Controls on leaf wax fractionation and δ2H values in tundra vascular plants from western Greenland

Accepted Manuscript Controls on Leaf Wax Fractionation and δ 2H Values in Tundra Vascular Plants from Western Greenland Melissa A. Berke, Alejandra Cartagena Sierra, Rosemary Bush, Darren Cheah, Keith O'Connor PII: DOI: Reference:

S0016-7037(18)30606-9 https://doi.org/10.1016/j.gca.2018.10.020 GCA 10981

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Geochimica et Cosmochimica Acta

Received Date: Revised Date: Accepted Date:

29 March 2018 16 October 2018 17 October 2018

Please cite this article as: Berke, M.A., Cartagena Sierra, A., Bush, R., Cheah, D., O'Connor, K., Controls on Leaf Wax Fractionation and δ 2H Values in Tundra Vascular Plants from Western Greenland, Geochimica et Cosmochimica Acta (2018), doi: https://doi.org/10.1016/j.gca.2018.10.020

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Controls on Leaf Wax Fractionation and δ2H Values in Tundra Vascular Plants from Western Greenland Melissa A. Berke1, Alejandra Cartagena Sierra1, Rosemary Bush1,2, Darren Cheah1,3, Keith O’Connor1 1

Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, Notre Dame IN 46556, USA 2

Department of Earth and Planetary Sciences, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3130, USA 3

Western Australian Organic & Isotope Geochemistry Centre, School of Earth and Planetary Sciences, Curtin University, Bentley, WA 6102, Australia Abstract Hydrogen isotope ratios of leaf waxes are used to reconstruct past hydroclimate because they are a reflection of meteoric water, but the interpretation of these signatures from ancient sedimentary archives relies on a thorough understanding of the drivers of modern isotope variability and controls on fractionation. These studies are particularly valuable in the high latitudes, regions especially vulnerable to rapid climate change and increasingly used for plantbased proxy reconstructions of past hydroclimate, but also where modern vegetation is understudied compared to the lower latitudes. Here we investigate δ2H values from leaf wax nalkanes of vascular tundra plants in the Kangerlussuaq area of western Greenland. We collected a variety of common tundra species to study possible interspecies variability in δ 2H values including dwarf shrubs (Betula nana, Empetrum hermaphroditum, Salix glauca, and Rhododendron lapponica), forbs and graminoids (Vaccinium uliginosum, R. tomentosum, and Calamagrostis lapponica), a horsetail species (Equisetum arvense), and a submerged aquatic macrophyte from a local lake (Stuckenia filiformis). Using previously measured leaf and stem waters to help constrain potential drivers of leaf wax n-alkane δ2H values, we find that the overall net fractionation (εapp) from the studied species is -75 ± 20‰. The εapp at Kangerlussuaq is consistent with other studies of Arctic vegetation that find smaller ε app than from the majority of lower latitude sites. The fractionation of leaf water and xylem water (ε lw/xw) and the fractionation of xylem water and precipitation (εxw/p) are both relatively constant, suggesting stable leaf and soil related fractionations across species. Estimates of biosynthetic fractionation (εbio), as evidenced from the fractionation of the δ2H values of n-alkanes and leaf water (εwax/lw), are not constant across species as sometimes assumed, and are small (average of εbio is -120 ± 1

27‰) compared to many published estimates. This supports a significant role in εbio shaping the εapp in this high latitude setting, where lipid biosynthesis may be driving differences in n-alkane δ2H values. This finding suggests that lipids in the Kangerlussuaq plants studied rely on the use of some proportion of different hydrogen sources during lipid synthesis, such as stored NADPH. The cumulative results of this survey of Kangerlussuaq area n-alkane δ2H values and water-wax fractionations suggest that fractionation in the high latitudes during the short summer growing season may play an important role in governing the small εapp compared to many low latitude sites. Better understanding of appropriate εapp and the importance of εbio in controlling plant wax fractionation from the high latitudes is necessary for future reconstructions of hydroclimate using leaf wax δ2H values in these regions.

Keywords: Hydrogen isotope fractionation, leaf wax, n-alkanes, Arctic plants, Greenland 1. INTRODUCTION Climate change is predicted to be faster and more severe in Arctic and Boreal regions than elsewhere due to positive feedback cycles that involve sea ice extent, changing albedo, and melting permafrost (IPCC, 2013; USGCRP, 2017). There is evidence of an overall intensification of the high latitude hydrologic cycle with warming climate, which is attributed to changes in atmospheric patterns and is predicted to have significant impacts on global ocean circulation (Held and Soden, 2006; Min et al., 2008; Peterson et al., 2002). Our framework for understanding modern environmental changes in the high latitudes often relies on stable isotopes of water (δ2H and δ18O) as tracers (Dansgaard, 1964; Edwards et al., 2004; Jouzel et al., 1997b; Jouzel et al., 2000). Though ice cores are the standard accepted archives for reconstructions of past climate in the high latitudes, capable of providing seasonally-resolved records but only spanning a few million years (Alley, 2000; Johnsen et al., 1995; Jouzel et al., 1997a; Schneider and Steig, 2008), hydrogen isotope ratios from leaf wax compounds are increasingly used and have the ability to provide past climate information corresponding to the integrated growing conditions recorded in sediments, potentially spanning tens of millions of years (e.g. Feakins et al., 2012; Porter et al., 2016; Thomas et al., 2016). Long chain length molecular compounds that compose the waxy surface coating on leaves are well-preserved over geologic time scales, with little diagenetic alteration at the low temperatures and pressures that typically occur during

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shallow sedimentary burial (Schimmelmann et al., 2006; Sessions et al., 2004). An improved understanding of high latitude modern plant wax compounds and their isotopic values can help constrain our records of past precipitation variability, providing context for a region with a highly dynamic hydroclimate system. A strong relationship between mean annual precipitation δ 2H and plant wax δ2H values has been observed from sedimentary reconstructions (e.g. Hou et al., 2007b; Huang et al., 2004; Sachse et al., 2004; Sauer et al., 2001). Although we know that there is temporal and spatial averaging of plant species isotopic composition into the sediments, this correlation often leads to the interpretation that waxes integrate the isotopic ratios of the annual precipitation in a given region, paving the way for their use as a proxy for mean annual precipitation in paleoreconstructions. However, in high latitudes, this relationship might also include integration of environmental water across multiple years, released from summer melt of permafrost or ice sheets (Jespersen et al., 2018; Kane et al., 1992) and supplied for uptake by the vegetation during the short growing season that dominates the region. And while the seasonality and preferential plant water use of precipitation may significantly influence soil water δ 2H values (Alstad et al., 1999; Leffler and Jeffery, 2013), it is often not constrained. Additionally, some recent research outside of the high latitudes suggests that leaf wax isotope ratios may be strongly influenced by only a narrow window of time surrounding initial leaf flush and development (Freimuth et al., 2017; Kahmen et al., 2011; Tipple et al., 2013). However, it is unclear how comparable this timing would be to the current study as the climate regimes are considerably different. These studies indicate that even in the presence of continual access to water, there may be a seasonal bias to the leaf wax isotope records, with plant wax compounds only reflecting the isotopic ratios of precipitation utilized prior to new leaf formation. While the use of meteoric water in formation of leaf waxes is of profound importance for modern plant studies, it also significantly affects paleoreconstructions using leaf wax δ2H values to reconstruct past precipitation δ2H values. The transformation of source water to wax δ2H values is the net effect of a series of isotopic fractionations that are influenced by plant physiology and the environment (Sachse et al., 2012). Net or apparent fractionation (εapp), determined using paired plant wax and plant source water, incorporates large offsets associated with the biosynthesis of leaf plant lipids (εbio) as well as fractionations associated with evaporation from leaves (transpiration) and from soil

