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Seasonal variation of leaf wax n-alkane 2

production and δ H values from the evergreen oak tree, Quercus agrifolia ab

c

Dirk Sachse , Todd E. Dawson & Ansgar Kahmen

cd

a

Helmholtz Centre Potsdam – GFZ German Research Centre for Geosciences, Section 5.1: Geomorphology, Potsdam, Germany b

Institute of Earth and Environmental Sciences, University of Potsdam, Potsdam-Golm, Germany

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c

Center for Stable Isotope Biogeochemistry, Department of Integrative Biology, University of California, Berkeley, CA, USA d

Department of Environmental Sciences – Botany, University of Basel, Basel, Switzerland Published online: 23 Feb 2015.

To cite this article: Dirk Sachse, Todd E. Dawson & Ansgar Kahmen (2015): Seasonal variation 2

of leaf wax n-alkane production and δ H values from the evergreen oak tree, Quercus agrifolia, Isotopes in Environmental and Health Studies, DOI: 10.1080/10256016.2015.1011636 To link to this article: http://dx.doi.org/10.1080/10256016.2015.1011636

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Isotopes in Environmental and Health Studies, 2015 http://dx.doi.org/10.1080/10256016.2015.1011636

Seasonal variation of leaf wax n-alkane production and δ 2 H values from the evergreen oak tree, Quercus agrifolia

Downloaded by [University of Victoria] at 21:34 08 April 2015

Dirk Sachsea,b∗ , Todd E. Dawsonc and Ansgar Kahmenc,d a Helmholtz Centre Potsdam – GFZ German Research Centre for Geosciences, Section 5.1: Geomorphology, Potsdam, Germany; b Institute of Earth and Environmental Sciences, University of Potsdam, Potsdam-Golm, Germany; c Center for Stable Isotope Biogeochemistry, Department of Integrative Biology, University of California, Berkeley, CA, USA; d Department of Environmental Sciences – Botany, University of Basel, Basel, Switzerland

(Received 12 October 2014; accepted 16 January 2015)

Dedicated to Professor Dr Hanns-Ludwig Schmidt on the occasion of his 85th birthday In order to understand the timing of leaf wax synthesis in higher plants, we analysed the variability in leaf wax n-alkane concentration, composition (expressed as average chain length (ACL)), and δ 2 Hwax values as well as plant source water δ 2 H values (xylem and leaf water) in the evergreen tree Quercus agrifolia over a period of 9 months, beginning with leaf flush. We identified three distinct periods of leaf development with the first month following leaf flush being characterized by de novo synthesis and possibly removal of n-alkanes. During the following 3 months, n-alkane concentrations increased sevenfold and δ 2 Hwax and ACL values increased, suggesting this period was the major leaf wax n-alkane formation period. During the remaining 4 months of the experiment, stable values suggest cessation of leaf wax n-alkane formation. We find that n-alkane synthesis in Q. agrifolia takes place over 4 months, substantially longer than that observed for deciduous trees. Keywords: hydrogen-2; isotope ecology; n-alkanes; leaf wax; oak tree

1.

Introduction

Stable isotope analysis in plant material is widely used as a tool to study plant–water relations at a variety of spatial and temporal scales [1,2]. At the leaf level some of the organic compounds derived from plants, for example lipids, are recalcitrant and can persist in soils and sediments on geological timescales [3]. Several of these lipids are source specific, that is, the source organism can be identified based on their molecular structure, which is why they are termed ‘biomarkers’. For example, long-chain n-alkanes and n-alkanoic acids with 24–34 carbon atoms are produced almost exclusively in the leaf waxes of higher terrestrial plants [4]. Specifically, these aliphatic hydrocarbons are constituents of the epicuticular wax [5] which forms a film on the surface of the leaf, protecting it from water loss. Epicuticular wax composition has been shown to be diverse among plant types [6,7] as well as dynamic with regard to leaf development [5] and environmental factors, such as temperature and water availability [8–10]. *Corresponding author. Email: [email protected] © 2015 Taylor & Francis

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D. Sachse et al.

A growing number of studies over the last more than 15 years have demonstrated that the hydrogen isotopic composition (expressed as a δ 2 H value) of these higher plant biomarkers is linearly related to the δ 2 H values of lake water and/or local precipitation [11]. As such, the hydrogen isotopic composition of these compounds (δ 2 Hwax ) can serve as a recorder of plant source water (i.e. precipitation) and the ecohydrological context in which the plants lived over geological timescales. This finding is increasingly being exploited for the reconstruction of paleo-climatic changes inferred from marine and lacustrine sediments where leaf waxes are preserved and at time very abundant in the record [12–15]. An important constraint for the robust interpretation of δ 2 Hwax paleoclimate data is the seasonal origin of the δ 2 Hwax signal. Earlier studies have suggested a pronounced seasonal variability in δ 2 Hwax of up to 30 ‰ exists in plant leaves from temperate climates [16,17]. However, the lack of replication in time or space and poorly resolved monitoring of environmental and plant water δ 2 H values in these studies makes it difficult to evaluate the effect of possible heterogeneity within a canopy (i.e. sun vs. shade leaves) or among tree individuals vs. real seasonal variations in δ 2 Hwax in the same individual. More recent studies that used a controlled sampling and monitoring strategy, either in a greenhouse or in the field, have found that δ 2 Hwax values of grasses and deciduous trees seem to become ‘locked in’ after the majority of leaf waxes are produced in the early growing season [18–21]. For example, Sachse et al. [18] have shown that δ 2 Hwax values of barley leaves did not vary within but did vary among leaf generations. This suggests that in each leaf generation δ 2 Hwax only records the environmental and physiological conditions of the formation period. This interpretation was supported by Kahmen et al. [19], who conducted a tracer experiment feeding deuterium-enriched water to greenhouse-grown poplar trees. The study revealed that the tracer was incorporated only in leaves that developed during the week-long tracer application, but not into leaves that were present before and that developed after the tracer application. Tipple et al. [20] showed that δ 2 Hwax values in field-grown poplar trees changed only during leaf flush (i.e. 1–2 weeks) and remained unchanged for the remainder of the growing season. Taken together, these studies provide compelling evidence that δ 2 Hwax values of deciduous tree leaves are produced only during leaf development and record as such only the environmental conditions of the early growing season. Translating the aforementioned findings directly into the sedimentary record remains a key research challenge, since only a limited number of plants have been investigated so far, and it is likely that these processes operate at different timescales in plants with different phenology. For example, no data exist for evergreen broadleaf plants, which keep their leaves for extended periods and are abundant in Mediterranean or tropical regions. Only one study so far has reported seasonal δ 2 Hwax values from an evergreen plant (Quercus agrifolia) from southern California [22], where no variability was observed. However, seasonal sampling did not encompass the full life cycle of the leaves, that is, sampling did not begin right after leaf flush. In the current study, we investigated the leaf wax development in an evergreen oak tree (Q. agrifolia) over the period of one year, beginning with the leaf flush and monitored source and leaf water δ 2 H values as well other climate parameters (T, relative humidity (rh)). The goal of our study was to assess the timing of n-alkane synthesis in Q. agrifolia to better understand the temporal integration of environmental conditions recorded in leaf wax n-alkane δ 2 H values.

