evaporative demands as

represented by the saturation vapor pressure deficit of the air of 1.7 1.2 and 0.8 0.8

kPa during the first sampling period and 1.8 1.1 and 1.0 1.1 kPa during the second

sampling period (mean 1 SD). Supplemental metal halide overhead lighting was used

to maintain minimum light levels from 0600 - 1800 h at approximately 600 umol m"2 s"1

photosynthetic photon flux density (PPFD) in both greenhouses. Fans were used to

circulate and mix air within each greenhouse. All saplings were randomly rotated every

3-6 d throughout the experiment to eliminate positional effects. Leaf water potential and

gas exchange were measured prior to and at the end of both sampling periods, while leaf

C and O isotope ratios were only measured prior to and at the end of the second sampling

period.

Leaf water potential

Leaf water potential was measured at pre-dawn (between 0400 - 0500 h) and at

mid-day (between 1100 and 1400 h) on representative shoots in the upper crown of each

sapling using a pressure chamber (Model 600, PMS Instruments Inc., Albany, OR, USA).

On each day, water potential measurements were made on a random selection of half of

the saplings in each treatment group. Measurements of pre-dawn water potential (fo)

made 1-3 d before treatments revealed no significant differences in water status among

saplings of each species in the different treatment groups. Baseline !ffc> for the first

sampling period was -0.19 0.01 and -0.20 0.01 MPa in S. giganteum and S.

11

sempervirens, respectively [mean 1 standard error (SE)]. Baseline Iffo for the second

sampling period was -0.09 0.01 and -0.10 0.01 MPa in S. giganteum and S.

sempervirens, respectively (mean 1 SE). !ffo and mid-day water potential ( *FMD) were

measured 17-18 d after treatments began in the first sampling period and 13-14 d after

treatments began in the second sampling period. Measurements of both !?PD and *FMD

alternated among species and treatment groups in each sampling period to eliminate

temporal bias.

Leaf gas exchange

Net photosynthesis (An), stomatal conductance (gs), and transpiration (E) per unit

leaf area were measured at mid-day (1100 - 1500 h) on representative shoots in the upper

crown of each sapling using a portable gas exchange system with a red-blue light source

(Model LI-6400, LiCor Inc., Lincoln, NE, USA). Photosynthetically active radiation

(PAR) and CO2 concentrations inside the chamber were maintained at 1200 |amol m" s" and 380 umol mol"1, respectively. Ta, RH, and air vapour pressure deficit inside the

chamber were maintained as close as possible to ambient levels within each greenhouse

at the time of measurement to approximate conditions influencing leaf 813C and 8180.

After gas exchange rates stabilized, six measurements were recorded over a period of 1

min and averaged for each shoot. Following measurement, the shoot within the chamber

was removed and digitally scanned for determination of shoot area via image analysis.

Each shoot was then dried at 60 C for 2 d and the dry mass recorded. In this study, we

define a shoot as a small diameter (

shoot mass per unit area (SMA), which was then converted to leaf mass per unit area

(LMA) using species-specific equations (Ambrose et al. 2009). LMA and dry mass were

then used to calculate leaf area-based gas exchange rates. Baseline measurements made

1-3 d before treatments revealed no significant differences in gas exchange among

treatment groups in each species. For the first sampling period, baseline An was 16.55

1.22 and 8.31 0.38 umol m"2 s"1, gs was 0.42 0.04 and 0.13 0.01 mol m"2 s"1, and E

was 3.56 0.27 and 1.54 0.07 mmol m" s" (mean 1 SE) for S. giganteum and S.

sempervirens, respectively. For the second sampling period, baseline An was 12.96

0.92 and 8.04 0.56 ^unol m"2 s"1, gs was 0.17 0.01 and 0.08 0.01 mol m"2 s'1, and E 9 1

was 2.02 0.16 and 1.04 0.09 mmol m" s" (mean 1 SE) for S. giganteum and S. sempervirens, respectively. Mid-day leaf gas exchange was measured 17-18 d after

treatments began in the first sampling period and 13-14 d after treatment began in the

second sampling period. Gas exchange measurements corresponded with leaf water

potential measurements on each sampling day and alternated among species and

treatment groups to eliminate temporal bias. Within each species, maximum gs values

were selected within 0.1 kPa categories, and the resulting data were fitted to the model gs =

gstet- In VPD, where gsref is a reference gs (defined as gs at 1 kPa VPD) and -Sis the sensitivity of the gs response to In VPD [i.e., the slope of gs versus InVPD (-dgs /dlnVPD)] (Oren et al. 1999).

