Production of long-chain n-alkyl lipids by heterotrophic microbes: New evidence from Antarctic lakes

Organic Geochemistry 138 (2019) 103909

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Production of long-chain n-alkyl lipids by heterotrophic microbes: New evidence from Antarctic lakes Xin Chen a,b, Xiaodong Liu a,b,⇑, Yangyang Wei a, Yongsong Huang b,⇑ a Anhui Province Key Laboratory of Polar Environment and Global Change, School of Earth and Space Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China b Department of Earth, Environmental and Planetary Sciences, Brown University, Providence, RI 02912, USA

a r t i c l e

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Article history: Received 28 June 2019 Received in revised form 31 July 2019 Accepted 9 August 2019 Available online 12 August 2019 Keywords: Antarctica Lacustrine sediments n-Alkanes n-Alkanoic acids Carbon isotopic ratios Vascular plant leaf waxes Microbial origin Long chain

a b s t r a c t Long-chain n-alkyl lipids are traditionally ascribed to an origin from terrestrial vascular plants because these compounds are major constituents of higher plant leaf waxes. Over the past half century, numerous studies have taken advantage of these sedimentary biomarkers and their isotopic ratios to reconstruct paleo-environmental and paleo-climatological changes at a variety of time scales. However, it is uncertain and extremely difficult to determine if these compounds can also derive from microbes because of the prevalence of higher plants in most environments around the globe. Here we show, for the first time from natural sediment samples, that long-chain n-alkyl lipids can predominantly originate from aquatic microbial sources at three high-latitude (>69°S latitude) Antarctic lakes, where no vascular plants are present in the surrounding land mass. The high carbon isotopic values (up to –12‰) of these longchain n-alkyl lipids exclude the possibility that these compounds are transported by wind from adjacent vegetated land masses. Instead, these isotope values are similar to lipids produced by aquatic microbial mats with an average bulk d13C value of –14.2 ± 1.7‰, indicating heterotrophic microbes are the likely source of these long-chain n-alkyl lipids. For comparison, we also show that when even small amount of vascular plants and mosses are present in the study region, for instance at Long Lake (
62°S latitude) in the Antarctic Peninsula, the carbon isotopic values of sedimentary long-chain n-alkyl lipids decline dramatically, suggesting a rapid proportional increase in the relative contribution of leaf wax sources to total long-chain n-alkyl lipid inventory in lake sediments. Ó 2019 Published by Elsevier Ltd.

1. Introduction Long-chain (>C25) n-alkyl lipids are among the most important biomarkers for reconstructing past environmental and climatic conditions. These compounds comprise the major components of terrestrial higher plant leaf waxes (Eglinton and Hamilton, 1967; Meyers, 2003). For example, long-chain n-alkanes are considered biomarkers for terrestrial inputs to remote sediments in the South Pacific of the Southern Ocean through long-range aeolian dust transport (Lamy et al., 2014; Jaeschke et al., 2017), and in Arabian Sea (Huang et al., 2007). Submerged and floating aquatic plants generally display enhanced mid-chain C23 and C25 n-alkanes, whereas emergent aquatic plants have n-alkane distributions sim⇑ Corresponding authors at: Anhui Province Key Laboratory of Polar Environment and Global Change, School of Earth and Space Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China (X. Liu) and Department of Earth, Environmental and Planetary Sciences, Brown University, RI02912, USA (Y. Huang). E-mail addresses: [email protected] (X. Liu), [email protected] (Y. Huang). https://doi.org/10.1016/j.orggeochem.2019.103909 0146-6380/Ó 2019 Published by Elsevier Ltd.

ilar to terrestrial vegetation, typically dominated by the long-chain homologues (>C27; Ficken et al., 2000; Gao et al., 2011; Aichner et al., 2017). Carbon isotopic ratios of long-chain n-alkyl lipids in sediment cores have been used extensively to reconstruct past vegetative assemblages (e.g., C3/C4 plants; Cerling et al., 1993; Huang et al., 2001; Schefuß et al., 2003; Huang et al., 2006, 2007; Eglinton and Eglinton, 2008; Thomas et al., 2014). Hydrogen isotope ratios of individual n-alkanes and n-alkanoic acids in lake and ocean sediments can be used to assess past hydrological changes (Huang et al., 2004, 2007; Hou et al., 2008; Tierney et al., 2011; Sachse et al., 2012; Thomas et al., 2012, 2016; Shanahan et al., 2013). Despite widespread applications, a central question that has never been conclusively addressed in the past 50 years is whether or not non-vascular plant sources may also contribute significantly to the long-chain n-alkyl lipids inventory in aquatic sediments and if so, by what percentage. Tentative evidence that non-vascular plants may account for a significant fraction of long-chain n-alkyl lipids in aquatic sediments abound. For example, the odd-overeven predominance, as evaluated using carbon preference index