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evaporation (Feakins and Sessions, 2010; Hou et al., 2008; Kahmen et al., 2013a; McInerney et al., 2011; Smith and Freeman, 2006). Since there is thought to be no fractionation of meteoric water in the uptake by plant roots and transport to the leaves for most plants (Ehleringer and Dawson, 1992; White et al., 1985), significant differences between ground water and xylem water, a proxy for meteoric source water, are attributed to soil water evaporation. This window into soil water δ 2H values used by plants can provide an estimate of integrated precipitation events and combined seasonal water (Ehleringer and Dawson, 1992; Ehleringer et al., 1998; Ehleringer et al., 1991). Soil water evaporation or differential use of evaporated shallow surface and more 2H depleted deep waters (Berke et al., 2015; Sachse et al., 2009; Sachse et al., 2006) is likely to create a significant difference between soil water and precipitation δ2H values. Evaporative 2H enrichment via transpiration, also a driver of εapp, can be seen in εwax/xw, the fractionation between xylem water and lipid δ2H values. The relative contribution of this enrichment to leaf wax δ2H values is known to vary regionally because the rate of transpiration depends in part on the relative humidity and temperature of the air surrounding the leaf (Hou et al., 2008; Kahmen et al., 2008; Sachse et al., 2012; Sachse et al., 2006; Smith and Freeman, 2006; Tipple et al., 2014). There is also some evidence that enhanced transpiration due to increased light exposure may affect δ2H values. Enhanced transpiration from near continuous light conditions such as those in high latitude settings are thought to yield smaller εwax/xw (Liu and Yang, 2008; Yang et al., 2011; Yang et al., 2009). Biosynthetic fractionation (εbio) is the fractionation that occurs during the formation of plant lipids from the biosynthetic leaf water pool (Sachse et al., 2012; Sessions et al., 1999). Investigating the isotope effects of leaf wax synthesis is often challenging due to difficulties constraining environmental effects on εbio in natural settings (Kahmen et al., 2011; Tipple et al., 2014). There have been studies that have suggested εbio is stable across a species (Sessions et al., 1999; Tipple et al., 2014). However, other studies have found variability in ε bio of up to 65‰ in sampled species (Kahmen et al., 2013b), which may contribute to large variability in εapp (Sachse et al., 2012). In light of data that suggests εbio variability can occur across the growing season (Newberry et al., 2015; Sachse et al., 2015), species (Kahmen et al., 2013b), or environments (e.g. Feakins and Sessions, 2010; Hou et al., 2007a), either through changes to biosynthetic

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fractionation or changes to cellular water δ 2H values (Kahmen et al., 2013a; Sessions, 2006), it may be inappropriate to assume εbio remains constant across plant types and environments. It is worth noting that while there may be fewer precipitation collections in western Greenland than elsewhere (IAEA/WMO, 2014), the Online Isotope Precipitation Calculator (OIPC, version 3.1) (Bowen and Revenaugh, 2003; Bowen and Wilkinson, 2002) July precipitation δ2H value estimate (-107‰) for Kangerlussuaq is similar to empirical data of summer precipitation of Lindborg et al. (2016) for the same region, significantly more 2H enriched than OIPC estimates of winter December-January-February (DJF) average of -157‰. OIPC annual δ2H value estimate (-141‰) for the Kangerlussuaq region is similar to annuallyintegrated lake water measurements of Leng and Anderson (2003). The intercept of the measured xylem water LEL and the LMWL, which incorporates snow and rain δ2H values, provides an estimation of the average meteoric water available and used by vegetation at Kangerlussuaq, with a δ2H value of -158‰ and δ18O value of -22‰ (Bush et al., 2017). The δ2H value of meteoric water determined by this intercept is more 2H-depleted than the other available estimates, and suggests a more 2H-depleted source water for local vegetation. The LMWL-LEL intersection at Kangerlussuaq, created from existing precipitation and plant water data, suggests a mixed-season plant source water δ2H value, reflecting a mixture of snow and frozen soil melt and summer precipitation (Bush et al., 2017), as has been seen elsewhere (Jespersen et al., 2018). Thus, this multi-season estimate of meteoric water is a more accurate representation of plant source water than either an annual-integrated δ2H value or summer precipitation δ2H value alone. There have been few studies that have focused on the modern plant wax δ2H values from the high latitudes (Daniels et al., 2017; Thomas et al., 2016; Wilkie et al., 2013), and fewer studies that have analyzed modern plant water fractionation (Daniels et al., 2017). To date, there have been no collections comparing plant waxes and waters (leaf and xylem) from Greenland to examine plant fractionations, though Thomas et al. (2016) analyzed modern plant waxes from another location in western Greenland, more than 200 km north of this study. Here, we use modern plant wax n-alkane δ2H values paired with previously described plant xylem and leaf waters (Bush et al., 2017) to examine the fractionations in high latitude plants common to the Kangerlussuaq region of western Greenland. We aim to refine the understanding of compound specific δ2H values and wax-water fractionations of high latitude vegetation to provide context

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and constraints for future reconstructions of past hydroclimate variability from western Greenland.

2. MATERIALS AND METHODS 2.1 Study Site Plant samples were collected near Kangerlussuaq (67°01’ N, 50°42’ W), located at the mouth of the Søndre Strømfjord of western Greenland (Fig. 1). The region experiences an arctic continental climate, moderated by the Atlantic Ocean to the west of the plant collection sites, and the Greenland Ice Sheet (GIS) to the east. Five months of the year (May – September) the region experiences mean monthly temperatures above freezing, with a mean annual temperature of 5.7°C and mean July temperature (the month of primary sample collection) of 10.9°C (Bush et al., 2017). Kangerlussuaq is 50 km north of the Arctic Circle and experiences 19-24 hours of sunlight in summer (Bush et al., 2017). The area falls within the zone of continuous permafrost (Jørgensen and Andreasen, 2007). The region receives mean annual precipitation of ~140 mm, with maximum precipitation occurring between July and September. The average relative humidity throughout the year is 62%, with a minimum of 24% (Bush et al., 2017). Samples for this study were collected in July 2014, during what was the warmest summer on record at the time (Jeffries et al., 2014). Studies of plant phenology at Kangerlussuaq suggest a short growing season, though research suggests progressively increasing growing season length due to a decline of Arctic sea ice (Kerby and Post, 2013).

2.2 Sample Collection Mature leaves from multiple individuals of common plant species found around Kangerlussuaq were sampled along the glacial valley between Kangerlussuaq and the edge of the GIS between 18 July and 3 August 2014 (Fig. 1) (Bush et al., 2017). Samples were collected at a significant distance from the road to circumvent possible disturbance of communities, water evaporation, and road contamination. Vegetation from the study region is classified as Low Arctic shrubland, with vegetation common to the tundra or steppe landscapes across the Arctic (Walker et al., 2005). This vegetation commonly includes dwarf shrubs, forbs, graminoids, aquatic vegetation in and around lakes, and abundant moss and lichen species. Eight abundant vascular plant species in the Kangerlussuaq region across the sampled sites were analyzed for 6

plant wax chain length distribution at 16 sites, between Sites 1-20 (B. nana, E. hermaphroditum, R. tomentosum, S. glauca, V. uliginosum, C. lapponica, S. filiformis, and E. arvense) (Table 1). Multiple individuals from four of these species between sites 1-10 were able to be measured separately for leaf and xylem water δ2H values based on available collected material (B. nana, R. tomentosum, S. glauca, V. uliginosum) and have been previously published (Bush et al., 2017). While mosses were common on the landscape, the difficulty in collecting dry samples precluded their sampling for this study. Leaf samples were collected from sun-exposed outer branches and xylem water samples were taken at the base of the branch from the main plant stem. Soil water measurements were not taken for comparison in this study. Collections for bulk δ13C and leaf wax δ2H analysis were made at the same time and from the same branches of these individuals and stored in brown paper bags. Leaf wax samples were allowed to dry to prevent mold development leading to possible wax degradation.

2.3 Lipid extraction and purification Whole dried leaves for each sample (~0.7 g material) were ground with a solvent-cleaned mortar and pestle to integrate isotopic variations across and within leaves. Dried samples were ultrasonically extracted using ~10 mL of 2:1 dichloromethane: methanol (v:v) for 20 min, repeated three times, removing the solvents and extracted lipids each time. The total lipid extract (TLE) was then dried under a gentle stream of ultrapure N2. The TLE was split in half by volume immediately after using a vortex to homogenize lipids and solvent. All results presented are based on total TLE. The working half of the TLE was transferred to a silica-gel column (solvent extracted silica gel, 70–230 mesh) for n-alkane fraction elution using 4 ml hexane. A blank carried through the entire procedure was analyzed to confirm contaminant-free procedural methods.