Carbon and oxygen isotopes

On each sampling date in the second sampling period, 4-8 representative leaves

were collected in the afternoon (1400 -1500 h) from different positions in the upper-

13

crown of each sapling for determination of leaf soluble sugar C isotope ratios. Leaves

were immediately frozen in liquid N2 and stored at -20 C. Leaves were then freeze-dried

and reduced to fine powder in the laboratory. A sub-sample of about 150mg of this

powder was used for soluble sugar extraction, following the procedure outlined by

Brugnoli et al. (1988) with minor modifications. Leaf soluble sugars were extracted in water in the presence of PVPP (Polyvinylpolypyrrolidone, 1:1, w/w), purified with Dowex-50 (FT1-) resin for the separation of amino acids from organic acids and sugars, and Dowex-1 (CI") resin for the separation of organic acids from sugars. The sugar-containing fractions were then freeze-dried for 2 d before analysis. Leaf soluble sugar

samples were analyzed via elemental analyzer/continuous flow isotope ratio mass

spectrometry (ANCA/SL elemental analyzer (Sercon, Cheshire, UK) coupled with PDZ Europa Scientific 20/20 (Manchester, UK)). The ratio of 12C to 13C was expressed in delta notation (8, in %o units) relative to the Vienna-Pee Dee Belemnite standard (V-PDB) as:

5 = I *V r .mta I sample

V -^standard * J xlOOO (7)

where i?sampie and i?standard are the 13C/12C ratio of the sample material and standard,

respectively. Three replicate source air samples in each greenhouse were collected in a

60 mL syringe and injected into pre-evacuated 10 mL exetainer tubes at 1100 and 1600 h on each sampling date. Air samples in each exetainer were then analyzed by isotope ratio

mass spectrometry in continuous flow mode (Finigan MAT Deltaplus XL, Finnigan MAT, Bremen, Germany) and expressed as 513C relative to V-PDB following Eqn 1. The mean 8 C value of all 6 samples in each greenhouse was used as source air 8 C on each

sampling date. Leaf soluble sugar C isotope discrimination (A13CLSS) was calculated as:

14

1 + 813C A13CLSS = " r\

1500 h through 5 mm-diameter high-density polyethylene (HDPE) tubing attached to a 4-loop trap submerged in dry ice-ethanol slurry. Condensed vapor in each trap was thawed,

immediately decanted into airtight plastic tubes, and stored at -20 C. Stem xylem water,

bulk leaf water, and greenhouse water vapor were equilibrated with CO2 of known

volume and isotopic composition for 3 d (Scrimgeour 1995) and analyzed for stable O isotope ratios by isotope ratio mass spectrometry in continuous flow mode (Finigan MAT DeltaPlus XL, Finnigan MAT, Bremen, Germany). The ratio of 160 to 180 was expressed in delta notation (8, in %o units) relative to the Vienna-Standard Mean Ocean Water standard (V-SMOW) using Eqn 7. Bulk leaf water O isotope enrichment above source

15t

water (A OL) was calculated as:

8 - 0 , - 8 - 0 , L l

+ 5 18 0 s where 8180i and 818Os are the 8180 of bulk leaf water and source water, respectively.

1 ft

Stem xylem water of each sapling was used as source 8 O at each sample date. Baseline

measurements of A18OL made 1-3 d before treatments revealed no significant differences

between species or among different treatment groups. Baseline A13CLSS was 19.75 0.48

%o for S. giganteum and 19.21 0.36 %o for S. sempervirens (mean 1 SE). All isotope

analyses were conducted at the Center for Stable Isotope Biogeochemistry at the

University of California, Berkeley. Long-term (3+ year) external precision for C and O

isotope analyses are 0.14 %o and 0.09 %o, respectively.

Measured A13CLSS calculated with Eqn 8 was compared with predicted A13CLSS

calculated with the linear model of Eqn 1 using Cj/ca estimates obtained from gas exchange measurements. Measured A18OL calculated with Eqn 9 was compared to

16

predicted steady-state A OL calculated with Eqns 2-6. For calculation of predicted i o

AlsOL, Ta and RH were provided by direct greenhouse microclimatic measurements; E, rs,

and r\> were estimated from gas exchange measurements; 818Os and 618Ov were directly

measured; and T\ was estimated from empirical relationships with Ta in saplings of each

species (7i = 0.9939(ra) + 2.7237, R2 = 0.96, P < 0.0001 for S. giganteum; Tx =

1.0237(ra) + 2.4454, R2 = 0.98, P < 0.0001 for 5. sempervirens). A sensitivity analysis 1 S

revealed that the closest fit between modeled and measured A OL was obtained using an

lvalue of 0.005 m.