2. 2.1.

Methods Sampling and study site

The study was conducted on the campus of the University of California, Berkeley. To monitor the seasonal variability in leaf wax n-alkane abundance and leaf wax n-alkane δ 2 H values, we

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3

selected a mature ( > 100 year old) Coast Live Oak (Q. agrifolia) tree in the vicinity of the Valley Life Sciences Building on the western side of the main campus. Sampling efforts started just before the flush of the 2007 leaf generation in the beginning of April 2007 and continued until the end of the year. Samples were collected at mid-day between 12:30 and 13:30 on a 2–4-week interval. Q. agrifolia typically produces a cohort of new leaves every spring. The leaves live for approximately 14 months. As such, two leaf generations typically overlap at the beginning of the growing season in April. Campus irrigation of the lawns (not the tree directly) may supply the tree with more water compared to an off-campus natural environment. However, the seasonal cycle of leaf growth is similar to off-campus trees, so that our study findings about the timing of leaf wax formation in this tree will also be applicable to naturally growing Q. agrifolia. To determine the variability in leaf water δ 2 H values, we sampled 3–5 sun-exposed leaves from four twigs, removed their mid-vein and stored them in 5 ml airtight plastic vials at − 20 °C. From the same four branches, we collected 5–20 leaves for n-alkane analyses. These leaves were collected in paper bags and dried at 60 °C for 48 h immediately after sample collection. On the first three sampling dates, leaves from the previous growing season (old leaves) and newly flushed leaves of the current growing season (young leaves) were present. On the first three sampling dates, we therefore sampled leaves from both leaf generations (old and young) and analysed these separately, and collected the freshly fallen leaf litter on sampling date 3. To assess variability in source water δ 2 H values, we collected xylem samples from the five branches on each sampling date. We removed the phloem from the sampled branches and stored the xylem at − 20 °C in 5 ml airtight plastic vials. For leaf and xylem water δ 2 H analysis, five replicate samples were analysed per sampling date. In Table 1, we report only the mean values and their standard deviation as a measure of natural inhomogeneity. At each sampling date, we also collected atmospheric water vapour for isotopic analysis. Vapour was trapped using polyethylene tubing that was looped three times, with the bottom two-thirds of the loops submerged in an ethanol-dry ice slurry (about − 80 °C), and attached to a small diaphragm pump that pulled air through the traps. The airflow through the cryogenic traps was monitored by flow metres and set at 0.5 L min−1 [23]. Our sampling system ran continuously from 11:00 to13:00 with the goal to collect the vapour that the leaves had experienced about two hours before sampling. To test the reproducibility of the set-up, we collected two replicate samples per sampling date on days 12 and 13. δ 2 H vapour values were virtually identical (Table 1). To monitor the climate in the canopy of the tree for the course of the growing season, we installed a climate sensor (rh/TempLog Datalogger, Oakton Instruments, Vernon Hills, IL, USA) in the canopy of the tree. Climate data were logged every 10 min from the beginning of April to the end of December 2007.

2.2.

Leaf water extraction and water isotope measurement

Bulk leaf lamina water was extracted from the leaves using cryogenic vacuum distillation at the Center for Stable Isotope Biogeochemistry (CSIB), UC Berkeley, as described in [23,24]. Leaf water was analysed for δ 2 H values using a Thermo Finnigan (Bremen, Germany) H– device interfaced with a Delta Plus XL isotope ratio mass spectrometer (IRMS) run in the dual inlet configuration. For a detailed description of the set-up, see also [19]. Briefly, water samples were reduced to H2 by injection onto chromium beads at 900 °C, then automatically measured after the gas was admitted into the IRMS. Calibration was performed with waters with two distinct isotope ratios to drift correct and normalize the analysis with long-term

Sampling date

Day of year (DOY)

Sample name

Mean δ 2 H xylem water (‰) vs. VSMOW

Replicate stdev

05 April 2007

95

SW1

− 40.9

1.2

12 April 2007

102

SW2

− 41.5

1.17

23 April 2007 07 May 2007 22 May 2007 07 June2007 26 June 2007 21 July 2007 03 August 2007 21 August 2007 18 September 2007 17 October 2007

113 127 142 158 177 202 215 233 261 290

SW3 SW4 SW5 SW6 SW7 SW8 SW9 SW10 SW11 SW12

− 42.6 − 43.1 − 43.7 − 43.0 − 45.7 − 49.1 − 50.8 − 53.4 − 56.2 − 55.2

13 November 2007

317

SW13

16 December 2007

350

SW14

Mean δ 2 H leaf water (‰) vs. VSMOW

Replicate stdev

1.33 0.89 0.98 1.44 0.46 0.99 0.71 0.90 1.30 1.98

− 4.0 − 8.2 20.1 13.8 0.0 20.6 15.3 3.5 4.5 − 6.4 − 3.8 − 2.6 − 4.8 − 5.9

1.8 1.0 1.1 5.8 1.0 1.7 1.9 2.3 3.2 3.3 2.5 1.1 1.7 1.3

− 52.9

1.36

− 3.7

0.8

− 50.3

0.83

− 6.4

0.9

Young leaves Old leaves Young leaves Old leaves

δ 2 H water vapour (‰) vs. VSMOW

Leaf water enrichment (‰) over xylem water

− 101.4

38

n.d

64

− 115.2 − 123.0 − 97.6 − 109.3 n.d − 94.6 − 96.7 n.d − 99.7 − 77.5 − 74.1 − 95.1 − 95.4 − 114.5

45 67 62 49 53 45 50 54 54 52 52 46

Note: Xylem and leaf water δ 2 H values are mean values of five replicate samples and the standard deviation (stdev) gives therefore the natural heterogeneity. Water vapour δ 2 H values are individual samples, except for DOY 290 and 317, where two samples were obtained for each day.