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(CPI), of long-chain n-alkanes is often significantly higher in living vascular plant samples (e.g., 4–40; Collister et al., 1994) than in sediments (e.g., 1.4–6.6; Sachse et al., 2004; Duan et al., 2011). Numerous studies have also inferred partial origin of long-chain n-alkyl lipids in marine and lake sediments from microbial sources based on compound specific d13C values (e.g., Gong and Hollander, 1997; Naraoka and Ishiwatari, 2000; Bovee and Pearson, 2014; Summons et al., 2013; van Bree et al., 2018). However, due to the relatively wide range of variability of CPI and d13C values in vascular plants (as well as in microbes), it is generally difficult to demonstrate conclusively if the observed CPI and d13C anomaly is indeed a result of partial microbial contributions in low and middle latitude marine and lake sediments. Previous studies have also shown that microbes (fungi and bacteria) can produce long-chain n-alkanes. For example, several studies reported that some fungal species and aerobic bacteria contain n-alkanes ranging from C14 to C37, with a predominance of n-C27, n-C29 and n-C31 (Oro et al., 1966; Jones, 1969), although these early studies may have significant uncertainties in their sampling approaches and analytical methodologies (as reviewed in Li et al., 2018). Enrichment cultures inoculated with fresh sediment suggest that certain diatoms may produce long-chain (e.g., C28) n-alkanoic acid (Volkman et al., 1980). Recent efforts to clarify this question applied in vitro stable isotope methodology (Tu et al., 2011; Zech et al., 2011; Li et al., 2018). For example, Li et al. (2018) applied a soil incubation experiment with deuterium-enriched water, which showed that anaerobic microbes only produce short-chain n-alkanes, but aerobic microbes can produce long-chain nalkanes (e.g., C29 n-alkane) by up to 0.1% per year, relative to the abundance of initial n-alkanes in soil. Accurate assessment of microbial contributions of long-chain nalkyl lipids to the total sedimentary inventory is important for paleoclimate interpretations. While the isotope approaches discussed above provide strong evidence for the production of longchain n-alkanes by microbes, there has been no study using natural environmental samples to provide conclusive evidence for microbial production of long-chain n-alkyl lipids. It is difficult, or even perhaps impossible, to answer this question from typical lake and marine sediments in low- and mid-latitude regions because of the abundant vascular plants in the surrounding environments which may readily overwhelm inputs of any n-alkyl lipids from non-vascular plant sources. Aquatic sediment samples from sites with little or no vascular plants in the surroundings would be thus ideal for detecting n-alkyl lipids of non-vascular plant sources. Matsumoto et al. (1981) found that long-chain n-alkanoic acids extending from C8 to C40 and with small even-carbon predominance (CPI19-33 ranging from 1.4 to 3.6) were abundant in soils from the Dry Valleys, Victoria Land, Antarctica. Subsequent studies also revealed abundant long-chain n-alkanes dominated by n-C23, n-C25, or n-C27 homologues with small odd-over-even predominance (CPI14-38 ranging from 1.98 to 2.59) and d13C values ranging from about –28‰ to –26‰ in McMurdo Dry Valley soils (Matsumoto et al., 2010). These long-chain n-alkyl lipids were proposed to originate from erosion of ancient sedimentary materials containing vascular plant debris formed during warmer periods in the deep geological past (e.g., Miocene-Pliocene; Matsumoto et al., 1990a, 1990b, 2010). However, the relatively low CPI values of the long-chain n-alkanes and n-alkanoic acids in these soils may signify partial origin from microbial sources, because several studies have shown long-chain n-alkanes from microbial sources do not display high CPI values (Clark and Blumer, 1967; Ladygina et al., 2006). There has been, however, no study of modern lake sediments and aquatic or terrestrial biomasses in the McMurdo Dry Valleys or other high latitude Antarctic sites to determine if longchain n-alkyl lipids are present. Because aquatic sediments, rather than soils, are generally the most common archive for studying