Field observations

Study sites and trees

Field observations of leaf C and O isotope ratios were made at 2 sites. The first

site was at Humboldt Redwoods State Park (HRSP) in northwestern California (4020' N,

1240' W, between 30 and 50 m above sea level). Trees at this site were located on the

alluvial floodplain of Bull Creek within an old-growth forest dominated by S.

sempervirens. The closed canopy in HRSP has high leaf area (Van Pelt & Franklin 2000)

and substantial vertical gradients in temperature and humidity (Ambrose, unpublished

data). HRSP experiences a moderate maritime-influenced climate throughout the year.

Annual precipitation at HRSP ranges from 1524 to 2032 mm, with most rainfall

occurring in the winter and occasional fog during the summer months (California State

Parks). The second site was at UC Berkeley's Whitaker Forest Research Station (WFRS)

in California's Sierra Nevada mountain range (3642' N, 11856' W, between 1600 and

1800 m above sea level). Trees at WFRS were located on moderate slopes in an all-aged

17

mixed-conifer forest consisting primarily of S. giganteum with a minor component of

Abies concolor, Calocedrus decurrens, and Pirtus lambertiana. The canopy at WFRS is

more open than at HRSP, with no significant vertical gradients in temperature or

humidity (Ambrose, unpublished data). WFRS experiences more variable and extreme

temperatures and vapor pressure deficits than HRSP. Precipitation at WFRS ranges from

1020 to 1140 mm annually, with a majority of the total precipitation falling as snow in

winter (Sequoia-Kings Canyon National Park). We selected 12 trees in 3 height classes

for sampling at each site. The crowns of each tree were accessed using single-rope and

arborist-style climbing techniques (Moffett & Lowman 1995; Jepson 2000). Trees

ranged from 25-98% and 34-95% of the maximum known height for S. sempervirens and

S. giganteum, respectively. All trees had fully exposed upper crowns and experienced

similar treetop light and microclimatic conditions within each site (Ambrose et al. 2009,

unpublished data).

Carbon and oxygen isotopes

We collected samples along vertical gradients in each tree for determination of

A13CLSS and A18OL- Leaf and stem samples were collected in the afternoon (1400 -1500

h) on clear days in July and November 2007 at the treetop, mid-crown, and base-of-live

crown positions of each tree. Approximately 4-8 representative leaves were collected at

each crown position, sealed in foil and plastic bags, and lowered to the ground. On the

ground, leaves were immediately frozen in liquid N2 and placed in coolers with dry ice

for transportation to the laboratory where they were stored at -20 C. Leaf samples were

then processed and analyzed for leaf soluble sugar 8 C following the procedure

18

described above for the greenhouse experiment. An additional 4-8 representative leaf

samples and 1 stem sample were also collected at each crown position, sealed in airtight

plastic tubes, and placed in coolers with dry ice for transportation to the laboratory where

they were stored at -20 C. Air and water vapor samples were collected at the same stem

and leaf sampling positions in 6 representative trees at each site for determination of

vertical gradients in source air 813C and water vapor 8180 following the procedures

described above for the greenhouse experiment. There were no significant differences

among trees or along vertical gradients in either air 8 C or vapor 8 O in the canopy at

either site (data not shown).

Statistics

Variation in leaf water potential, gas exchange, A13CLSS, and A18OL among control

and water-stressed saplings of each species in the greenhouse experiment was examined

using analysis of variance (ANOVA). Comparisons of interest were made using Tukey's

HSD test. Relationships among measured and modeled variables in both the greenhouse

and field experiments were examined using ordinary least squares and reduced major axis (RMA) regression. All statistical analyses were performed with JMP (version 8.0, SAS Institute, Cary, NC, USA).

19

RESULTS

Greenhouse experiment

Treatment effects on leaf water potential and gas exchange

Significant differences in pre-dawn water potential (k>) were observed between

control and water-stressed saplings of both species at both humidity levels (Table 1).

Control saplings at both humidity levels maintained *FPD at pre-treatment levels. Among

water-stressed saplings, fo was more negative in S. sempervirens than in S. giganteum

and was more negative under low than high humidity in both species. Differences

between !fpD and mid-day water potential (for>) in the two species were similar,

averaging 0.66 0.05 and 0.68 0.04 for S. giganteum and S. sempervirens, respectively

(mean 1 SE). Significant differences in most leaf gas exchange variables between

control and water-stressed saplings were observed in both species at both humidity levels

(Table 1). In all treatment groups except water-stressed saplings at low humidity, S.