D. Sachse et al.

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Table 1. Xylem water, leaf water (measured), and water vapour δ 2 H values during the study period.

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external precision recorded with a third standard. Long-term (since 2001) external precision is ± 0.26 ‰.

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2.3.

Lipid biomarker extraction and quantification

We performed lipid biomarker extractions on three replicate leaf samples for each sampling date. About 0.4 g of the dried leaf samples were ground and extracted immersing the sample in a 10:1 mix of dichlormethane/methanol (DCM/MeOH). The samples were sonicated for 15 minutes, and the solution was filtered through a glassfiber filter to remove particulate material. A quantification standard (5-α-androstane) was added to each sample. The extract solution, containing soluble lipids, was then applied to a silica gel-filled SPE column following the established protocols [9,25]. The fraction containing n-alkanes was eluted using n-hexane as a solvent. The polar fraction was collected eluting the SPE column with DCM/MeOH in a 10:1 mixture. This fraction was not analysed further and archived. After evaporation of excess solvent, the F1 fraction was transferred into gas chromatograph (GC) vials and analysed for n-alkane quantification on an Agilent 7890A GC equipped with a 5975C Series mass spectrometric detector (MSD) system and an additional flame ionization detector (FID) at the Institute for Earth and Environmental Sciences at the University of Potsdam. The GC was equipped with an Agilent J&W HP-5 ms column (30 m long, 0.25 mm diameter, 0.25 µm film thickness) and a PTV injector, set at an initial injection temperature of 70 °C for 0.85 min. After injection, the injector was heated with a rate of 72 °C min−1 to 300 °C held constant for another 2.5 min. The temperature programme of the GC oven was held for 2 minutes at 70 °C, subsequently heated to 320 °C at a rate of 12 °C min−1 . The final temperature of 320 °C was held for 15 minutes. n-Alkanes were identified using their mass spectra and retention times of an external n-alkane standard mixture. Quantification was achieved by comparing compound peak areas of the FID trace with the peak areas of the internal standard (5α-androstane). We report all data from the three replicate samples in Tables 2 and 3. For interpretation we use the mean values of these replicates per sampling date and report their standard deviation as a measure of natural heterogeneity. 2.4.

Compound-specific isotope measurement of n-alkanes

The hydrogen isotope composition of n-alkanes was analysed using an IRMS (Delta V Plus, ThermoFisher) coupled to a GC (Trace GC Ultra, ThermoFisher) in the Physiological Plant Ecology Laboratory at ETH Zurich. Compounds were separated in the GC on a 60 m DB5 column. The injector was operated in splitless mode at a temperature of 270 °C. In all, 1 µl of each sample with a concentration of 300 ng µl−1 of the most abundant n-alkane was injected in triplicates, yielding peaks >20 Vs. The GC oven temperature programme started at 90 °C (held for 2 minutes), then raised to 150 °C at 10 °C min–1 , finally reaching 320 °C at a rate of 4 °C min−1 . This temperature was held for 10 minutes. After separation on the GC column, individual compounds were converted to H2 gas in an aluminium oxide reactor at 1420 °C. An alkane standard mixture (A3, provided by A. Schimmelmann, Indiana University) was run at three different concentrations (100, 200, and 400 ng µl−1 ) at three times during each sequence. Injecting standards in different concentrations revealed that peak sizes below an area of 20 Vs produced unreliable δ 2 H values. As such, sample compounds with peak sizes smaller than 20 Vs were omitted from the analyses. The linear relationship of known and measured δ 2 H values from the A3 mixture was used to derive sample δ 2 H values relative to the VSMOW scale. The H+ 3 factor was stable with 2.04 (1σ standard deviation of 0.1). Long-term average precision, monitored through triplicate analysis of a laboratory standard (nC29 alkane), is below 2 ‰.

Table 2. nC29 δ 2 H values, concentration and ACL index for the young leaves obtained from Q. agrifolia over the study period. 6

Sampling date 05 April 2007

95

12 April 2007

102

23 April 2007

113

07 May 2007

127

22 May 2007

142

07 June 2007 26 June 2007

158 177

21 July 2007

202

03 August 2007

215

21 August 2007

233

18 September 2007 17 October 2007

261 290

13 November 2007

317

16 December 2007

350

δ 2 H nC29 (‰) vs. VSMOW Sample name SW1-3y SW1-4y SW1-5y SW2-3y SW2-4y SW2-5y SW3-3y SW3-4y SW3-5y SW4-3y SW4-4y SW4-5y SW5-3y SW5-4y SW5-5y SW6 SW7-3y SW7-4y SW7-4y SW8-3y SW8-4y SW8-5y SW9-3y SW9-4y SW9-5y SW10-3y SW10-4y SW10-5y SW11 SW12-3y SW12-4y SW12-5y SW13-3y SW13-4y SW13-5y SW14-3y SW14-4y SW14-5y

− 142.0 − 140.9 − 143.0 − 142.6 − 154.4 − 155.3 − 155.8 − 153.2 − 145.9 − 146.1 − 140.6 − 140.8 − 135.0 − 135.1 − 136.9 No leaf wax data − 127.6 − 133.1 − 128.1 − 131.0 − 129.1 − 126.5 − 128.4 − 125.6 − 126.2 − 128.5 − 130.2 − 132.3 No leaf wax data − 130.1 − 127.0 − 129.9 − 129.9 − 133.7 − 136.7 − 130.0 − 133.3 − 132.0

Analytical stdev 0.3 0.2 0.5 0.0 0.6 0.6 0.7 0.6 0.3 0.6 1.1 0.7 0.8 0.5 1.4 0.8 0.2 0.8 4.1 1.1 0.8 1.0 1.2 0.5 0.1 0.2 2.0 4.5 1.1 1.8 0.9 0.2 1.2 0.8 0.2 0.5

Mean δ 2 H nC29 (‰) vs. VSMOW stdev

− 142

1

− 151

7

− 152

5

− 142

3

− 136

1

− 130

3

− 129

2

− 127

2

− 130

2

− 129

2

− 133

3

− 132

2

µg/g dry weight 507 538 409 501 444 420 262 385 357 244 302 299 381 258 315 535 517 715 929 764 909 1364 1260 1190 2354 1941 1682 2237 2338 2415 1710 2494 2170 1928 2556 1872