2.4 Lipid identification and quantification n-Alkanes were identified and quantified using a Thermo Trace Ultra ISQ gas chromatograph with flame ionization detector (GC-FID) and mass spectrometer (MS, 90:10 flow split). Analyses were done in splitless injector mode at 300°C with a fused silica column (Agilent J&W DB-5, 30m length, 0.25mm ID, 0.25μm film thickness) with helium as the carrier gas. The GC oven temperature program utilized began with a 1 minute hold at 60°C followed by 7

a ramp from 60 to 320°C at 6°C/min with an isothermal for 12 minutes. Compounds were identified through comparison of retention times with n-alkane standards and through MS fragmentation patterns. All samples were run in hexanes and spiked with internal standard (5αandrostane) with a comparable concentration to the most abundant n-alkanes prior to analysis for quantification by signal intensity comparison. Leaf wax n-alkane concentrations are reported as μg n-alkane/g dry leaf. The average chain length (ACL), the weighted concentration of longchain n-alkanes is here defined as:

where the m and n are the shortest and longest chain lengths, respectively, and i is the number of carbon atoms in each homologue. For n-alkanes, the odd-numbered homologues span from C21 to C35. 2.5 δ2H analyses of n-alkanes Compound-specific hydrogen isotope analyses were performed using a Thermo Trace 1310 GC coupled to a Finnegan Delta V Plus isotope ratio mass spectrometer (IRMS) with a Conflo IV interface and a high-temperature (pyrolysis) conversion reactor set at 1450°C. Much like the GC-FID, the GC-IRMS injector was run in splitless mode at 300°C with a fused silica, 30m DB-5 phase column (Agilent J&W DB-5, 30m length, 0.25mm ID, 0.25μm film thickness) with helium as the carrier gas. The GC oven temperature program utilized began with a 1 minute hold at 60°C followed by a ramp from 60 to 320 °C at 6°C/min with an isothermal for 15 minutes. The H3+ factor was determined daily prior to standard and sample analyses and averaged 2.8 ± 0.05 ppm/mV during the period of analysis for these samples. A standard suite of n-alkanes C16-C30 with known δ2H composition were run 6-10 times at the beginning of each new HTC reactor (A. Schimmelmann, Indiana University, Mix A6). All δ2H values are expressed relative to VSMOW using standard delta notation in per mil (‰), normalized by calibrating the H2 reference gas δ2H values using Mix A6 for each new reactor (see Polissar and D’Andrea, 2014). Lab standards (purified plant extract, androstane/squalane mix, and A6) were run at the beginning and ends of daily runs (five-six measurements) and every 4-5 injections to help monitor instrument precision and the condition of the pyrolysis reactor through time. All samples 8

were measured at least in duplicate and an internal standard co-injected in samples (squalane) to monitor reproducibility was consistently <5‰.

2.6 Leaf bulk carbon isotope analysis Dried, homogenized leaves from a subset of plant species were analyzed for bulk δ13C values. Leaf mid-ribs were removed where possible prior to homogenization. A Costech ECS4010 Elemental Analyzer (EA) interfaced with a Delta V Advantage IRMS was used for analysis. The combustion reactor was run at 1000°C, reduction reactor at 650°C, with the column at 65°C. On average, 2.5g of homogenized leaves were analyzed. The δ13C values were normalized to VPDB scale by comparison to a calibration curve (r2 = 0.999) from standards (peach leaves, acetanilide, sulfanilamide, sorghum flour, and protein powder) run before, during, and after the samples. Working standards throughout the run (acetanilide, n= 4 and sulfanilide, n = 3) varied <0.1‰, with δ13C values within 0.5‰ of expected values.

2.7 Calculation of net fractionation Isotopic fractionations are reported here based on the following calculation:

ɛa/b =

-1

Net (apparent) fractionation is discussed in terms of hydrogen isotope fractionation between a, where a is typically a leaf wax compound such as an n-alkane as presented here and b is the water used by the plant. Often, this fractionation is calculated between empirical precipitation values, soil water, xylem water, leaf water, or model interpolated precipitation (using OIPC version 3.1) (Bowen and Revenaugh, 2003; Bowen and Wilkinson, 2002). We note that OIPC estimates significantly differ for this region using OIPC version 3.1 (for example, the annual precipitation estimate of -141‰) compared to version 2.2 (annual precipitation estimate of 120‰) used in Bush et al. (2017). Here we look at variety of these potential source waters to assess net fractionation, while estimating soil evaporation, transpiration, and biosynthetic fractionation.

3. RESULTS 9

3.1 Wax n-alkane abundances and distributions The n-alkanes from all species exhibit odd over even chain length predominance (Fig. 2). None of the plants had sufficient concentrations of n-alkanes < C21 for measurement. The nalkane distributions and abundances were different between the eight species analyzed, and nalkane abundances were not evenly distributed among odd chain lengths for any species. We observe very low abundances of total n-alkanes in some species. Chain lengths are variable within and across species. Total average n-alkane abundances are E. arvense (28.5 ± 17 μg /g dry leaf, n = 10), V. uliginosum (60.5 ± 23 μg /g dry leaf, n = 8), S. filiformis (106.7 ± 28 μg /g dry leaf, n = 2), C. lapponica (93.3 ± 82 μg /g dry leaf, n = 2), B. nana (392.1 μg /g dry leaf, n = 14), E. hermaphroditum (2172.4 ± 1099 μg /g dry leaf, n = 2), R. tomentosum (1341.8 ± 559 μg /g dry leaf, n = 5), S. glauca (266 ± 364 μg /g dry leaf, n = 15). In three species, C31 is the predominant chain length (B. nana, R. tomentosum, and E. hermaphroditum) while C27 is the predominant chain length in four species (V. uliginosum, C. lapponica, E. arvense, and S. glauca). S. filiformis has little C27 or C29 and is instead composed primarily of C23. ACL across species ranged from 24.4 (S. filiformis) to 30.5 (R. tomentosum). 3.2 Bulk δ13C values of leaf material Four of the most abundantly sampled Arctic plant species (B. nana, S. glauca, R. tomentosum, and V. uliginosum) were run for bulk δ13C values. Measurements yielded an average of δ13C values of -29‰ ± 1.4 (n = 16, analyzed in duplicate) for the 5 sampled sites (Sites 9, 8, 6, 7, 3). There is no correlation between bulk δ 13C values and n-alkane chain length weighted δ2H values among all the species (r2 = 0.06, n = 16). However, linear regressions through individual species suggest correlations, though there are few individuals measured. B. nana (r2 = 0.4, n = 5), S. glauca (r2 = 0.01, n = 5), and V. uliginosum (r2 = 0.8, n = 4) are shown (Fig. 3). 3.3. Leaf wax n-alkane δ2H values All n-alkane chain lengths that were abundant enough for δ2H analysis (C23, C25, C27, C29, and C31) were measured and are reported in Table 1. Given isotopic offsets between chain lengths and differing abundances of chain lengths, comparison between sites and species is facilitated here by using a chain length amount-weighted average δ2H value. The amount10

weighted δ2H values across all species, sites, and individuals range between -171‰ and -282‰. For species averages across all sites, the most 2H-enriched value is from forb V. uliginosum (-190 ± 8.3‰). E. arvense showed similar 2H enrichment, though wider variability between individuals (-198 ± 16.9‰). The species with the most 2H-depleted average is C. lapponica (-267 ± 22.2‰), though this is only based on two individuals. There is no observed spatial trend in δ2H values (Table 1) or fractionation (Table 2) across all plants.