giganteum had significantly higher net photosynthesis (Aa), stomatal conductance (gs),

and transpiration (E) rates than S. sempervirens. There were also greater absolute but

lower proportional reductions in Aa, gs, and E in water-stressed S. giganteum compared to

water-stressed S. sempervirens at both humidity levels. There was a greater proportional

reduction in An in water-stressed compared to control saplings at low humidity than at

high humidity in both species, with less decline in S. giganteum (66% versus 45%) than

in S. sempervirens (71% versus 58%). There was also a greater proportional reduction in

gs in water-stressed compared to control S. giganteum at low humidity compared to high

humidity (71% versus 54%), but there was a similar reduction in gs in S. sempervirens at

both humidity levels (72% versus 71%). Water-stressed saplings of both species had

20

lower E compared to control saplings at both humidity levels, although these differences

were not significant at high humidity. Control and water-stressed saplings of both

species generally had lower E at low humidity compared to high humidity. Control

saplings of S. giganteum had 18% lower An and 39% lower gs at low humidity compared

to high humidity, while control saplings of S. sempervirens had 38% lower An and 49%

lower gs at low humidity compared to high humidity. Control saplings of S. giganteum

had 7% higher E at high humidity compared to low humidity, while control saplings of S.

sempervirens had 16% lower E at high humidity compared to low humidity. There were

no significant differences in instantaneous Cj/ca between water-stressed and control

saplings in either species at either humidity level (Table 1).

Relationship of leaf gas exchange with leaf water potential and vapour pressure deficit

When data were combined for each species, leaf gas exchange significantly

declined as fo and WMD became more negative, with steeper declines associated with

decreasing fo in both species (Fig. 1). At a given fo and *FMD, S. giganteum had

higher An, gs, and E rates than S. sempervirens, although differences between the species

diminished as fc> and YMD values became increasingly negative. Correlations of !ffc>

and *FMD with leaf gas exchange characteristics were stronger in S. sempervirens than S.

giganteum. When data were combined for each species, there was a significant decline in

maximum gs with increasing leaf-to-air vapour pressure deficit (VPD; Fig. 2).

Sequoiadendron giganteum had a higher gSK{ but also a greater sensitivity to increasing 0 1

VPD (-S) than S. sempervirens (0.58 versus 0.21 mol m" s" for gsref and 0.54 versus 0.15

21

mol m'2 s"1 kPa"1 for -8, respectively), leading to increasingly similar gs values in both

species at VPD values approaching 2.5 kPa (Fig. 2).

Correlations among leaf gas exchange and carbon and oxygen isotopes

A CLSS was positively correlated with AR and gs in both species at low humidity,

positively correlated with An in S. giganteum at high humidity, and not correlated with

AJgs in either species (Fig. 3). When data from both humidity levels were combined,

A13CLSS in both species was positively correlated within (R2 = 0.60, P < 0.0001 in 5.

giganteum and R2 = 0.25, P = 0.009 in 5. sempervirens) and gs (R2 = 0.60, P = 0.001 in S.

giganteum and R = 0.20, P = 0.002 in 5. sempervirens). Although there was a greater

range in^4n and gs in S. giganteum compared to S. sempervirens (18.1 versus 11.8 umol 9 1 ") 1

m" s" for AQ and 0.30 versus 0.17 mol m" s" for gs, respectively), there was a smaller

range in A13CLSS in S. giganteum compared to S. sempervirens (3.5 versus 4.8%o,

respectively). Mean values of A CLSS did not differ significantly among water-stressed

and control saplings or between species at either humidity level (P > 0.05). A18OL was

negatively correlated with gs and E in S. giganteum at low humidity, negatively correlated

with gs in S. sempervirens at high humidity, and positively correlated with VPD in S.

sempervirens at low humidity (Fig. 4). When data from both humidity levels were

combined, A18OL was negatively correlated with gs in both species (R2 = 0.33, P - 0.004

in S. giganteum and R = 0.40, P = 0.0007 in S. sempervirens), negatively correlated with

E in S. sempervirens (R2 = 0.25, P = 0.009), and positively correlated with VPD in both

species (R2 = 0.55, P < 0.0001 in S. giganteum and R2 = 0.58, P = 0.009 in S. 1 ft

sempervirens). Similar to gs, there was a greater range of both E and A GLUIS . 22

9 1

giganteum compared to S. sempervirens (4.0 versus 2.8 mmol m" s" for E and 18.8 1R

versus 16.9%o for A OL, respectively), even though S. giganteum experienced a smaller

range of VPD than S. sempervirens (1.6 versus 2.1 kPA, respectively). Mean values of

A OL did not significantly differ among water-stressed and control saplings or between

species at either humidity level (P > 0.05).