µg/g dry weight

stdev ACL

485

67

455

42

335

64

282

33

318

62

589

110

867

90

1271

87

1992

339

2330

89

2125

394

2119

380

28.51 28.51 28.53 28.63 28.62 28.59 28.43 28.53 28.50 28.49 28.48 28.53 28.59 28.37 28.40 28.67 28.70 28.89 28.69 28.71 28.81 28.77 28.76 28.77 28.80 28.77 28.81 28.82 28.85 28.82 28.83 28.84 28.81 28.88 28.88 28.89

Mean ACL

stdev

Period Leaf formation

28.52

0.01

28.61

0.03

28.49

0.05

28.50

0.03

28.45

0.12 Major leaf wax formation

28.75

0.12

28.73

0.06

28.77

0.01

28.79

0.02 No leaf wax formation

28.83

0.02

28.83

0.02

28.88

0.01

Note: Three replicate leaf samples were taken each sampling day. In addition, we report the values for each sample, and mean values for each sampling day (underlined, which were used for data interpretation).

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nC29 concentration Mean concentration Day of year (DOY)

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Table 3. nC29 δ 2 H values, concentration and ACL index for the old leaves obtained from Q. agrifolia over the study period.

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Sample name SW1-3o SW1-4o SW1-5o SW2-3o SW2-4o SW2-5o SW3-2o SW3-4o SW3-5o SW3-litter

δ 2 H nC29 (‰) vs. VSMOW

Analytical stdev

− 131.4 − 128.6 − 129.2 − 133.1 − 136.5 − 135.6 − 138.4 − 146.4 − 134.8 − 140.6

0.9 0.3 1.1 0.4 0.3 2.2 1.1 0.9 0.9 0.7

nC29 Mean δ 2 H concentration nC29 (‰) vs. µg/g dry VSMOW stdev weight

− 130

1

− 135

2

− 140

6

2441 1658 2099 1793 2081 3345 2462 n.d. 2142 1771

Mean concentration stdev ACL

2066

2406

2302

28.81 28.78 393 28.79 28.75 28.82 826 28.89 28.86 n.d. 226 28.89 28.79

Mean ACL

stdev

28.79

0.02

28.82

0.07

28.87

0.02

Note: Three replicate leaf samples were taken each sampling day (except for the litter sample). In addition, we report the values for each sample, and mean values for each sampling day (underlined, which were used for data interpretation).

2.5.

Modelling of leaf water δ 2 H values

Leaf water δ 2 H values from modern leaves vary strongly in response to daily fluctuations in climate, atmospheric vapour δ 2 H values and leaf physiology. We therefore used the Peclet-modified Craig–Gordon model [26–30] to estimate average daily (9:00–17:00) leaf water δ 2 H values for April–December 2007. The model we used builds on the original Craig–Gordon equation as modified by Dongmann [27]: δ 2 He = δ 2 HSW + ε∗ + εk + (δ 2 HWV − δ 2 HSW − εk )

ea , ei

(1)

where δ 2 He is the isotope composition of leaf water at the sites of evaporation, δ 2 HSW is the hydrogen isotope composition of the plant’s source or xylem water, ε∗ is the equilibrium fractionation between liquid water and vapour at the air–water interfaces [31]. εk is the kinetic fractionation that occurs during water vapour diffusion from the leaf intercellular air space to the atmosphere [32]. δ 2 HWV describes the hydrogen isotope composition of water vapour in the atmosphere, and ea /ei is the ratio of ambient-to-intercellular vapour pressures [28]. This basic Craig–Gordon model typically overestimates the evaporative 2 H enrichment of leaf water. Farquhar and Lloyd [26] have thus suggested that the discrepancy between the predicted leaf water enrichment based on Equation (1) and the observed values of mean leaf water is due to isotopic gradients within the leaf. These gradients form as a result of the mixing of the transpirational stream of non-enriched (source) water with enriched water moving backwards by diffusion in the opposing direction from the sites of water evaporation and therefore 2 H enrichment. The ratio of transpirational flow over back-diffusion is described by the Péclet number (℘ after Farquhar and Lloyd [26]), which relates the mean lamina mesophyll leaf water isotopic enrichment over source water (δ 2 HLW ) to δ 2 He as δ 2 HLW =

δ 2 H(1 − e−℘ ) , ℘

(2)

where δ 2 HLW is the hydrogen isotope composition of mean lamina leaf water and the Péclet number (℘) is defined as ELm . (3) ℘= CD

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D. Sachse et al.

In Equation (3), E is the transpiration rate (mol m−2 s−1 ), C is the molar concentration of water (mol m−3 ), D is the diffusivity of H2 O in water (m2 s−1 ), and Lm is the effective path length for the transpirational flow of water from the xylem veinlets through the mesophyll (m) to the site of evaporation [23,33]. In the aforementioned model, δ 2 HLW values of mean lamina leaf water are driven by the primary climatic variables ea , T air , δ 2 HSW and by the secondary variables δ 2 HWV , T leaf , gs , and E [30]. Under field conditions, the secondary variables respond to changes in the primary climatic variables. For our simulations, we therefore used a model where the relationships between the primary climatic drivers and the secondary model variables were accounted for so that the input variables for the model were reduced to ea , T air , and δ 2 HSW . Continuous data for ea and T air were available from the data logger. As it was our goal to simulate mean daytime leaf water δ 2 H values, we averaged ea and T air values for each day from 9:00 to 17:00. Seasonally continuous data for δ 2 HSW were obtained by interpolating between the measured δ 2 HSW that we collected in 2–4-week intervals. As δ 2 HWV was not in equilibrium with δ 2 HSW , we used a mean seasonal δ 2 HWV of − 105 ‰ that we obtained from averaging our measured δ 2 HWV over the entire growing season. The fit between the modelled data and the measured data was high (δ 2 HLW measured = 0.9522 × δ 2 HLW modelled + 1.1992, R2 = 0.79). 2.6.

Data analysis

We report all δ 2 H values vs. VSMOW in permil (‰) and calculate enrichment factors, or isotope fractionation values (ε) between two pools (a and b) according to the formula: εa/b =

D/Ha δ 2 Ha + 1 −1= 2 − 1, D/Hb δ Hb + 1

ε values are also reported in permil (‰), implying a factor of 1000.

3. 3.1.