lipid biomarkers, it is particularly important to directly examine distributions and carbon isotopic values of sedimentary longchain n-alkyl lipids. The primary objective of this study is to investigate long-chain n-alkyl lipids in surface sediments from three lakes at high latitude (69–74°S), ice-sheet free sites in East Antarctica. The study sites are located in a region often referred to as a cold desert due to their exceptionally low precipitation (Wynn-Williams, 1990). These aquatic ecosystems are dominated by cyanobacteria and algae as the primary autotrophic organisms (Fumanti et al., 1997; Jungblut et al., 2009), whereas terrestrial ecosystems are characterized by the absence of vascular plants or, at slightly warmer sites, scant presence of moss and lichen. However, our warmest study site (Long Lake, 62°S) from the Antarctic Peninsula differs from our other sites in that scattered moss and lichen, as well as two species of vascular plant (Deschampsia antarctica, or Antarctic hair grass and Colobanthus quitensis) are present, especially in coastal areas (Alberdi et al., 2002). We also performed the first systematic analyses of n-alkyl lipids and their d13C values in modern plant samples, including microbial mats, terrestrial moss and lichen. Four soil samples from our two highest latitude sites are also analyzed as well for comparison. Our data suggest that heterotrophic microbes can be a source of long-chain n-alkyl lipids in aquatic environments.

2. Materials and methods 2.1. Study site and sampling Four study sites including IIL9 and IIL3 ponds in Inexpressible Island (IIL), Daming Lake (DM) and Long Lake (LL) were selected for studying n-alkyl lipids in sediment and aquatic microbial biomass samples (Fig. 1). These lakes and ponds are not influenced by penguin guano based on the field investigation and bulk d13C values. Penguin guanos are characterized by low d13C values of
–29‰ (Liu et al., 2013; Lorenzini et al., 2014), which is much lower than those observed in our study lakes. Two additional ponds, IIL2 and IIL5, were only sampled for aquatic microbial biomass in pond water but not sediments (Fig. 1; Table 1). Three of the sites (IIL9, IIL3 and DM) are located in East Antarctica and have little or no visible terrestrial vegetation around the lakes. All lakes and ponds on Inexpressible Island contained abundant visible algae and microbial biomass at the time of sampling collection. Long Lake (62°120 18.5600 S, 58°580 2.4900 W) is located on Fildes Peninsula, King George Island, South Shetland Islands (Fig. 1a). It is about 250 m long, 60 m wide, 4 m deep and 16 m above present sea level. The annual mean air temperature is –2.1 °C, with an average summer temperature of 1–3.5 °C in December to February. This location has an average annual precipitation of 634.9 mm, and an average annual relative humidity of 88% (Wang and Peter, 2002). The low elevation sites around Fildes Peninsula are covered by abundant lichens and mosses (Supplementary Fig. S1). Penguins including Adélie (Pygoscelis adeliae), Gentoo (P. papua) and Chinstrap (P. antarctica) penguins as well as marine mammals (especially Pinnipedia) are present in the coastal areas (Wisnieski et al., 2014; Roberts et al., 2017). Moss (Sanionia uncinata) and lichen (Usnea aurantiacoatra) samples around Long Lake were also collected for analysis in this study. IIL9 and IIL3 on Inexpressible Island (74°530 18.9800 S, 163°430 26. 3800 E) are small ponds with a maximum depth of about 50 cm during sampling. These ponds are always wet during the summer season due to melt-water stream input. However, they are frozen in the winter time due to extremely low temperatures (average winter temperatures are 21.8 °C in May to October, and average summer temperature were 6.5 °C in December to February, and the

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Fig. 1. Locations of study lakes and sampling sites in Antarctica. (a): Long Lake, Fildes Peninsula, Antarctic Peninsula; (b): IIL9, IIL5, IIL3, and IIL2 ponds, Inexpressible Island, Ross Sea (sediments were collected from IIL9 and IIL3 ponds, and microbial mats were collected from IIL9, IIL5 and IIL2 ponds); (c): Daming Lake, Prydz Bay, East Antarctica. Lake photos of Long Lake and Daming Lake were courtesy of Libin Wu and Yanjun Mei, respectively.