Covariation in leaf gas exchange and carbon and oxygen isotopes 152

Across all treatments in each species, mean A OL was negatively related to mean

A CLSS, and mean An was positively related to mean gs (Fig. 5). In both species the

relative change in A OL was greater than the relative change in A CLSS and the relative

change in gs was greater than the relative change in Aa. In S. giganteum, there was a

0.14%o change in A CLSS for a l%o change in A OL, which corresponded to a 50.51 9 1 9 1

umol m" s" change in An for a 1 mol m" s" change in gs. In S. sempervirens, there was a

0.12%o change in A13CLSS for a l%o change in A18OL, which corresponded to a 70.88

umol m" s"1 change inAn for a 1 mol m"2 s"1 change in gs. Thus, there was a larger 1 "X 1 Jt

change in A CLSS relative to A OL but a smaller change in An relative to gs in S.

giganteum compared to S. sempervirens.

Comparison of predicted versus observed carbon and oxygen isotopes

When all data were combined, there was no significant correlation between

measured and predicted AI3CLSS estimated using cjca values from gas exchange

measurements and Eqn 1, although the data were centered on a 1:1 relationship (data not

shown). There was a larger range in predicted than measured A CLSS (8.6 versus 5.3%o, 23

respectively). Correlations between measured and predicted A CLSS did not improve

when considering species or treatment groups separately. When all data were combined,

there was a significant positive correlation between measured and predicted A18OL

estimated using gas exchange and microclimate measurements and the steady-state leaf

water model incorporating the Peclet effect (Eqn 5; data not shown). The best fit

between measured and predicted A18OL, based on proximity to the 1:1 line, was obtained

when assuming an L value of 0.005 m. The correlation between measured and predicted -to 1 Q

A OL improved when analyzing each species separately: predicted A OL = 11.04 +

0.52-measured A180L, R2 = 0.89, P < 0.0001 for S. giganteum; predicted A180L = 11.96 +

0.57-measured A180L, R2 = 0.84, P < 0.0001 for S. sempervirens. In contrast to A13C,

there was a smaller range in predicted than in measured A18OL (9.4 versus 18.7%o,

respectively).

Field observations

Vertical gradients in carbon and oxygen isotopes

When data for all crown positions were combined in each species, there was a

significant decrease inA1JCLSS with increasing height in different-sized mature S.

giganteum and S. sempervirens trees (Fig. 6a). The slope of the regression with height

was similar in both species, but S. giganteum had a lower A CLSS at a given height than

S. sempervirens. When crown positions were analyzed separately, significant decreases

in A13CLSS with increasing height were only observed at the mid-crown and treetop

positions of both species (Table 2). Across all heights, S. giganteum had generally higher

A18OL than S. sempervirens. When data from all crown positions were combined, there

24

was a significant increase in bulk leaf water A OL with increasing height in S.

sempervirens but not in S. giganteum (Fig. 6b). When crown positions were analyzed

separately, significant increases in A I 8OL were observed at all 3 crown positions in S.

sempervirens but only at the treetop position in S. giganteum (Table 2). Regression

coefficients of determination of A13CLSS and A18OL against height were stronger for

combined data than for most individual crown positions separately (Fig. 6; Table 2).

Covariation in carbon and oxygen isotopes

When mean data for all crown positions were combined in each species, there was

a significant negative correlation between A CLSS and A OL in S. sempervirens but not

in S. giganteum (Fig. 7). There were no significant correlations in either species when

individual crown positions were analyzed separately. Thus, the negative relationship

observed in S. sempervirens resulted from differences among the crown positions. There I D 1 -J

was also a significant negative correlation between A OL and A CLSS across the two

species (A13CLSs = 21.47 - 0.20-A18OL, R2 = 0.48, P = 0.009).

25

DISCUSSION

Results of our greenhouse experiment indicate that S. sempervirens and S.

giganteum saplings possess different levels of physiological sensitivity to short-term soil

water deficit and lowered atmospheric humidity. Soil water deficit and low humidity had

a greater combined effect on leaf water potential and gas exchange than either of the

treatments individually, suggesting that climate change scenarios that involve both

reduced plant water availability and increased evaporative demand due to higher

temperatures will have a potentially large impact on the water and carbon relations of

these species. However, the relative influence of each greenhouse treatment on gas

exchange characteristics differed between the two species and will determine their

potential response to these changes. In all treatments, S. giganteum maintained higher

leaf water potentials and leaf gas exchange rates, maintained higher gas exchange rates at

a given leaf water potential and VPD, and had higher reference gs and exhibited greater

stomatal sensitivity to decreasing water potential and increasing VPD than S.