Results Climatic variability during the study period

On a daily basis, air temperature varied between 6.4 and 23.6 °C between July and December 2007 on the UC Berkeley campus. The mean value of 14.7 °C during the study period agrees well with the long-term average temperature of 14.5 °C (http://www.usclimatedata.com/ climate/berkeley/california/united-states/usca0087). Temperature during the summer months (June–September) was slightly higher than that during the winter months (Figure 1). Relative humidity varies strongly from day to day (between 26.7 and 95.1 %, mean of 74.2 %) and no clear seasonal trend was observed (Figure 1). 3.2.

Variability in xylem water δ 2 H values over the study period

Xylem water δ 2 H values varied over the study period by 16 ‰, ranging from − 40.9 ‰ at the beginning of the experiment (DOY 95) to − 56.2 ‰ (DOY 261) (Table 1). Xylem water δ 2 H values remained stable around − 40 ‰ for the first 60 days (until DOY 158), before they decreased to − 56.2 ‰ after 166 days. For the remaining 89 days, values increased slightly to − 50.3 ‰. Overall, xylem water δ 2 H values were relatively stable, with a mean value over the 251 days of sampling of − 47.7 ‰ and a 1σ standard deviation of 5.4 ‰. We note that as the UC Berkeley campus (but not the tree directly) is irrigated, the variability in xylem water δ 2 H values may

35 0

9 30 0

25 0

20 0

15 0

50

10 0

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100 90 80 70

15

60 50 40 30

leaf flush leaf formation

no leaf wax formation 07

07

2. .1 06

06

.1

1.

07 0. .1 06

06

.0

9.

07

07 06

.0

8.

07 06

.0

7.

07 06

.0

6.

07 06

.0

5.

07 4. .0 06

3. .0

major leaf wax formation

20

07

5

06

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10

relative humidity [%]

temperature [°C]

20

Figure 1. Variability in temperature (red) and relative humidity (blue) over the study period from April to December on the Berkeley campus from a climate logger placed within the tree. The thin line represents a data point every 30 minutes; the thick lines represent daily average values.

not necessarily represent the isotopic composition of local precipitation. This is, however, not relevant to our study, as we directly monitored xylem water, the H source water for lipids. 3.3.

Variability in measured and modelled leaf water δ 2 H values over the study period

Measured mid-day leaf water δ 2 H values ranged from − 8.2 ‰ (DOY 95) to + 20.6 ‰ (DOY 127) (Table 1). Measured mid-day leaf water δ 2 H values were more variable and more positive during the first 60 days of the experiment compared to the remaining period. After DOY 158, measured mid-day leaf water δ 2 H values varied only from 4.5 ‰ (DOY 177) to − 6.4 ‰ (DOY 202 and 350). The mean δ 2 H value of measured mid-day leaf water over the entire growing season was 2 ‰ with a 1σ standard deviation of 9.9 ‰. Measured mid-day leaf water δ 2 H values from the old leaves sampled during the first two sampling dates (DOY 95 and 102) were more negative compared to young leaves sampled on the same day (Table 1). Modelled mean daily leaf water δ 2 H values varied over the growing season between + 26.7 ‰ and − 11.8 ‰ (Figure 2). We observed a seasonal trend in leaf water δ 2 H values with a decrease by about 25 ‰ during the first 3 months of the experiment (DOY 95–202), relatively stable values until DOY 250 and strong fluctuations and a general increase in leaf water δ 2 H values until DOY 350. We calculated mean leaf water δ 2 H values for the periods of leaf wax synthesis, as identified from the biomarker data (see the later text) to estimate integrated biosynthetic fractionation values (εbio ), see discussion in the following. 3.4.

Variability in leaf wax n-alkane concentrations

We identified n-alkanes with carbon numbers of 21–32 in the analysed leaf samples. A strong predominance of odd over even carbon numbered n-alkanes was observed for all samples. In young leaves, the concentration of n-alkanes with carbon numbers below 27 and above 31 was always below 6 µg g−1 dry leaf material, in the old leaves it was below 30 µg g−1 . The dominant

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35 0

30 0

25 0

20 0

15 0

50

10 0

day of year (DOY)

15

5 –30 –5 –40

–50 xylem water (measured)

–125

–60

–135 –140

d 2H nC29 (old) d 2H nC29 (litter)

–145

d 2H nC29 (young)

d 2H nC29 [‰] vs VSMOW

–130

–150 -155

nC29 concentration (old) nC29 concentration (litter) nC29 concentration (young)

2000

ACL (old) ACL (litter)

28.9

ACL (young)

1500

28.8

28.7

ACL

1000

28.6 500 28.5

07 2. .1 06

06

.1 1.

07

07 06

.1

0.

07 06

.0

9.

07 .0 06

28.4

no leaf wax formation

8.

07 7. 06

06

.0

6.

07

major leaf wax formation

.0

5. 06

.0

4. 06

.0

3. .0

leaf formation

07

leaf flush

07

0

07

n-alkane concentration [µg/g dry weight]

2500

06

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–15

d 2H xylem water [‰] vs VSMOW

midday leaf water (measured) midday leaf water (modelled) daily mean leaf water (modelled) weekly mean leaf water (modelled) mean leaf water between samplings (modelled)

2

H leafwater [‰] vs VSMOW

25

Figure 2. Variability in leaf water, xylem water and nC29 δ 2 H values, nC29 concentration and ACL in old and young leaves from the evergreen tree Q. agrifolia over the study period. The three identified periods of leaf wax development are indicated.