average annual temperature is 16.1 °C with an annual average relative humidity of 42.1%; Ding et al., 2015). Inexpressible Island has an area of approximately 50 km2, and is located in Terra Nova Bay, Ross Sea, East Antarctica (Fig. 1b). The western coast of Inexpressible Island is adjacent to the Nansen Ice Sheet, while the northeastern coast connects with the Hell’s Gate Ice Shelf. Along the western margins of Inexpressible Island there are raised beaches with elevations ranging from 20 m to 30 m above sea level, and are truncated to the west by the Nansen Ice Sheet (Baroni and Orombelli, 1994). A large number of abandoned Adélie penguin colonies can be found on Inexpressible Island (Baroni and Orombelli, 1994; Emslie et al., 2007), and an active colony of about 21 thousand pairs occupies ice-free terrain surrounding Seaview Bay (He et al., 2017). Moss and lichen are extremely rare around the IIL9 and IIL3 ponds, whereas algae and microbial biomass are visibly abundant in the pond water. Some photos of the microbial mats are given in Supplementary Fig. S2. Daming Lake is located on Millor Peninsula, Larsemann Hills, East Antarctica (69°120 S-69°280 S, 76°E-76°300 E; Fig. 1c) and has an area of 0.2 km2, a maximum water depth of 4 m and an altitude of 30 m above sea level. The average annual temperature at this site is 9.8 °C (average temperature is
0 °C in December to February), and the average annual precipitation is 250 mm (Ding et al., 2013). The lake microbial community is dominated by abundant algae, primarily cyanobacteria (Liang et al., 1999), diatoms and green algae (Cynan Ellis-Evans et al., 1998; Sabbe et al., 2004; Hodgson et al., 2005). There are no visible mosses and lichens around the lake. Sediment samples were collected from three sampling trips in February 2008, August 2011 and January 2016, respectively (Table 1). Short sediment cores LL2 (47 cm), DM (63 cm), IIL3

(54 cm) and IIL9 (48.5 cm) were collected from Long, Daming, IIL3 and IIL9 lakes, respectively. IIL3 and IIL9 cores were collected in PVC tubes (about 12 cm diameter) and DM in a 7 cm diameter tube which were pushed or hammered into the sediment. At the time of sampling, Long Lake was frozen with an ice thickness of
1 m. A Jiffy Ice Drill was used to drill ice on Long Lake. Core LL2 was collected using a KC Model-B sediment sampler. After transport back to the laboratory, sediment cores (IIL3, IIL9 and LL2) were sectioned at 0.5 cm intervals, whereas the DM core was sectioned at 1 cm intervals. We choose the top 6 cm of IIL3 and IIL9 cores including 4 discrete samples at 0–0.5, 1.5–2, 3.5–4 and 5.5–6 cm sections for analyses, the top 5 cm of the DM (1–2, 2–3, 3–4, and 4–5 cm) and LL2 (0.5–1, 1–1.5, 1.5–2, 2.5–3 and 4.5–5 cm) sediment cores including four and five discrete samples, respectively for analyses in this study. All the subsamples were kept at 20 °C prior to analysis. For comparison with our sediment samples, we also collected and analyzed various aquatic and terrestrial biomass, as well as soils from the different sites (Table 1). These end-member environmental samples were collected from two sampling trips in January 2014 and January 2016, respectively (Table 1). Specifically, four moss samples (Sanionia uncinata, Syntrichum magellanica and Bryum argenteum) and four lichen samples (Usnea aurantiacoatra) were collected from Fildes Peninsula, Campo Icarus and Cape Irizar (Table 1). Soil under moss and natural soil samples were also collected at Inexpressible Island, Campo Icarus and Cape Irizar (Table 1). Microbial mats samples were collected from IIL9, IIL5 and IIL2 ponds, Inexpressible Island (Table 1). The microbial samples formed dense mats (see photos in Supplementary Fig. S2) are a mixture of cyanobacteria and algae (primary producers) on the water surface and were deposited along the lake ashore as

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Table 1 The location, collection time and the type of environmental samples collected around the study areas. Sample i.d.