sempervirens. This is in agreement with previous observations in a wide range of

systems showing that species with high gs at low VPD also have higher sensitivity to

increasing VPD than species with lower gs, consistent with the functional role of stomata

in regulating leaf water potential (Oren et al. 1999). Overall, reductions in gas exchange

in water-stressed saplings compared to controls were proportionally greater in S.

sempervirens than in S. giganteum at both low and high humidity. Thus, S. giganteum

saplings have greater stomatal control of leaf-level water loss and are able to maintain

higher leaf gas exchange rates than S. sempervirens saplings under conditions of both soil

and atmospheric drought. Water-stressed S. giganteum saplings showed greater declines

26

in gs than in An at both humidity levels compared to control saplings (Table 1), indicating

that they may increase their water-use efficiency in response to soil and atmospheric

drought. In contrast, water-stressed S. sempervirens saplings showed greater declines in

gs than in An only at high humidity compared to control saplings (Table 1), indicating that

water-use efficiency in saplings of this species will be substantially compromised if

reduced soil water availability is accompanied by greater atmospheric drought.

We found positive relationships between An, gs, and A13CLSS in both S. giganteum

and S. sempervirens saplings, although the strength of the relationships differed between

species and humidity levels (Figs. 3 & 5). Plants commonly respond to reduced soil or

atmospheric moisture with simultaneous decreases in both gs and An (Farquhar & Sharkey

1982). If the "supply function" of photosynthesis (i.e., gs) decreases at a faster rate than

the "demand function" (i.e., biochemical capacity) then both cjc& and A C will decrease.

We observed a greater decline in gs than in An, leading to positive correlations with

A CLSS in both species. Stronger correlations were observed at low humidity with

distinct differences in An and gs between water-stressed and control saplings, indicating

that the combination of soil and atmospheric drought produced a stronger effect on Cj/ca and A13CLSS than soil drought alone. This conclusion is consistent with chlorophyll

fluorescence measurements in S. sempervirens saplings experiencing short-term soil

water deficits indicating that variation in An is primarily due to variation in gs in response

to declining water potential and not damage to or downregulation of photosystem II

(Simonin et al. 2009). The lower photosynthetic rates we observed are therefore likely

the result of lower gs and consequently lower CO2 supply rather than impaired

photosynthetic capacity.

27

There are several possible reasons why there was not a stronger relationship

betweenA13CLss and cjca or AJgs in our greenhouse experiment. First, ci/ca or AJgs

estimated from instantaneous measurements reflect gas exchange at only one time point I T

in the afternoon while the A C values of leaf soluble sugars reflect photosynthetic

activity throughout the day (Brugnoli et al. 1988; Farquhar et al. 1989). The

instantaneous measurements therefore do not account for diurnal variation in gas

exchange that is reflected in A CLSS, which likely varies among species and saplings

experiencing different environmental conditions and water status. Second, cjca or Aa/gs

only reflects the influence of stomatal resistance, whereas A13CLSS reflects the ratio of

chloroplast and atmospheric CO2 concentrations (cc/ca) influenced by both stomatal and

mesophyll CO2 resistances which do not necessarily scale with each other or with An

(Warren & Adams 2006; Flexas et al. 2008; Siebt et al. 2008). Finally, logistical

constraints prevented us from measuring leaf gas exchange on more than one leaf per tree

but we were able to obtain multiple leaf samples for A13CLSS analysis. Although we

attempted to sample foliage of similar condition and from similar crown positions for

each type of measurement, it is possible that differences arose from slight variation in

environmental conditions and leaf physiology between samples.

Our greenhouse results support current theory (Farquhar & Lloyd 1993; Farquhar,

Cernusak & Barnes 2007) and numerous studies in a range of species (e.g., Barbour et al.

2000a; Barbour & Farquhar 2000; Cernusak et al. 2003, 2005; Brandes et al. 2007) 1 St

suggesting that plants with more open stomata should have lower leaf water A O. Plants

with higher gs tend to have lower leaf temperatures, which lowers e\ and equilibrium

fractionation (e ), and produces leaf water less enriched in O. Higher transpiration rates 28

associated with greater gs should also increase the Peclet number (p), thereby

reinforcing the decrease in enrichment. Consistent with this, we observed negative

relationships between gs and E with A18OL, although the strength of the relationships

differed between species (Fig. 4). These differences are likely due to the fact that

maximum gs but not all gs values are strongly controlled by VPD in S. giganteum and S.

sempervirens (Fig. 2), similar to recent observations in Pinus sylvestris (Brandes et al. 1 8