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n-alkane in all samples was nC29 , with concentrations between 282 µg g−1 dry leaf material (DOY 127) and 2330 µg g−1 dry leaf material (DOY 290) for young leaves, and between 2066 and 2406 µg g−1 dry leaf material in the old leaves (Table 3, Figure 2), making up as much as 90 % of all n-alkanes present. The concentration of nC27 as the next abundant n-alkane varied in young leaves between 4 and 42 g g−1 dry leaf material, followed by nC28 (between 1 and 18 µg g−1 dry weight), nC31 (1–11 µg g−1 dry leaf material) (data not shown). In the old leaves, the concentrations of these n-alkanes were slightly higher and varied between 118 and 162 µg g−1 leaf material for nC27 , between 123 and 143 for nC28 , and between 47 and 55 for nC31 . In addition, in the old leaves the concentration of nC25 varied between 14 and 17 µg g−1 dry leaf material (data not shown). nC29 concentration of the young leaves varied substantially over the growing season (Figure 2). During the first 60 days of the study, nC29 alkane concentrations were low and decreased slightly (from 485 µg g−1 dry leaf material on DOY 95 to 282 µg g−1 dry leaf material on DOY 127). After DOY 142, nC29 concentrations increased almost 10-fold to values of around 2000 µg g−1 at DOY 233. After DOY 233, concentrations remained relatively constant until the end of the growing season. Since concentrations of all n-alkanes except nC29 were extremely low, and we base our assessment exclusively on nC29 , we do not show data from the other homologues. Briefly, the short chained n-alkanes (nC21 , nC22 , and nC23 ) showed low but decreasing concentrations over the course of the study period. In contrast, all other n-alkanes (i.e. with carbon numbers above 23) showed relatively low but constant concentrations in the beginning of the growing season and strongly increased until DOY 233. After that date until the end of the study, concentrations remained again relatively constant. The compositional change in the leaf waxes of the plants leaves was also reflected in variable average chain length (ACL) values: ACL values of young leaves were around 28.5 until DOY 127, after which they increased to values of around 28.9 until DOY 233. After that date until the end of the experiment they remained constant. All old leaves, including the litter sample taken on DOY 113, showed similar ACL values (mean 28.9, 1σ standard deviation of 0.02, litter sample ACL: 28.8). 3.5.

Variability in leaf wax n-alkane δ 2 H values

Leaf wax n-alkane δ 2 H values were only analysed for nC29 as the low concentration of the other compounds did not permit stable isotope measurements. δ 2 H values of nC29 extracted from old leaves (including the litter sample) varied over the study period from − 130 ‰ on DOY 95 to − 140 ‰ on DOY 113 (Figure 2, Table 3). δ 2 H values of nC29 from young leaves showed a larger variability that ranged from − 152 to − 127 ‰ (mean of − 136 ‰, 1σ standard deviation of 9 ‰). δ 2 H values of nC29 extracted from young leaves also decreased by 10 ‰ from − 142 ‰ at the beginning of the experiment DOY 95 to values of − 152 ‰ on DOY 113 (Figure 2). After that date they increased until DOY 177 by 20 ‰ to values of around − 130 ‰. For the remainder of the experiment, δ 2 H values of nC29 from the young leaves did not change significantly and varied between − 127 and − 133 ‰. δ 2 H values of nC29 extracted from the litter sample taken on DOY 113 were similar to δ 2 H values of the fresh leaves sampled that day ( − 140 and − 141 ‰). 4.

Discussion

The aim of our study was to identify the time window in which the majority of leaf wax nalkanes are formed on the leaves of the evergreen-leaf oak, Q. agrifolia. Based on n-alkane

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concentrations alone, the timing of leaf wax synthesis is difficult to predict. This is because a period of constant concentration can either be a consequence of no leaf wax production, or that leaf wax development and removal (by wind and/or rain) are in equilibrium. However, when during the same period changes in leaf wax composition (ACL) and/or leaf wax δ 2 H values are observed, it is more certain that new leaf waxes have been formed. On the other hand, if all three parameters remain unchanged, it is likely that no significant leaf wax production occurred. Based on these assumptions, the variability in leaf wax n-alkane concentration, leaf wax n-alkane composition (i.e. ACL) and leaf wax n-alkane δ 2 H values over the study period allowed us to identify three distinct periods of leaf wax development in Q. agrifolia (Figure 2):

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(a) Leaf flush and initial leaf formation (DOY 95–142, 5 April – 22 May). (b) Major phase of leaf wax formation (DOY 142–233, 22 May– 21 August). (c) Ceased leaf wax n-alkane formation (DOY 233–350, 21 August– 16 December). In what follows, we will discuss the dynamics of leaf wax composition in old leaves and leaf wax development during the three periods individually. 4.1.

Leaf flush and initial leaf formation (DOY 95–142, 5 April–22 May)

Leaf wax n-alkane concentrations in the young leaves just after leaf flush decreased slightly until DOY 142. ACL values also declined slightly, and nC29 δ 2 H values varied by up to 25 ‰. These observations suggest significant dynamics in the newly established leaf wax in the first 6 weeks after leaf flush, where n-alkanes seem to be continually produced and possibly also removed. For the data we collected, it is impossible to estimate the percentage of leaf wax n-alkanes, which were produced de novo or removed. However, the large variability we observed in δ 2 Hwax (with the largest changes during the study period of approximately 20 ‰ in just 4 weeks) suggests significant changes and possibly complete turnover of leaf wax n-alkanes. If we assume that the majority of leaf wax n-alkanes sampled on a particular day was synthesized in the weeks before that sampling, we then can estimate the mean biosynthetic isotope fractionation (εbio ) during n-alkane synthesis for the time between two samplings by using a mean (modelled daily average) leaf water δ 2 H value during the period before the leaf sampling and a mean nC29 δ 2 H value for that period (Table 4). We are aware of the limitations of this approach, as we have no control on the actual percentage of n-alkanes synthesized during that period. We note that the significant dynamics observed in leaf wax composition and leaf wax δ 2 H values during the initial 6 weeks after leaf flush suggests major n-alkane synthesis during this period. However, the calculated fractionation effect is not the result of an actual biosynthetic process, but rather represents an integrated value for the leaf formation period. We therefore refer to this fractionation as εbio . For the three sampling dates where modelled leaf water δ 2 H values for the preceding 2 week period could be determined, we obtained a mean εbio value of − 151 ± 12 ‰, which is within the range of values previously reported εbio values for terrestrial vascular plants [19,22,34] as well as aquatic organisms [35–39]. Based on measured xylem water δ 2 H values, we also calculated mean values for the net or apparent fractionation (εapp ) of − 107 ± 8 ‰ during this period, a value on the lower end of the range of previous reports, but consistent with data from dryer regions [22] where significant leaf water isotope enrichment occurs. 4.2.