4. DISCUSSION 4.1 Leaf wax n-alkane distribution Sedimentary ACL variability has been used to infer past changes in climate, vegetation type, and environment. The concept is based on the idea that different plant groups (for example, photosynthetic pathways C3 vs C4), or climate parameters (for example, temperature or water stress from increased aridity), may preferentially produce different compound chain lengths (e.g. Hanisch et al., 2003; Meyers, 2003; Meyers and Ishiwatari, 1993; Zhang et al., 2006). Previous studies of modern vegetation, aeolian dust, and sediments have theorized a connection between n-alkane chain lengths and temperature, aridity, latitude, vapor pressure deficit, and sun exposure (Bush and McInerney, 2015; Dodd and Poveda, 2003; Eglinton and Hamilton, 1967; Eley and Hren, 2018; Poynter et al., 1989; Rommerskirchen et al., 2003; Sachse et al., 2006; Tipple and Pagani, 2013). However, a large meta-analysis of thousands of assembled observations found that plant type was not a significant driver of chain length for terrestrial plants, though there was some control on aquatic plants and moss chain length preference (Bush and McInerney, 2013). Observations of chain length distributions from species at Kangerlussuaq are in keeping with the few other available studies of modern leaf lipid distributions from Arctic settings. Overall abundances of total n-alkanes varied widely across plant life forms, agreeing with prior studies that have analyzed growth form of modern vegetation and found that “fingerprinting” plant type by chain length abundances was not possible due to the significantly varied distributions (Bush and McInerney, 2013; Eglinton and Hamilton, 1967). We observe large differences between species and plant form, such as across all dwarf shrubs (ACL of 27, 1σ = 1.4 n = 31) compared to other plant types, such as forbs and graminoids (ACL of 26.4, 1σ = 2.2 n = 27). However, there is also considerable variability within the same plant morphology, such as ACL variability across forbs V. uliginosum (ACL of 25.8, 1σ = 0.2, n = 8) and R. tomentosum 11

(ACL of 30.5, 1σ = 0.3, n = 5) (Fig. 4). Individuals of the same species show remarkably similar distributions of n-alkanes and a consistent dominant chain length (Fig. 2). Multiple individuals of one species measured in Kangerlussuaq, Greenland, B. nana, were also measured in another high latitude site, Toolik Field Station, Alaska (Daniels et al., 2017). Comparison between Daniels et al. (2017) and results from this study at Kangerlussuaq show similar ACL (~28) and chain length distribution, where C31 is the most dominant, followed by C27, and then C29. While ACL has been observed to vary across sampled transects of some studies (e.g. Tipple and Pagani, 2013), the species here show no spatial variability, likely due to the similar environmental conditions across a small spatial range. Investigation of one submerged lacustrine plant species, S. filiformis, from Bird Lake, also known as Sea Tomato Lake (Fig. 1), conforms to prior observations of aquatic chain length distributions (Ficken et al., 2000). Namely, C23 is the dominant chain length for measured individuals, and there are low abundances or absent long chain lengths (C29, C31, C33). However, C23 also occurs in similar large abundances in other vegetation at Kangerlussuaq. The total μg/dry g of C23 in S. filiformis matches that seen in the terrestrial shrub B. nana at Kangerlussuaq. Additionally, E. arvense has overall low n-alkane abundances, but a relatively high proportion of C23 relative to other chain lengths (Fig. 2). C23 has also been identified as a significant component of Sphagnum mosses (Bush and McInerney, 2013; Pancost et al., 2003), such as those commonly found in this region. In summary, these findings suggest that while aquatic vegetation n-alkanes may be dominated by C23, as in S. filiformis, inputs to the sedimentary record of C23 may be derived from other sources, including nearby terrestrial vegetation. Thus, C23, an n-alkane chain length often generalized as characteristically aquatic, may also be a poor indicator of vegetation type in the high latitudes, depending on other nearby vegetation, abundances, or transport to the sedimentary archive. 4.2 n-Alkane δ2H Values We examine the bulk foliar δ13C values of vegetation at Kangerlussuaq for evidence of the influence of water use efficiency (WUE) (Hou et al., 2007a), the ratio of carbon assimilation via photosynthetic uptake to water loss via leaf transpiration (Farquhar et al., 1989). WUE varies by plant type and species, but also importantly for the high latitudes, is driven by environmental parameters such as sunlight exposure and temperature (Hou et al., 2007a; Rawson et al., 1977). 12

We see no significant correlations between bulk δ 13C values and n-alkane δ2H values across all species, and individual species correlations are not significant (Fig. 3). Correlation between bulk δ13C values and n-alkane δ2H values for V. uliginosum and B. nana (n = 4, r2 = 0.78, p = 0.117 and n = 5, r2 = 0.36, p = 0.285, respectively) might suggest some potential water loss control through differences in WUE. If additional sampling showed a significant correlation, it might suggest that these species could be regulating stomatal behavior as a response to aridity or perhaps extended daylight, ultimately influencing their εapp and δ2H values. WUE might have been influenced by what was an unusually warm summer in Kangerlussuaq, however it is still interesting to note that response was not identical among species. Poor correlation between bulk foliar δ13C and n-alkane δ2H values sampled in July may reflect the integration of different times with dissimilar environmental conditions. Additionally, differences in stomatal conductance and species-specific water use strategies likely play a role in the varied responses between plants of this study or in correlations seen here and a lack of consistent relationship between δ 13C and δ2H values seen elsewhere (Bi et al., 2005; Chikaraishi and Naraoka, 2007; Feakins and Sessions, 2010; Hou et al., 2007a). The weighted δ2H values of n-alkanes from Kangerlussuaq reveal considerable variability, both between individuals and across different species (Table 1, Fig. 5). The interspecies variability in δ2H values we observe at Kangerlussuaq is large, more than 70‰. Intraspecies 1σ standard deviation is less than 10‰ for most species with more than two individuals (1σ of B. nana = 4‰, S. glauca = 7‰, R. tomentosum = 8‰, V. uliginosum = 8‰). Only one species with more than two sampled individuals showed a significant intraspecies variability (E. arvense, 1σ = 17‰). The ranges of δ2H values described here between individuals and species is typical for those seen in other studies of modern plant leaf waxes (e.g. Chikaraishi and Naraoka, 2003; Daniels et al., 2017; Feakins and Sessions, 2010; Sachse et al., 2012; Sachse et al., 2006). Often, studies focusing on plant type or morphology observe a link between life form and δ2H values of leaf waxes (e.g. Liu et al., 2006; Sachse et al., 2012; Smith and Freeman, 2006). A variety of sites, species, and morphologies were targeted for analyses at Kangerlussuaq, and although all plant types could not be sampled across all sites, we find no clear variability in δ2H values with location or correspondence to plant type. However, we do observe δ2H values within the range previously observed for shrubs and graminoids, with graminoids known to be more 2H-depleted than other plant morphologies (Sachse et al., 2012). 13

Despite large variability in modern integrated plant wax δ2H values, there is often a strong correlation to local environmental and climatic variables (e.g. Feakins and Sessions, 2010; Garcin et al., 2012; Sachse et al., 2012), which supports their use in paleoclimate studies. Observations from regional or sediment integrated wax studies often show better correlations to environmental parameters than individual plants or plant species (Sachse et al., 2012). An average of all plant wax values yields a value of -217‰. This value is also roughly similar to surface sediment leaf wax δ2H wax values from n-alkanes and n-alkanoic acids that have been reported from other arctic locations ranging from -240‰ to -160‰ (Shanahan et al., 2013; Wilkie et al., 2013). 4.3 Isotopic fractionation during biosynthesis, εbio (εwax/lw) For wax biosynthesis, bulk leaf water δ2H values from the time of leaf collection provide an upper constraint of maximum 2H enrichment on possible cellular formation waters. While calculation of biosynthetic fractionation (εbio) using wax δ2H values and bulk leaf water δ2H values (εwax/lw) has significant uncertainty, it can serve as a field-based approximation using the pool of water found in the leaves at the time of collection and can help us to understand the metabolic effects that control plant δ2H values (Cormier et al., 2018). We observe considerable differences in calculated εbio (εwax/lw) from the survey of available Greenland vegetation. The average calculated εbio value for B. nana, R. tomentosum, V. uglinosum, and S. glauca is -120 ± 27‰ (Fig. 6). This value is considerably more 2H-enriched compared to other studies that have reported εbio, ranging from ~-180‰ to -150‰ (Feakins and Sessions, 2010; McInerney et al., 2011; Sachse et al., 2004; Sessions et al., 1999). There are a lack of εbio estimations from similar high latitude settings, but this difference hints at the importance of εbio as a significant influence of εapp at high latitudes that has not been previously explored. Though only an approximation of this fractionation, it is interesting to note that εbio varies between species. We calculate εbio of -107 ± 20‰ (B. nana), -156 ± 13‰ (R. tomentosum), and -128 ± 19‰ (S. glauca) and -72‰ (V. uglinosum) (Fig. 6, Table 3), with significant (p<0.05, one way ANOVA) differences noted between B. nana and R. tomentosum. Use of a stable biosynthetic fractionation for lipid formation across plant types has been fairly common (Chikaraishi and Naraoka, 2003; McInerney et al., 2011; Sachse et al., 2004; Sessions et al., 1999; Smith and Freeman, 2006). However, in addition to this study, other recent 14