Type

Collection time

Location

D1 D2 D4 D5 TX TY T13 TX19 IIL9 IIL5 IIL2 IIS4 IIS5 T10S TX19S Long Lake Daming Lake IIL3 pond IIL9 pond

Lichen Lichen Lichen Lichen Moss Moss Moss Moss Microbial mat Microbial mat Microbial mat Soil Soil Soil under moss Soil under moss sediments sediments sediments sediments

2014/January 2014/January 2016/January 2016/January 2014/January 2014/January 2016/January 2016/January 2016/January 2016/January 2016/January 2016/January 2016/January 2016/January 2016/January 2011/August 2008/February 2016/January 2016/January

Fildes Peninsula, Antarctic Peninsula Fildes Peninsula, Antarctic Peninsula Cape Irizar, Ross Sea, Antarctica Cape Irizar, Ross Sea, Antarctica Fildes Peninsula, Antarctic Peninsula Fildes Peninsula, Antarctic Peninsula Campo Icarus, Ross Sea, Antarctica Cape Irizar, Ross Sea, Antarctica Inexpressible Island, Ross Sea, Antarctica Inexpressible Island, Ross Sea, Antarctica Inexpressible Island, Ross Sea, Antarctica Inexpressible Island, Ross Sea, Antarctica Inexpressible Island, Ross Sea, Antarctica Campo Icarus, Ross Sea, Antarctica Cape Irizar, Ross Sea, Antarctica Fildes Peninsula, King George Island, Antarctic Peninsula Millor Peninsula, Larsemann Hills, east Antarctica Inexpressible Island, Ross Sea, Antarctica Inexpressible Island, Ross Sea, Antarctica

pond water retreated (Fumanti et al., 1997; Jungblut et al., 2009). We hand-picked microbial mats from the water surface and placed them in a plastic sample collection bag. No visible algal biomass was present in Long Lake at the time of sampling so we did not collect algal samples from this lake. 2.2. Sample analysis 2.2.1. Lipid biomarker extraction Lipid analysis in this study follows the general procedure described in Hou et al. (2008). Briefly, all samples were freezedried, then homogenized and ground after removing rock fragments. Samples (
1–2 g) were weighed prior to extraction. Lipids were extracted three times with a Dionex accelerated solvent extractor (ASE 200) using dichloromethane (DCM): methanol (9:1, v/v) at 120 °C and 1200 psi. The total lipid extract was separated into acid and neutral fractions using aminopropylsilyl gel columns. Neutral compounds were eluted with DCM/isopropanol (2:1, v/v), followed by carboxylic acids with 4% acetic acid in ether. The neutral fraction was dried using nitrogen at room temperature and ultrasonically dissolved in hexane. The samples were eluted with hexane, DCM, hexane/ethyl acetate (3:1, v/v), and MeOH, respectively. The alkane fraction eluted with hexane was directly analyzed using gas chromatography with flame ionization detection (GCFID). The carboxylic acid fraction was methylated in anhydrous 5% HCl in methanol at 60 °C overnight. Hydroxyl-carboxylic acids were removed from the methylated acid fractions via silica gel flash column using DCM as the eluent (Thomas et al., 2014). Methyl esters of carboxylic acids were analyzed using GC-FID. Compounds were quantified using an internal standard (hexamethylbenzene) and by the proportional relation of the peak areas for each biomarker. The absolute concentrations of long-chain n-alkanes and nalkanoic acids are presented in Supplementary Tables S1–S4. 2.2.2. GC and GC–MS analyses Samples were quantified using an Agilent 6890 + GC-FID system with an Agilent DB-1 column (30 m  3.2 mm  0.17 lm film thickness). The GC oven temperature program was as follows: initial temperature was 50 °C, followed by heating at 20 °C min1 to 255 °C, then heating at 3 °C min1 to 300 °C, and finally heating at 10 °C min1 to 320 °C, where it was held for 15 min. Hydrogen was used as the carrier gas, with a flow rate of 36 cm s1. The same GC column and oven temperature program were used for GC–MS analyses of selected samples for compound identification, which were performed on an Agilent 6890 GC interfaced to a 5973N