Major phase of leaf wax formation (DOY 142–233, 22 May– 21 August)

During the 13-week period from DOY 142–233, we observed major changes in leaf wax concentration and composition as well as smaller changes in nC29 δ 2 H values. Leaf wax concentration

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Period

DOY

Leaf formation 95–142 Major leaf wax formation 142–233 No leaf wax formation 233–350 Seasonal mean (young leaves) Seasonal mean (old leaves)

Mean δ 2 H xylem water (‰) vs. VSMOW stdev − 42.1 − 45.1 − 50.7 − 46.7

1.1 3.2 4.3 5.4

Mean δ 2 H leaf water (‰) vs. VSMOW (modelled)

stdev

7.9 − 1.5 − 0.8 0.4

6.1 4.5 8.5 7.6

Mean δ 2 H nC29 (‰) vs. VSMOW − 145 − 129 − 131 − 136 − 135

stdev

Mean nC29 concentration μg/g dry weight

stdev

mean ACL

stdev

εbio

7 2 3 9 5

375 909 2141 1097 2136

89 343 140 820 282

28.51 28.74 28.83 28.68 28.83

0.06 0.03 0.03 0.15 0.03

− 151 ± 12 − 107 − 128 ± 6 − 88 n.a. n.a. − 136 − 94

εapp

n (for εbio and εapp ) 5 4

Note: In addition, we report values of the integrated biosynthetic fractionation (εbio ) and the apparent fractionation εapp (between xylem water δ 2 H and nC29 δ 2 H values) for the two periods were we assume major leaf wax turnover or production.

Isotopes in Environmental and Health Studies

Table 4. Average xylem water δ 2 H values, modelled leaf water δ 2 H values, as well as nC29 δ 2 H values, concentration, and ACL values for the three periods of leaf wax n-alkane formation.

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increased sevenfold from around 300 μg g−1 dry leaf material to around 2100 μg g−1 dry leaf material, ACL values increased from 28.5 to 28.8 and nC29 δ 2 H values increased from − 142 to − 130 ‰. Interestingly, from DOY 233 onwards until the end of the growing season the concentration and composition of young leaves n-alkanes as well as their nC29 δ 2 H values reached values that were indistinguishable from those observed for the old leaves sampled before their senescence at the beginning of the sampling period (the mean concentration of nC29 in the old leaves was 2136 μg g−1 leaf material, mean ACL was 28.8, and mean nC29 δ 2 H was − 135 ‰, Table 4). The observed changes between DOY 142 and 233 were the largest compositional changes in the leaf wax of Q. agrifolia observed during our study period and suggest that during this period the vast majority of leaf wax n-alkanes was produced. The observation that nC29 δ 2 H values increased only by 12 ‰ from − 142 to − 130 ‰ during this period was likely a consequence of the relatively low variability of leaf water δ 2 H values during this period with variations on the order of only 10 ‰ for weekly averaged values (Figure 2). Although leaf water δ 2 H values actually decreased by 10 ‰ between DOY 177 and 202, we do not see an effect on n-alkane δ 2 H values despite a twofold increase in n-alkane concentration during this time. We would, however, only expect a maximum decrease of 5 ‰ in nC29 δ 2 H values, after a doubling of n-alkane abundances, an effect possibly masked by the natural heterogeneity and analytical uncertainty. Since leaf water δ 2 H values showed a 5 ‰ decrease (as a result of a 5 ‰ decrease in xylem water δ 2 H values during this period, Table 1), an alternative explanation for the observed increase in δ 2 Hwax values during the major leaf wax formation period could be due to changes in εbio . Evidently, we estimated the integrated biosynthetic isotope fractionation (εbio , see discussion above) during these 6 weeks to be on average –127 ± 6 ‰, significantly lower than observed during the initial 6 weeks after leaf flush, but still within the range of reported εbio values [20]. Estimated smaller εapp values during this period of major leaf wax development of − 84 ± 5 ‰ may therefore not be a consequence of changes in leaf water δ 2 H values but of changes in εbio . In the following we discuss possible causes for seasonally variable εbio values.

4.3.

Ceased leaf wax formation (DOY 233–350, 21 August– 16 December)

After DOY 233 (21 August) leaf wax n-alkane concentration, ACL and nC29 δ 2 H values did not change significantly and remain indistinguishable from the values observed for the old leaves and the litter sample (Figure 2). During this period we observed the largest variability in δ 2 H values of leaf water, as a consequence of declining temperatures and extended week-long dry periods in the fall (Figure 1). The observation that all three studied parameters remain unchanged, despite significant weather changes, suggests that no significant leaf wax n-alkane production took place during late summer and fall. Although our sampling did not encompass a full annual cycle, the striking similarity in leaf wax concentrations, composition as well as nC29 δ 2 H values between the fall and the old leaves sampled in the preceding spring, strongly suggests that no further leaf wax n-alkane production occurred in the winter. As such, we refrain from estimating εbio or εapp values for this period, since we hypothesize no significant new leaf wax n-alkane production took place. This finding confirms earlier studies that the leaf wax n-alkane δ 2 H signal becomes ‘locked in’ during the phase of leaf wax formation during the early growing season and remains unchanged afterwards [18–21]. These previous studies, which have investigated either annual grasses or deciduous riparian trees suggested short periods of leaf wax formation (weeks). In contrast, we

Isotopes in Environmental and Health Studies

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find that the phase of leaf wax formation was significantly longer in the evergreen tree Q. agrifolia and extended from April to mid-August. The period during which the n-alkane δ 2 H signal recorded the plant–water environment in this plant was thus 3 months (May–August).

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4.4.

Low variability in leaf wax composition in old leaves

Leaf wax n-alkane concentration and composition of the old leaves sampled before their senescence at around DOY 113 did not change significantly indicating little, if any, formation of n-alkanes during this period. However, nC29 δ 2 H values decreased by 10 ‰ during the 18 days when these leaves were sampled. The relatively high variability of nC29 δ 2 H values for the sample taken on DOY 113 (1σ standard deviation of 6 ‰) indicates a relatively large heterogeneity in old leaves. Considering the analytical uncertainty (approximately 2–3 ‰) and the heterogeneity among replicate samples (by between 2 and 6 ‰), a 10 ‰ change in nC29 δ 2 H values does not necessarily indicate significant de novo formation of n-alkanes in the old leaves. The litter samples on DOY 113 is not distinguishable from the old leaves sampled that day, based on its similar leaf wax n-alkane concentration, composition and δ 2 H value. This suggests that no alterations in n-alkane concentration, composition and δ 2 H values occur during the final stages of leaf senescence and litter fall.

4.5.