studies using empirical observations and modeled εbio have seen interspecies εbio variability (Kahmen et al., 2013a; Tipple et al., 2014; Yang et al., 2009; Zhou et al., 2011), which suggests caution in application of a constant εbio value for ancient environmental reconstruction. For example, the estimated differences in ε bio may reflect variability in cellular water used in wax production, as it is known that empirical leaf water values do not always accurately reflect the biosynthetic pool for lipid formation. The cellular pool used for wax formation involves some mix of 2H enriched leaf water closer to the site of stomatal evaporation, more 2H depleted xylem water, or water from other locations such as mesophyll or bundle sheath cells (Cormier et al., 2018; Kahmen et al., 2013b; Tipple et al., 2014). Research has found that the mismatch lies in the fact that biochemical hydrogen sources for lipid synthesis can come from directly from water (or H exchange with water), from biosynthetic precursors (such as acetate or acetyl-CoA ), or from NADPH (Kazuki et al., 1980; Sachse et al., 2012; Sessions et al., 1999). Thus, variability in εbio between plant species is likely due to some combination of biochemical traits, that likely vary by plant form, as well as environmentally-influenced factors, such as transpiration (Gamarra et al., 2016). At Kangerlussuaq, we observe plant εbio less negative than most global plant εbio which may be due to variations in use of source NADPH, potentially relying on a larger proportion of heterotrophically produced reserve NADPH (Kahmen et al., 2013a; Kahmen et al., 2013b; Schmidt et al., 2003; Zhang et al., 2009), which for plants growing in inland Greenland may be a metabolic response of using stored photosynthates due to growing conditions beginning in late summer and continuous low summer light intensity (Cormier et al., 2018; Sessions, 2006). The critical role of εbio in determining net fractionation is only more recently being recognized and is an important avenue for future study. 4.4 Isotopic fractionation from transpiration (εlw/xw) The relative importance of leaf evaporative processes are key in understanding what alterations to meteoric water, beyond possible variations in εbio, are important in shaping lipid δ2H values. Examination of fractionation between leaf and xylem water δ2H values (εlw/xw) allows us to assess the impact of transpiration on leaf wax δ 2H values (Fig. 6). The average εlw/xw across all species with available water data is 35 ± 12‰ (Table 2). We observe that calculated fractionation from leaf associated transpiration is relatively constant across all species (Fig. 6),

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unlike εbio. We observe similar calculated εlw/xw for B. nana (31 ± 14‰) and S. glauca (36 ± 12‰) and individuals of R. tomentosum (37‰) and V. uliginosum (43‰). Early research suggested low transpiration rates in high latitude settings associated with low temperatures and vapor pressure deficit (Bliss, 1960, 1962; Hygen, 1951). While cold temperatures might suggest low transpiration rates, increased aridity, windiness, and cloud cover have all been linked to increased transpiration in high latitude vegetation (Bliss, 1960; Wilson, 1954). And while the demands of transpiration can often be met with available water for tundra vegetation with little water stress, not all high latitude plants have similar drought tolerance or seasonal physiological sensitivity (Cahoon et al., 2016; Patankar et al., 2013). Recent studies of Arctic vegetation observed enhanced transpiration and attributed the response to leaf water 2H enrichment from continuous sunlight (Yang et al., 2011; Yang et al., 2009). Yang et al. (2009) conducted greenhouse simulations of low intensity, continuous arctic sunlight and observed deciduous conifer leaf water 2H enrichment. A 40‰ 2H enrichment of the leaf wax n-alkane δ2H values was attributed to enhanced transpiration (Yang et al., 2009). However, another high latitude study found 2H enrichment of leaf water over xylem water, but using a simplified evaporative enrichment model, found no significant impact of continuous daylight on εlw/xw (Daniels et al., 2017). Roden and Ehleringer (1999) showed that the site of constant leaf evaporation was quick to reach isotopic equilibrium, and thus Daniels et al. (2017) posited that enhanced transpiration might not lead to significantly altered leaf water δ2H values. The values of fractionation from transpiration at Kangerlussuaq are lower than arid plant studies that calculated εlw/xw average values of 74 ± 20‰ (Feakins and Sessions, 2010), but are still 2Henriched (35 ± 12‰, this study), on order with 2H enrichment seen at other high latitude sites (Daniels et al., 2017). While transpiration-influenced εlw/xw cannot be directly attributed to continuous summer sunlight, cold temperatures, or wind-driven fractionation at this high latitude site, it is clear that 2H enrichment due to transpiration would influenceδ2H values of leaf wax compounds (Bush et al., 2017; Feakins and Sessions, 2010; Tipple et al., 2014; Yang et al., 2011).

4.5 Isotopic fractionation from soil evaporation (εxw/p) Study of the relationship between xylem water and empirical environmental water data as a proxy for average precipitation at Kangerlussuaq (εxw/p) allows us to examine a critical piece of 16

net fractionation, soil water evaporation (Sachse et al., 2012; Sachse et al., 2009; Smith and Freeman, 2006). We find 2H enrichment due to soil water evaporation, with an average of 14 ± 17‰ across all species, significantly different from the mean of fractionation from transpiration, εlw/xw (two tailed unpaired t-test p = 0.0001) (Table 2). Mean εxw/p are similar for each species, with no statistically significant difference between species means (one way ANOVA p = 0.7346), making fractionation due to evaporation appear constant regardless of plant species or morphology. While interspecies comparisons show some variability, there is more significant variability within species (Table 3). Relatively similar εxw/p across species suggests that a broad generalization in amount of evaporative 2H enrichment impacting these plants during the growing season may be applicable. Variations between species may support the use of pools of soil water with different amounts of evaporative 2 H enrichment. Previous studies have predicted that for some locations, soil water evaporation significantly contributes to εapp. Using a greenhouse and modeling based approach, Hou et al. (2008) theorized that soil water evaporation would have the largest influence on leaf wax δ 2H values, exceeding the fractionation by leaf transpiration or biosynthetic fractionation. However, subsequent field studies and Craig-Gordon leaf water simulations (Feakins and Sessions, 2010; Kahmen et al., 2013a; Kahmen et al., 2013b) have shown significant variability due to transpiration, particularly in arid settings, is likely. Assessment of surficial soil water in comparison to ground water in these locations suggested plants were accessing deeper water sources and were thus not affected by surface soil 2 H enrichment (Feakins and Sessions, 2010; Kahmen et al., 2013a; Kahmen et al., 2013b). In the case of Kangerlussuaq, it is an arid region with limited growing season and soil water usage cannot penetrate below existing permafrost (Bush et al., 2017). Shallower rooting depths would necessarily emphasize the importance of surficial soil evaporation (Shanahan et al., 2013). At Kangerlussuaq, we see larger fractionation associated with transpiration than evaporation that is significant for S. glauca (two tailed unpaired t-test p = 0.0227) though not significant for B. nana (two tailed unpaired t-test p = 0.0870). If transpiration associated fractionation does have a larger role in 2H enrichment of leaf wax δ2H values for some species at Kangerlussuaq, it might suggest that the roots of some species are accessing water below the influence of surface evaporation despite shallow soil depths or perhaps are accessing varying amounts of winter melt. The importance of transpiration at Kangerlussuaq may also be related in 17