quadruple mass spectrometer. MS ionization energy was 70 eV. The scan range was m/z 50–600. Standard n-alkanes (C21, C23, C25, C27, C29) and fatty acid methyl esters (FAME: C16, C18, C22, C24, C28) were analyzed alongside sample compounds and used for verification of compound identification. Further confirmation of compound identification was carried out using GC–MS by comparison of mass spectra with those in the library of National Institute of Standards and Technology (NIST), as reported previously (Gao et al., 2011, 2012). 2.2.3. Carbon isotope analysis The d13C values of individual n-alkanes and FAMEs were determined using an HP 6890 gas chromatograph interfaced to a Thermo Finnigan Delta V plus isotope ratio mass spectrometer through a combustion reactor (Thomas et al., 2014). Standards with known d13C values (mixture of C16, C18, C22, C24 and C28 FAMEs for n-alkanoic acids) were measured after every sixth sample injection to monitor instrument stability. The accuracy of carbon isotopic values was verified by measuring the commercially available Indiana n-alkane isotopic standard mixture (Gao et al., 2014). The d13C values for FAMEs were corrected for the isotopic contribution of the carbon in the methyl group added during methylation (Huang et al., 2002). Stable isotope abundances were expressed in d notation as the deviation from the standard in parts per thousand (‰), d13C = [(Rsample / Rstandard) – 1]  1000, where R is the 13C/12C ratio, and the Rstandard value was based on Vienna Pee Dee Belemnite (V-PDB). The average standard deviation of duplicate analyses was < 0.4‰. Organic carbon isotope analysis of acid-treated (1 mol L1 HCl) bulk sediment and plant samples was performed using the sealed tube combustion method. Sediment and plant samples and standards were fully combusted and CO2 gas was separated by a ‘‘purge and trap” adsorption column and passed to an isotope ratio mass spectrometer (IRMS) for analysis. The analytical precision (standard deviation) for the organic carbon isotopic measurements was better than ± 0.1‰. 3. Results 3.1. Distribution of n-alkyl lipids in modern plant biomass and soil samples 3.1.1. Microbial mats We analyzed a total of three microbial mat samples collected from IIL9, IIL5 and IIL2 ponds, respectively (Table 1). The

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n-alkanes in these three microbial mat samples have similar carbon chain length distributions ranging from C14 to C29, peaking at C17 short-chain length (Fig. 2a; Supplementary Fig. S3). Longchain (>C23) n-alkanes are characterized by relatively low carbon preference index (CPI23-29, shown as odd-over-even predominance) values, with a mean value of 3.9 ± 2.6. The n-alkanoic acids display similar distributions among the three microbial mat samples. All samples are dominated by C16 n-alkanoic acid (palmitic acid), followed by C18 n-alkanoic acid, and then C14 n-alkanoic acid. Long-chain n-alkanoic acids, primarily C24, C26 and C28, are generally low in abundance (Fig. 2b). In the three samples containing long-chain n-alkanoic acids, the average CPI24-30 (even-over-odd predominance) value for C24 to C30 n-alkanoic acids is
6.2 ± 1.8. 3.1.2. Lichens Four lichen samples from Cape Irizar and Fildes Peninsula were analyzed (Table 1). The n-alkanes in lichen are characterized by a bimodal distribution with maximal carbon number at C22 and C27 and C29, and carbon number range from C17 to C33 (Fig. 2c; Supplementary Fig. S3). There is no obvious odd-over-even carbon preference; the average CPI index for C23 to C33 n-alkanes is 1.5 ± 0.3. This n-alkane distribution, with predominance of longer chain

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n-alkanes in the lichen samples, differs from those in microbial mats. The n-alkanoic acids in lichen are similarly dominated by C16 and C18 n-alkanoic acids as microbial mats, but the longer chain n-alkanoic acids are more abundant (Fig. 2d). The long-chain nalkanoic acids are dominated by C24 and C28 homologues, with strong even-over-odd predominance (CPI24-32
5.0 ± 1.8). 3.1.3. Moss and soil under moss Four moss samples from Cape Irizar, Campo Icarus and Fildes Peninsula, and two soil samples from moss covered sites on Cape Irizar and Campo Icarus were analyzed (Table 1). The n-alkanes in moss and soil under moss are similar, characterized by bimodal distribution with maximal carbon number at C27 and C29, and the carbon number ranges from C17 to C33 (Fig. 2e and g; Supplementary Fig. S3). The average CPI index for C23 to C33 n-alkanes in moss and soil under moss was 8.3 ± 5.2 and 4.1 ± 0.6, respectively, which is higher than those lichen samples. The n-alkanoic acid distributions in moss and soil under moss were similar and dominated by C16 and C18 n-alkanoic acids as in lichen, but the long-chain nalkanoic acids were relatively more abundant in moss than in lichen (Fig. 2d, f, h). The longer chain n-alkanoic acids are dominated by C24 and C28 homologues, with predominant even-over-odd carbon