2007). This finding, combined with the fact that differences in A OL were substantially 1 8

larger among than within humidity levels (Fig. 4), indicates that variation in A OL in redwoods may be more strongly related to variations in evaporative conditions (ea/ei) than to variation in gs. These results suggest that the degree to which the full operating range

of gs is controlled by VPD in any particular species must be carefully considered when 1 o i o

interpreting variation in A OL. The model we applied to predict A OL (Eqn 5)

confirmed that in addition to eje\, a Peclet effect driven by E and scaled effective path 1 8

length (!) has to be taken into account when estimating leaf water A OL. The best model results were obtained when L was assumed to be 0.005 m. This value is low but within

the range observed or modeled for other tree species, including conifers (Wang, Yakir & Avishai 1998; Cernusak et al. 2005; Farquhar & Cernusak 2005; Pendall, Williams &

Leavitt 2005; Brandes et al. 2007; Kahmen et al. 2008).

When variation in A1JC is driven by changes in gs, a negative relationship

between A C and A O (i.e., a positive relationship between 8 C and 8 O) is predicted

(Yakir & Israeli 1995). This is because decreases in gs will tend to decrease C[/ca and

A13C as well as A180, while decreases in An will tend to increase C[/ca and A13C but not 1 8

affect A O. If both gs and An change simultaneously, then the decrease in Cj/ca caused by 29

decreasing gs will be partially offset by the increase in Ci/ca associated with the increased

Aa, and the change in A180 for each unit change in A13C should increase (Barbour,

Walcroft & Farquhar 2002). Because gs tended to decline at a greater rate than An, a

generally negative relationship between A CLSS and A180L was still observed across

treatment groups in both S. giganteum and S. sempervirens (Fig. 5). The stronger

relationship between A18OL and A1 CLSS in S. giganteum saplings is likely due to greater

stomatal sensitivity to VPD in this species compared to S. sempervirens (Fig. 2). These

results are consistent with observations of the relationship between A C and A O in

organic matter from a number of other systems (e.g., Sternberg, Mulkey & Wright 1989;

Saurer et al. 1997; Barbour & Farquhar 2000; Barbour et al. 2000; Barbour et al. 2002;

Sullivan & Welker 2007). Our results are also generally in agreement with the

conceptual models proposed by Scheidegger et al. (2000) and Grams et al. (2007).

However, an important requirement of these models is that gs be strongly controlled by

RH or VPD. We found that this assumption is not entirely valid in the saplings we

examined, thereby limited our ability to make strong inferences about the relative

influence of gs and Aa on A13C. The results of our greenhouse experiment therefore

suggest that the usefulness of these conceptual models will be compromised in species or

systems where this assumption is not met, and that in these cases measurement of

maximum gs rather than ambient gs may permit more robust mechanistic interpretations

of C and O isotopes.

We observed a large degree of isotopic variation within and among different-sized

1 ^ 1 R

mature redwood trees in the field, with A CLSS varying 6.4%o and A OL varying 19.3%o

along the height gradient in S. giganteum, and A13CLss varying 8.2%o and A180L varying

16.1%o along the height gradient in S. sempervirens (Fig. 6). The large variation we

observed reflects the influence of substantial vertical gradients in water stress,

microclimate, physiology, leaf morphology, and canopy structure in tall trees and forests.

Leaf water potential {) decreases with increasing height due to the constant hydrostatic

effects of gravity and is further reduced by increasing hydraulic path-length resistance

during transpiration (Zimmermann 1983). An increase in leaf-level water stress caused

by lower with increasing height leads to reduced gs and consequently decreased An and

carbon uptake (Ryan & Yoder 1997). Lower also induces lower leaf turgor, resulting

in reduced cell expansion, slower cell division, and higher leaf mass per unit area (LMA;

Koch et al. 2004; Woodruff, Bond &Meinzer 2004; Meinzer, Bond & Karanian 2008).

Lower LMA may in turn reduce leaf mesophyll CO2 conductance (gm), further lowering

carbon assimilation (Parkhurst 1994; Niinemets 1999; Flexas et al. 2008; Mullin et al.

2009). In many closed forest canopies, there are also substantial changes in light,

temperature, and humidity with height, typically leading to higher VPD in the upper

canopy (Parker 1995). While acclimation to higher irradiance towards the treetop tends

to enhance photosynthetic capacity of foliage, evidence suggests that decreasing !Pand

increasing VPD offset the positive effects of higher light on photosynthetic gains in the

upper canopy (Pearcy 1999; Niinemets, Sonninen & Tobias 2004; Niinemets &

Valladares 2004; Koch et al. 2004; Ishii et al. 2008; Ambrose et al. 2009; Mullin et al.