part to the near continuous light cycle in the summer as theorized for other high latitude sites (Shanahan et al., 2013; Yang et al., 2009), however the empirical data collected here cannot distinguish the relative importance of near constant sunlight on transpiration. 4.6 Net Fractionation (εapp) The summed total fractionation between meteoric water and plant lipids (εapp) is still one of the largest sources of uncertainty in global paleohydrological reconstructions using leaf waxes (Polissar and D’Andrea, 2014; Sachse et al., 2012). This uncertainty is particularly evident when comparing different regions. Indeed, a point of continued debate regarding plant wax fractionation from high latitude sites has been whether εapp is similar or smaller than those from plants at the lower latitude sites (Daniels et al., 2017; Feakins et al., 2012; Porter et al., 2016; Sachse et al., 2012; Shanahan et al., 2013; Thomas et al., 2016; Wilkie et al., 2013; Yang et al., 2011). At Kangerlussuaq, εapp was calculated using n-alkane chain length abundance-weighted δ2H values and the precipitation calculation based on LEL-LMWL intersection (εwax/p). We find that the mean net fractionation value across all plants was -75 ± 20‰ (εwax/p). To examine how net fractionation estimates would vary using other sources of water for lipid formation, we first observe εwax/xw, using xylem water measurements as a proxy for the soil water used by each plant. The net fractionation εwax/xw = -86 ± 25‰ (Table 2), which is not significantly different from εwax/p used for εapp (two tailed unpaired t-test, p = 0.0717). Further, estimated εapp is small at Kangerlussuaq compared to estimates using OIPC values of precipitation. Using the July OIPC precipitation estimate of -107‰, we calculate εwax/OIPC, July = -128 ±19‰ and using the annual OIPC precipitation estimate of -141‰, we calculate εwax/OIPC, Annual = -91 ±17‰. Calculated net fractionation is significantly different between (εwax/p) and modeled annual OIPC estimates of precipitation (two tailed unpaired t-test, p = 0.0021) and July OIPC (two tailed unpaired t-test, p <0.0001). However, using DJF OIPC estimated precipitation, we find no significant difference between εwax/DJF OIPC and εwax/p (two tailed unpaired t-test, p = 0.8578). This supports the importance of winter precipitation when considering meteoric water used by plants in the Kangerlussuaq area. The εapp for the surveyed Kangerlussuaq plants is small compared to the global average of εapp, 121 ± 32‰ (n = 316 using C29 n-alkane) (Sachse et al., 2012), but is comparable to most previously reported high latitude n-alkane based εapp estimates. A global synthesis suggests a 18

trend towards less negative net fractionation values in drier conditions, as defined by low relative humidity (<0.7) and evapotranspiration (<1000 mm/yr) (Sachse et al., 2012). At Kangerlussuaq, this study suggests that εbio may be a primary driver of the less negative εapp in the high latitudes, perhaps implicating the importance of timing of water availability for εapp in this region. Observation of relatively small high latitude εapp values compared to the global average has been previously theorized to be controlled by continuous summer sunlight driving fractionation (Feakins et al., 2012; Porter et al., 2016; Shanahan et al., 2013; Wilkie et al., 2013; Yang et al., 2011; Yang et al., 2009). Using lake water as a proxy for precipitation and sedimentary nalkanoic acids at Baffin Island Shanahan et al. (2013) estimated εapp of -61 ± 20‰. Porter et al. (2016) observed a similarly small εapp (-59 ± 10‰) for n-alkanoic acids (C28), using precipitation δ2H values determined from relict ice across East Beringia. Wilkie et al. (2013) studied nalkanoic acids from modern vegetation, including common grasses, forbs, and low-lying shrubs, and collected precipitation from the Lake El’gygytgyn region, Siberia, and observed a significantly larger εapp than other high latitude sites (-107 ± 12‰), but still small compared to global εapp estimates. This estimate was similar to an integrated fractionation calculated using lake surface sediment C30 n-alkanoic acids samples spanning the last 200 years (-96 ± 8‰) (Wilkie et al., 2013). Another study from western Greenland, using summer (May-June) OIPC (111‰) and C23, C25, and C27 n-alkanes from four dominant shrub species on the landscape, found εapp values ranging from -103 ± 15‰ to -107 ± 35‰ (Thomas et al., 2016). Not all studies of plants from the high latitudes observe small εapp estimates. The overall εapp estimated at Kangerlussuaq was significantly smaller than that of the Toolik Field Station region in Alaska (-132‰ using n-alkanes and xylem water) (Daniels et al., 2017). This is also seen at the individual species level, where B. nana from this region of Alaska were found have an εapp of -89‰ for n-alkanoic acids and -105‰ for n-alkanes (Daniels et al., 2017). At Kangerlussuaq, B. nana n-alkane εapp is significantly different than at Toolik, Alaska (>30‰) (Daniels et al., 2017) (Table 3). The other sampled species from Daniels et al. (2017), Eriophorum vaginatum (cottongrass), had larger fractionation than any other high latitude plant study to date (-182 ± 10‰ and -154 ± 26‰ for n-alkanes and n-alkanoic acids, respectively) (Daniels et al., 2017). Larger fractionations in grasses compared to forbs and shrubs are commonly observed globally, often attributed to grass physiology or biochemical differences (Sachse et al., 2012). However, the other striking εapp differences, particularly for B. nana in the 19

two high latitude locations of Kangerlussuaq, Greenland and Toolik Field Station, Alaska, may be due to differences in estimated plant water usage, as recent research has highlighted that a significant fraction of winter moisture is used and transported in the xylem of B. nana (Jespersen et al., 2018). Recent research at Toolik Field Station found that deciduous shrubs obtain more of their water from snow meltwater than evergreen shrubs or graminoids, between 30-60% snowmelt usage (Ebbs, 2016; Jespersen et al., 2018). It was theorized that snowmelt contribution through the growing season varied among high latitude plants due to deeper rooting depth and deep-soil water use of deciduous shrubs like B. nana than evergreen shrubs or graminoids, especially in the early to middle summer growing season (Jespersen et al., 2018). Additionally, differences could relate to plant growth forms, as B. nana is known to exhibit morphological variation due to hybridization with other species of Betula where multiple members of the genus have overlapping range (Thórsson et al., 2007), including northern Alaska and Greenland (de Groot et al., 1997; Furlow, 1997), which could cause physiological or biosynthetic differences visible in water-wax fractionations.

5. Conclusions In this study, we assessed the importance of vegetation type, plant water use, transpiration, evaporation, and biosynthesis in controlling fractionation between water and nalkane δ2H values in plants from Kangerlussuaq, western Greenland. Using n-alkane and precipitation estimated δ2H values at Kangerlussuaq (Bush et al., 2017), the mean net fractionation εapp (εwax/p) was -75 ± 20‰, which is small in comparison to global averages. However, εpp at Kangerlussuaq is similar to most other εapp estimates from high latitude settings, the majority of which find similarly small net fractionations. These findings suggest that hydroclimate reconstructions at high latitude regions like Kangerlussuaq cannot use similar εapp to those at lower latitude sites and emphasize the need for water fractionation information specific to the environmental conditions, plants, and precipitation of the high latitudes when reconstructing these environments using plant waxes. Our modern plant collections in the Kangerlussuaq region of western Greenland provides information about wax δ2H values and isotope fractionations. The δ2H values of leaf wax nalkanes showed large variability across species (>70‰), but exhibited relatively similar values within each species. Comparing plant leaf wax δ2H values of n-alkanes to plant waters (xylem 20

and leaf) from the same individuals allows estimation of the controls on fractionation that are a part of εapp. The fractionation associated with leaf transpiration (ε lw/xw), is relatively constant across measured species, on average 35 ± 12‰. Transpiration-associated 2H enrichment is similar to that seen at other high latitude sites (e.g. Daniels et al., 2017). Soil evaporation associated 2H enrichment appears relatively constant regardless of plant species or morphology, with εxw/p on average 14 ± 17‰. We observe large variability in biosynthetic fractionation (εbio) between species and suggest that a constant εbio value which is often theorized may not accurately capture differences between species in high latitude settings. The estimated εbio has an average of -120 ± 27‰, and significantly influences εapp for the surveyed plants. The εbio determined from this study is considerably smaller than the εbio range of -150 to -175‰ commonly reported elsewhere (e.g. Feakins and Sessions, 2010; Sachse et al., 2006; Smith and Freeman, 2006). Varying estimated εbio suggests differential usage of 2H enriched leaf water and stored plant waters. Overall, these findings suggests that εbio values in high latitudes warrant further study as a potential primary driver shaping εapp. In summary, the majority of available high latitude net fractionations of modern plants, including this new study from western Greenland, seem to suggest an εapp significantly different from that of low latitudes. Generally, high latitude sites show significantly smaller ε app than global averages, with some evidence that near-constant light exposure during the growing season plays a significant role in this evaporative control and 2H enrichment (Yang et al., 2009). Ranges in εapp between high latitude sites are most likely due to variability in empirical or estimated precipitation due to seasonality or rooting depth, and to a lesser degree differences in wax chain lengths measured or plant growth forms. We contend that while we may understand aspects of isotope signatures of leaf wax compounds and wax-water fractionation (Sachse et al., 2012), we do not fully grasp plant seasonal water usage or biosynthesis using stored NADPH in the high latitudes, which could lead to overconfidence in paleoprecipitation estimates.