Changes in εbio during leaf and leaf wax formation (April–August)

Earlier we hypothesized that the observed increase in δ 2 Hwax values during the major leaf wax formation period, when at the same time leaf water δ 2 H values decreased, could be due to changes in εbio . Environmental factors, such as temperature and light intensity, have been suggested to affect εbio as well [40,41]. For example, in green algae an increase in temperature resulted in an increase in εbio [41], while no change in εbio was observed for greenhouse-grown C3 and C4 plants [42]. In our experiment, the temperature was slightly higher by 5 °C between DOY 142 and 233 compared to the period of leaf flush. However, εbio became smaller under higher temperatures, opposite to the effect observed in green algae. Also, no significant changes in light intensity have likely occurred during spring and summer. Therefore, we suggest that neither light intensity changes nor the observed temperature change were responsible for the difference in εbio . Lastly, εbio has been suggested to be sensitive to different pathways of NADPH production [43–46]. While overall NADPH is strongly depleted in 2 H ( − 200 ‰ on average [11]), NADPH from different metabolic sources differ in their isotopic composition. While direct measurements of NADPH δ 2 H values are rare, it is estimated that NADPH produced directly in the light reaction of photosynthesis is characterized by δ 2 H values as low as − 600 ‰ [44,47]. In contrast, NADPH produced via the oxidative pentose phosphate cycle is less depleted in 2 H [44,48]. Indeed, an increase in δ 2 H values of plant material from aquatic and terrestrial plants has been observed and interpreted to be caused by increased reliance on stored carbohydrates [48,49], in particular during the early growing season [46]. As such, the observed decrease in εbio between leaf flush and the major leaf wax formation phase may indicate that the plant started to use stored carbohydrate reserves during the period of major leaf wax formation, but not during the initial leaf flush phase. This is in contrast to results from deciduous trees or aquatic plants, where such a process was reported during the initial stages of leaf growth in spring [46,48]. Evergreen plants on the other hand photosynthesize year-round and may tap their reserves only during periods of water stress or increased NADPH demand, such as the formation of new leaves.

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4.6.

D. Sachse et al.

Consequences for the study of plant–water relationships in modern plants and paleoenvironments using leaf wax n-alkane δ 2 H values

In general, our data corroborate a growing number of greenhouse and field studies, suggesting that leaf wax n-alkane δ 2 H values record environmental conditions during only a finite period in the early growing season and remain almost unchanged after the leaf wax is fully developed. That means the isotope values of the leaf wax biomarkers become ‘locked in’ after a short, welldefined period [18–20]. This is in contrast to some earlier studies which had observed significant seasonal variation in deciduous tree and evergreen needle derived leaf wax n-alkane δ 2 H values [16,17]. The more sophisticated sampling strategy and the analysis of replicate samples in recent field and greenhouse studies suggest that earlier studies have either underestimated natural heterogeneity in leaf wax n-alkane δ 2 H values (i.e. sampling of shade vs. sun leaves, sampling of different leaf generations) or investigated plants which have indeed synthesized n-alkanes de novo in the late season, due to environmental stress [8]. The data we present here suggest that the duration of the period, during which n-alkane δ 2 H values track variation in leaf water δ 2 H values, varies significantly among different plants. Previous studies on grasses and deciduous tree leaves suggested that this period is short (days to weeks) and occurs during spring [18–21,50] while our findings from Q. agrifolia suggest that this period is up to 3 months long. Since we report here the first continuous seasonal δ 2 H nalkane data from an evergreen species, including leaf water isotope monitoring, we cannot assess how representative our results are for evergreen trees in general. We note that this oak species is evergreen in that it has green leaf area year-round. But, leaves never live more than 2 years and more commonly are replaced every year, which is different from most tropical evergreen plants, for example. However, our data agree with results from epicuticular wax removal experiments conducted on the evergreen plant Prunus laurocerasus, where n-alkane concentrations increased over the whole measurement period of 60 days, suggesting de novo formation during at least these 2 months [5]. Our data confirm that leaf wax n-alkane δ 2 H values are recorders of the plant–water environment, during the period of leaf wax formation and can as such be used to assess plant ecohydrological conditions. If our hypothesis that a variable εbio during seasonal leaf growth is a consequence of a differential use of stored carbohydrate and photosynthetically produced NADPH is valid, then investigating changes in εbio during leaf development may shed light onto the conditions under which stored carbohydrates are used by the plant. Further investigation of differences in εbio among different plants may also assess the variability of εbio and its causes. With regard to the interpretation of sedimentary records of n-alkane δ 2 H values for paleoclimate studies, our data suggest that the recorded signal does not represent the whole season, but only the period of leaf wax formation. From the existing limited data, it seems that sediment records consisting of mainly deciduous tree or grass-derived leaf waxes integrate only over a few weeks in the early growing season (i.e. spring to early summer in the temperate northern hemisphere), whereas records with dominant input of evergreen plants may integrate over a few months. Clearly, further investigations on a variety of different plants under different climatic conditions are needed to assess magnitudes of εbio variability on seasonal scales as well as among different plant types.

5.

Conclusions

The data we show here give new insights into seasonal development of leaf wax n-alkane concentration, composition and δ 2 H values from an evergreen oak (Q. agrifolia) on the campus of the University of California at Berkeley. Our findings contribute to a more robust interpretation

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of leaf wax n-alkane δ 2 H values from sedimentary records and indicate patterns of changing within-plant substrate use for leaf wax formation in the growing season. Specifically, we find: (1) Leaf wax n-alkane δ 2 H values from the investigated evergreen oak Q. agrifolia only changed during the initial phases of leaf flush and leaf development but remained unchanged for the larger part of the year. As such leaf wax n-alkane δ 2 H values from this evergreen oak recorded the environmental conditions only during the first months of the life of the leaf. (2) The phase of major leaf wax formation was with 3 months during summer significantly longer in this evergreen tree compared to previously investigated annual grasses and deciduous trees. (3) Our data suggest substantial shifts in εbio during the 3-month period of leaf wax formation, with larger values during and shortly after leaf flush, compared to the major leaf wax formation phase. We hypothesize that increased usage of stored carbohydrates during the major leaf wax formation phase was responsible for this change. Our data also suggest that leaf wax n-alkane δ 2 H values from different plants integrate over different seasonal scales, highlighting the importance to consider climate seasonality and the vegetational composition in interpreting sedimentary biomarker records. Clearly, further studies on the timing of seasonal wax development and the assessment of εbio from a variety of different plant types under different climatic conditions are needed to provide a framework for a quantitative interpretation of leaf wax n-alkane δ 2 H values. Acknowledgements The authors would like to thank Johanna Menges (University of Potsdam) for help with lipid extraction and quantification.

Disclosure statement No potential conflict of interest was reported by the authors.

Funding This work was supported by the German Research Foundation (DFG) through an Emmy-Noether Research Grant (DS1889/1–1) to D.S. and by an ERC Starting Grant (279518 COSIWAX) to A.K.

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