Fig. 2. Normalized relative abundance of n-alkanes and n-alkanoic acids in environmental samples. Samples of three lake microbial mats, four lichens, four mosses, two soils under moss and two natural soil samples were collected from the Antarctic Peninsula and Ross Sea region, East Antarctica. The error bars are the 1r standard deviations of measured relative abundances in the environmental samples. The long-chain n-alkanes (>C21) has been enlarged 5 and 10 times in soil and microbial mat samples, respectively for easy visualization. Similarly, the long-chain n-alkanoic acids (>C20) has been enlarged 10 in lichen and 20 in soil and microbial mats.

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preference (CPI24-32
7.3 ± 6.0 and 7.4 ± 3.5 for moss and soil under moss). 3.1.4. Additional soil samples We analyzed two additional soil samples from Inexpressible Island having little terrestrial vegetation (Table 1). The n-alkanes in soil have carbon chain lengths ranging from C17 to C29, with maximal carbon numbers at C17 and C20 (Fig. 2i; Supplementary Fig. S3). Long-chain (>C23) n-alkanes are generally lower in relative abundance than in the soil under moss. The n-alkanoic acids in soil are also dominated by C16 and C18 n-alkanoic acids, but long-chain n-alkanoic acids are minor (Fig. 2j). The long-chain even n-alkanes and odd n-alkanoic acids were extremely low in abundance (virtually invisible in Fig. 2j). 3.2. Characteristics of n-alkyl lipid distributions in lake sediments 3.2.1. Long Lake sediments The top 5 cm of LL2 core (47 cm long) including five separate sediment samples were analyzed separately for compound distributions. Because we found that the top 5 cm sediment subsamples display similar compound distributions and isotopic ratios, we averaged all results for Long lake samples and provided 1r standard deviations (Fig. 3). The same averaging was also done for samples from all other lakes discussed below. The n-alkanes in Long Lake show an abundance of C23 with no predominant oddover-even carbon preference (Fig. 3a; Supplementary Fig. S4). The average CPI index for C23 to C33 n-alkanes is 1.6 ± 0.2. The nalkanoic acids in Long Lake sediments are characterized by a bimodal distribution with maximal carbon numbers at C16 and

C28, respectively (Fig. 3b; Supplementary Fig. S4). Long-chain n-alkanoic acids are abundant with a strong even-over-odd predominance (CPI24-30
4.8 ± 0.7). 3.2.2. IIL3 and IIL9 lake sediments The n-alkanes in IIL9 were characterized by a bimodal distribution peaking at short-chain (C17 and C19) and C27, respectively (Fig. 3c; Supplementary Fig. S4). However, n-alkanes in IIL9 had a different chain length distribution than those from IIL3, as shown by the relatively lower abundance of long-chain n-alkanes (Fig. 3e; Supplementary Fig. S4). Samples from these two ponds display similar CPI values for long-chain (C23 to C29) n-alkanes in IIL3 (CPI23-29
3.2 ± 0.6) and IIL9 (CPI23-29
3.8 ± 0.4). The nalkanoic acids in both lakes have similar distribution patterns, with carbon chain lengths ranging from C14 to C30 and maximizing at C16 and C24 n-alkanoic acid (Fig. 3d and f; Supplementary Fig. S4). Long-chain n-alkanoic acids are also abundant with strong evenover-odd predominance (CPI24-30
4.7 ± 0.8 and 4.1 ± 1.2 for IIL3 and IIL9, respectively), the CPI for n-alkanoic acids in IIL3 and IIL9 is similar to LL. 3.2.3. Daming Lake sediments The distribution patterns of n-alkanes in Daming Lake differ from the other three lakes, with carbon chain length maximizing at C23 and C27, and short-chain (C17–C19) n-alkanes are in low abundance (Fig. 3g; Supplementary Fig. S4). The n-alkanoic acids in Daming Lake are similarly dominated by C16 and C24 as in IIL3 and IIL9 lakes (Fig. 3h; Supplementary Fig. S4). The CPI values for long-chain (C23–C33) n-alkanes and n-alkanoic acids (C24–C30) were 2.1 and 1.7 ± 0.1, respectively, and lower than the other three lakes.