Acknowledgements This manuscript was greatly improved by the thorough and thoughtful comments of three reviewers and GCA Associate Editor Sarah Feakins. The authors wish to thank Andrew Jacobson for providing the opportunity for field collections and the staff of the Kangerlussuaq

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International Science Support facility for field support. Support provided by Dennis Birdsell and the Notre Dame Center for Environmental Science and Technology. Thank you to University of Notre Dame undergraduate researchers Christa Costello, Teresa Muldoon, and Michael Tillema for assistance in the lab. Funding for this project was provided by the Clare Booth Luce Foundation and by the Notre Dame Environmental Change Initiative.

Figures: Figure 1: Map of the Kangerlussuaq, Greenland region (square denoted by arrow) with plant sampling locations (16 sites total, non-sequential numbering). Bird Lake, referenced in the text, is obscured by sampling points 1-7 around the lake.

Figure 2: Chain length concentrations of n-alkanes (C21 – C35) for collected species at Kangerlussuaq, Greenland, in μg/g dry leaf weight. Error bars are standard error (1σ) of the mean and n is the number of individuals of each species that was measured.

Figure 3. Crossplot of bulk plant leaf δ 13C values and chain length abundance-weighted n-alkane δ2H values for B. nana (n = 5, r2 = 0.36, p = 0.285; open squares), S. glauca (n = 5, r2 = 0.01, p = 0.848; red diamonds), R. tomentosum (n = 2, gray triangles), and V. uliginosum (n = 4, r2 = 0.78, p = 0.117; blue circles). Linear regressions for species with more than two samples are shown. Linear regression of all samples is r2 = 0.06 (n = 16, p = 0.362).

Figure 4: Box and whisker plot of the average chain length (ACL) for all measured individuals of shrubs, B. nana (n = 14), S. glauca (n = 15), and E. hermaphroditum (n = 2), forbs and graminoids R. tomentosum (n = 4), V. uliginosum (n = 8), C. lapponica (n = 2), herb E. arvense (n = 10), and aquatic macrophyte S. filiformis (n = 2). ACL was calculated using n-alkane concentrations from C21 – C35. Boxes show median, 25th and 75th percentiles, whiskers extend to minimum and maximum values, and points represent outliers.

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Figure 5: Chain length abundance-weighted n-alkane δ2H values for all individuals of shrubs (B. nana (n = 14), S. glauca (n = 15), and E. hermaphroditum (n = 2), forbs and shrubs R. tomentosum (n = 15), V. uliginosum (n = 8), C. lapponica (n = 2), herb E. arvense (n = 9), and aquatic macrophyte S. filiformis (n = 2). Boxes show median, 25th and 75th percentiles, whiskers extend to minimum and maximum values, and points represent outliers.

Figure 6: Calculated fractionations for B. nana, S. glauca, V. uliginosum, and R. tomentosum from Kangerlussuaq, Greenland. All symbols represent calculated mean values from individuals, with error bars indicating the standard deviation (1σ) of the mean. Square symbols are calculated fractionation between leaf water and xylem water (εlw/xw), used to study leaf transpiration: B. nana (n = 6), S. glauca (n = 7), R. tomentosum (n = 1), V. uliginosum (n = 1); triangle symbols are calculated fractionation between xylem water and precipitation (ε xw/p), used to study soil evaporation: B. nana (n = 10), S. glauca (n = 8), R. tomentosum (n = 2), V. uliginosum (n = 2); diamond symbols are calculated fractionations between wax n-alkanes and xylem water (εwax/xw): B. nana (n = 10), S. glauca (n = 8), R. tomentosum (n = 2), V. uliginosum (n = 2); star symbols are calculated fractionation between wax and precipitation, used to discuss net fractionation (εwax/p, εapp): B. nana (n = 10), S. glauca (n = 8), R. tomentosum (n = 4), V. uliginosum (n = 2); circle symbols are calculated fractionations between wax n-alkanes and leaf water (εwax/lw), used to discussed biosynthetic fractionation (εbio) B. nana (n = 6), S. glauca (n = 7), R. tomentosum (n = 2), V. uliginosum (n = 1). The shaded rectangle highlights all plant averages for calculated fractionations and the standard deviation (1σ) of the mean.

Tables: Table 1: Chain length abundance weighted δ 2H values and propagated uncertainty of cumulative measured chain lengths. Mean δ2H values from replicate analyses of each individual and 1σ standard deviation for C23 – C31 n-alkanes, where abundances allowed for isotopic analysis. Fractional abundances are measured chain length concentrations for individuals. Weighted δ 2H values are used throughout the text. Site numbers correspond to map site numbers shown in Figure 1. Bulk δ13C values were analyzed for samples with excess material. 23

Table 2: The δ2H values of leaf water (lw) and xylem water (xw) (Bush et al., 2017), and nalkane δ2H values for each individual. Calculated fractionations of xylem water/precipitation (εxw/p), leaf water/xylem water (εlw/xw), wax/leaf water (εwax/lw), wax/precipitation (εwax/p), and wax/xylem water (εwax/xw). Precipitation (-158‰) from Bush et al., (2017) empirical estimate using precipitation and snow δ2H values. Site numbers correspond to map site numbers shown in Figure 1. Table 3: Mean fractionations across all sites for species with available data and 1σ standard deviation (of individual plant values across all sites) where possible. Each individual was from a separate site (Sites 1-10) listed in Table 2.

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Table 1

Species Site 1 Salix glauca Betula nana Rhododendron tomentosum Equisetum arvense Empetrum hermaphroditum Site 2 Salix glauca Betula nana Vaccinium uliginosum Stuckenia filiformis Site 3 Salix glauca Betula nana Rhododendron tomentosum Vaccinium uliginosum Empetrum hermaphroditum Equisetum arvense Stuckenia filiformis Site 4 Salix glauca Betula nana Rhododendron tomentosum Vaccinium uliginosum Equisetum arvense Calamagrostis lapponica Site 5 Salix glauca Betula nana Equisetum arvense Vaccinium uliginosum Rhododendron lapponica Site 6 Salix glauca Betula nana Vaccinium uliginosum Equisetum arvense Calamagrostis lapponica Site 7 Betula nana Equisetum arvense Salix glauca Site 8 Betula nana Equisetum arvense Vaccinium uliginosum Rhododendron tomentosum Site 9 Salix glauca Betula nana Site 10 Betula nana Salix glauca Site 11 Salix glauca Site 14 Betula nana Equisetum arvense Salix glauca Site 15 Betula nana Salix glauca Site 16 Equisetum arvense Salix glauca Vaccinium uliginosum Site 18 Betula nana Salix glauca Site 20 Betula nana Salix glauca Vaccinium uliginosum