2009). These combined effects result in increased foliar 813C with increasing height in a

large number of trees and ecosystems (Buchmann et al. 1997a, 1997b; Berry, Varney &

Flanagan 1997; Yoder et al. 1994; Hubbard et al. 1999; McDowell et al. 2002,2005;

Scartazza et al. 2004). Significant vertical gradients in bulk leaf water 8180 resulting

31

from changes in environmental, structural, and physiological conditions have also been

observed in both temperate and tropical forest trees (Sternberg, Mulkey, & Wright 1989;

Saurer, Siegwolf & Scheidegger 2001; Ometto et al. 2005). All of these influences must

therefore be considered in order to understand the causes of variation in A C and A O

throughout forest canopies.

We found that A13CLSS was lower at a given height in S. giganteum than in S.

1 ^

sempervirens and that A CLSS significantly declined with height in both species at a 1 &

similar rate (Fig. 6a). In contrast, we found that A OL was lower at a given height in S.

sempervirens than in S. giganteum, and that A18OL significantly increased with height in

S. sempervirens but not in S. giganteum (Fig. 6b). The height gradient led to a significant

negative relationship between A CLSS and A OL in S. sempervirens but not in S.

giganteum (Fig. 7). According to the conceptual models of Scheidegger et al. (2000) and

Grams et al. (2007), these patterns would indicate a decrease in VPD and/or gs but no

change in An (or a greater relative change in VPD and/or gs than in An) with increasing

height in S. sempervirens, and an increase in Aa but no change in VPD and/or gs (or a

greater relative change in An than in VPD and/or gs) with increasing height in S.

giganteum. Sapflow measurements in S. sempervirens trees of different height show

significantly lower average treetop area-based stomatal conductance (Gs) rates in taller

trees (Ambrose et al. 2010). If treetop Gs declines at a greater rate than treetop

photosynthesis in these trees, these results are consistent with the generally negative

relationship between A13CLSS and A18OL we observed at the tops of different-sized S.

sempervirens trees in the present study. We do not have comparable sapflow data to

evaluate the isotopic patterns of variation in S. giganteum. Gas exchange measurements

from both species show no significant height trends in maximum area-based An or gs but

do show significant decreases in maximum mass-based Aa and gs with increasing height

(Grulke & Miller 1994; Koch et al. 2004; Ishii et al. 2008; Mullin et al. 2009; Ambrose

et al. 2009; Ambrose, unpublished data). The decrease in maximum mass-based gas

exchange rates was attributed to increases in LMA and decreases in gm with height.

Collectively, these results suggest that lower A13CLSS with increasing height reflects the

influence of stomatal and mesophyll resistance and hence cjc&, highlighting the

importance of considering leaf structure and its effects on gm when interpreting variation

in A C within or among species. These effects were not evident in our greenhouse

experiment because all saplings of a given species had similar LMA values. However,

the lower A13CLSS in S. giganteum saplings compared to S. sempervirens saplings likely

reflects the greater LMA and hence lower gm in S. giganteum.

Our S. sempervirens study trees were located in a dense and closed canopy with

substantial increases in VPD with height, while our S. giganteum study trees were located

in a more open and sparse canopy with no discernible trend in VPD with height 1 o

(Ambrose, unpublished data). The fact that we observed a significant increase in A OL

with increasing height in S. sempervirens but no significant change in A OL with height

in S. giganteum is probably largely the result of differences in canopy structure and

evaporative conditions between the two sites. Both area- and mass-based gs rates in S.

giganteum are higher than in S. sempervirens at all heights (Ambrose et al. 2009),

suggesting that the higher A18OL observed at all heights in S. giganteum compared to S.

sempervirens is due to much higher VPD at the former compared to the latter site. These

33

data therefore highlight the importance of considering canopy structure and VPD in

addition to gs when interpreting variation in A180 within forest canopies.

In conclusion, our greenhouse experiment showed that the combination of soil

and atmospheric drought had a much greater impact on leaf water potential and gas

exchange than either one effect individually, but that the physiological response to these

effects differed between the two species. Under high VPD conditions, S. giganteum

appears to have greater stomatal control of leaf-level water loss than S. sempervirens, but

is equally sensitive to soil water deficits. Observed and predicted reductions in

springtime rainfall and fog frequency on the California coastline and reduced snowpack

and spring ground water recharge in the Sierra Nevada will therefore likely have large

impacts on the carbon and water relations of these species. Our greenhouse experiment

and field observations both indicate that the analysis of A CLSS and A OL can be a

promising tool for assessing physiological responses to changing environmental

conditions in trees, but adequate consideration of species-level differences in stomatal

sensitivity, leaf morphology, mesophyll conductance, and canopy structure is critical for

correct interpretation of isotopic variation resulting from these changes.

34

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