C23 1σ Mean

C25 1σ Mean

δ2H C27 1σ Mean

-222

1.2

-223

0.5

-219

0.8

-198

2.2

-192

0.2

-199 -234

1.4 0.9

-217

-225

0.6

0.2

-223 -214 -192 -215

0.6 1.3 0.9 0.8

-227 -212 -197

0.8 0.8 0.1

-239 -217

1.8 0.7

-186

1.5

-241 -219 -235 -194

0.8 1.0 0.0 0.4

0.0 0.9

-193 -212

0.4 0.2

-211

0.5

-221

0.2

-225

0.9

-231 -224

0.5 0.1

-201 -215 -249

0.1 0.5 2.3

-231 -209 -203 -184

0.1 0.5 1.1 0.9

-216 -191

0.3 0.2

-225

0.4

-205

0.0

-209 -174

-217 -189

-223

-195 -220

-158

1.9 0.1

0.6 0.1

4.0

0.4 0.2

-218 -209 -182 -183

0.2

0.1 0.6 2.3 0.4

C31 1σ Mean

-226 -208 -237

0.3 0.1 0.4

-228 -210 -234

0.7 0.2 1.5

-239

0.4

-240

0.4

-200 -206

0.4 0.1

-207

-211 -244

0.6 0.1

-212

1.0

-252

0.8

-251

0.8

Fractional Abundance C25 C27 C29 C31

0

0

0

1

0

0 0

0

0 0 0 0

1 0 1

0 0

1 0 0 1

0.8

0

-213 -213 -242

0.5 0.7 0.4

-221 -252

0.8 0.9

-213 -201 -197

0.3 0.4 1.6

-202

0.1

-222

0.1

-204

0.0

-211

0.7

-212

0.8

-273

0.4 1.6

-214 -245

0 1

0 0

0

0

0

1 0

0.2 0.4 0

-232 -222 -186 -198 -266

1.8 0.7 0.5 0.0 0.4

-230 -218 -188 -216 -289

0.5 0.5 1.8 0.5 0.3

-210 -174 -219

1.5 1.6 0.1

-210 -177 -220

1.2 0.7 0.2

-200

-222 -205 -193

0.3 0.5 0.7

-202 -227 -194

1.0 1.1 0.4

-219 -221

0.1 1.0

-219

-259

0.5

-252

0.3

0 0

0 0

-202

0.8

0 0

0.0 0 0

-239 -226

0.8 0.7

-239 -220

0.2 1.0

-214

0.5

-215

0.1

-233 -228

0.7 0.5

-223 -226

0.2 0.4

-211 -191

0.2 1.2

-208

0.8

-234

0.5

-230

0.3

-217 -193 -223

1.1 0.0 0.2

-215 -205 -221

1.3 0.3 0.4

-204

1.4

-204

-217 -219

1.2 0.4

-218 -217

0.4 0.3

-212

-233 -194

1.0 1.3

-188 -227 -202

2.8 0.7 0.8

3.7

C23

1

-197 -221

-189

C29 1σ Mean

0

0.3 0 0

0.3

-214

0.4

0 0 0 0

1 0 0 1 0 0 1

1 0 0

0 0 1

0

1

0 0

0

0 1

0

0

1

0 0 0

0 1

1 1 0 0 0

0

1

0

0

0

δ2H (weighted)

-223 -210 -195 -221 -241 -214 -243 -191 -252 -203 -218

-28.5 -29.5 -29.7 -29.0

0.0 0.0 0.0 0.1

-231 -218 -187 -208 -282

-24.5 -29.0 -29.0

0.0 0.0 0.1

-207 -175 -219

-27.0

0.0

-27.8

0.1

-30.6

0.0

-29.1 -28.8

0.4 0.1

-27.7

0.1

-29.3 -29.0 -29.4

0.0 0.0 0.0

-228 -216 -244 -197 -218 -251 -227 -208 -195 -184 -216

1 0 1 0 1

0 0 0

0 0 1

0

0 0 1

0 0 0

0 0

0

0

1

-215 -219 -193 -256 -239 -219

0

0 0

1 0

0

0

0 0

0 0

0 0

0

0

1

0 0 0

0 0 0

0

0

-210 -198 -222

0 0

0 1

0

0

-215 -218

0 0

0 1 1

1

δ13C (bulk) 1σ

-227 -214 -235 -197 -239

0 0 0 0 0

0

Propagated error

-217 -225 -232

-171 -229 -199

-220

0.6

-216 -226

0.8 0.2

-211 -222

0.0 0.1

-198

0.0

-198

0.0

0

0 0

0 1

0

0

-208 -224

-209 -208

0.5 1.1

-207 -220 -174

0.6 0.1 0.2

-213 -222 -172

0.7 0.1 0.7

-207 -207

0.3 0.2

-210

0.6

0 0

0 0 0

0 1 1

0 0

0

-209 -219 -173

Table 2

Species lw Site 1 Salix glauca Betula nana Rhododendron tomentosum Site 2 Salix glauca Betula nana Site 3 Salix glauca Betula nana Rhododendron tomentosum Vaccinium uliginosum Site 4 Salix glauca Betula nana Rhododendron tomentosum Site 5 Salix glauca Betula nana Vaccinium uliginosum Site 6 Salix glauca Betula nana Site 7 Betula nana Site 8 Betula nana Rhododendron tomentosum Site 9 Salix glauca Betula nana Site 10 Betula nana Salix glauca Mean 1σ

δ2H xw wax alkane

xw/p

lw/xw

Fractionation (ε, ‰) wax/lw wax/xw

wax/p

-107 -136 -102 -138 -103 -135

-227 -214 -235

26 24 27

34 42 37

-134 -125 -147

-105 -88 -116

-82 -66 -92

-138 -181 -126 -166

-223 -210

-27 -10

53 48

-99 -96

-52 -52

-78 -61

-137 -150 -141 -138 -128 -164

-241 -214 -243 -191

10 20 24 -7

15

-120

43

-72

-107 -85 -122 -32

-98 -67 -101 -39

-101 -132 -97 -127 -94

-228 -216 -244

31 37

36 34

-141 -132 -165

-110 -102

-83 -69 -102

-91 -130 -124 -131

-227 -208 -184

33 40 32

45

-150

-112 -96 -61

-82 -60 -31

-137 -162 -158

-231 -218

-5 0

30

-108

-82 -71

-86 -71

-137 -149

-207

11

14

-81

-68

-58

-154 -144

-215 -256

5 17

-72 -130

-67 -116

-114 -147 -114 -144

-239 -219

13 17

39 35

-141 -118

-108 -88

-96 -72

-137 -150 -162

-217 -225

10 -5

15

-93

-79 -75

-70 -80

-116 -146 18 14

-221 17

14 17

35 12

-120 27

-87 25

-75 20

Table 3

Species Salix glauca Betula nana Rhododendron tomentosum Vaccinium uliginosum

xw/p 10 15 23 12

1σ 21 16 5 28

n 8 10 2 2

lw/xw 36 31 37 43

1σ 12 14 n/a n/a

n 7 6 1 1

Fractionation (ε, ‰) wax/lw 1σ n -128 19 7 -107 20 6 -156 13 2 -72 n/a 1

wax/xw -94 -80 -123 -46

1σ 22 15 7 20

n 8 10 2 2

wax/p -86 -66 -103 -35

1σ 8 5 10 6

n 8 10 4 2

Figure 1

8 9 16

10

11 15 4 7 5 2 3 16 18

14

20

/ 00

4

88

16 Kilometers Kilometers Kilometers 16 16

Figure 2

160

180

Betula nana (n = 14)

µg/g dry leaf weight

Salix glauca (n = 15)

160

140

140

120

120

100

100 80 80 60

60

40

40

20

20

0

0 21

23

25

27

29

31

33

35

1600

21

23

25

27

29

31

33

35

31

33

35

29

31

33

35

29

31

33

35

800 Empetrum hermaphroditum (n = 2)

Rhododendron tomentosum (n = 5)

µg/g dry leaf weight

1400 1200

600

1000 800

400

600 200

400 200 0

0 21

23

25

27

29

31

33

21

35

40

23

25

27

29

120 Vaccinium uliginosum (n = 8)

Calamagrostis lapponica (n = 2)

µg/g dry leaf weight

100 30 80 20

60 40

10 20 0

0 21

µg/g dry leaf weight

20

23

25

27

29

31

33

21

35 60

Equisetum arvense (n = 10)

23

25

27

Stuckenia filiformis (n = 2)

50 15 40 30

10

20 5 10 0

0 21

23

25

27

29

31

n-alkane chain length

33

35

21

23

25

27

n-alkane chain length

Figure 3

-160 B. nana S. glauca R. tomentosum V. uliginosum

r ² = 0.78

δ2H (‰)

-180

-200 r ² = 0.36 -220 r ² = 0.01

-240

-260 -31

-30

-29

-28

-27 13

δ C (‰)

-26

-25

-24

m ap

C

V.

S. is

se

m

rv en

ca

um

ni

fil ifo r

m

um

os

po

a

ca

itu

au

an

to s

in

ap

ig

en

E. a

.l

ul

gl

hr od

.t om

er

R

E. h

S.

B. n

ACL

Figure 4

32

30

28

26

24

22

m ap

C

V.

S. is

se

m

rv en

ca

um

ni

fil ifo r

m

um

os

po

a

ca

itu

au

an

to s

in

ap

ig

en

E. a

.l

ul

gl

hr od

S.

.t om

er

R

E. h

B. n

δ2H (‰)

Figure 5

-160

-180

-200

-220

-240

-260

-280

-300

Al

ul

la

nt

(a

ve r

um

ag

e)

um

os

to s

in

ca

na

au

na

gl

en

ig

.t om

lP

R

V.

S.

B.

Fractionation (ε, ‰)

Figure 6

100

50

0

-50

-100

εlw/xw εxw/p εwax/xw εwax/p εwax/lw

-150

-200