Fig. 3. Normalized average relative abundance of n-alkyl lipids in lake sediments. The top 5 cm of surface sediment at Long and Daming Lakes contain five and four discrete samples, respectively; the 6 cm surface sediments at IIL9 and IIL3 ponds contain four discrete samples. The error bars are the 1r standard deviations of measured relative abundances in the discrete lake sediment samples. The long-chain n-alkanes (>C21) has been enlarged 5 in IIL3 Lake sediments for easy visualization.

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3.3. Carbon isotopic values of n-alkyl lipids in environmental samples and lake sediments 3.3.1. Microbial mats We analyzed bulk d13C values for the three microbial mat samples collected at Inexpressible Island (–12.6‰, –14.1‰ and –16.0‰; Table 2). We were unable to obtain d13C values for n-alkanes from these microbial samples because the long-chain n-alkane concentrations in these samples were too low, and short-chain n-alkanes co-eluted with large amounts of unresolved compound mixtures (UCM; Supplementary Fig. S3). The n-alkanoic acids of microbial mat samples were dominated by C16 and C18 n-alkanoic acids, but long-chain n-alkanoic acids were present in sufficient concentration for isotopic measurements. We measured short and long-chain n-alkanoic acids in separate sample injections in order to adjust signal amplitudes and ensure best measurement accuracy (The 1r standard deviations of duplicate analyses were < 0.4‰). The d13C values of n-alkanoic acids in these three microbial mat samples differ among IIL9, IIL5 and IIL2 (Table 2; Supplementary Table S5). C14, C16 n-alkanoic acids were about –18‰ at IIL2 and IIL9, but –26‰ to –24‰ at IIL5. C18 n-alkanoic acid had d13C values of approximately –26‰ at IIL2 and IIL9, but 3‰ lower in IIL5. C20 to C24 n-alkanoic acids had similar d13C values ranging from –22‰ to –18‰ for IIL2 and IIL9, but the values for C20 to C28 n-alkanoic acids were from –29‰ to –26‰ for IIL5. The d13C values of long-chain C26 to C30 n-alkanoic acids in IIL2 and IIL9 had the highest d13C values of all n-alkanoic acids, ranging from –18‰ to –13‰ (Table 2; Supplementary Table S5). 3.3.2. Modern terrestrial biomass (lichen and moss) These samples represent the majority of terrestrial photosynthetic plants and biomass at our study sites. We measured the bulk d13C values of two lichen and two moss samples, respectively, with average values: –24.9‰ and –26.3‰ (Table 2). We were able to measure d13C values for the entire suite of n-alkanes from C17 to C33 (Fig. 4a; Supplementary Fig. S3) and n-alkanoic acids from C14 to C32 (Fig. 5b; Supplementary Fig. S3). However, we were unable to obtain the d13C values of n-alkanoic acids in the two lichen samples due to the concentrations being too low. The n-alkane d13C values in lichen and moss are broadly similar and both display a trend of decreasing d13C values with increasing carbon chain length (Fig. 4a). The short-chain C19 and C20 n-alkanes have an average d13C value of about –30‰, C21 to C23 n-alkanes
–33‰ to –32‰, whereas longer chain n-alkanes range from about –38‰ to –34‰ (Fig. 4a; Table 2; Supplementary Table S6). The n-alkanoic acids in lichen and moss also exhibit carbon isotopic differences with longer chain n-alkanoic acids showing lower d13C values (Fig. 5b; Supplementary Table S5). Specifically,

C14–C20 n-alkanoic acids have d13C values of about –32‰ to –30‰, whereas long-chain (C22–C32) n-alkanoic acids have average values of
–37‰ to –34‰ (Table 2). 3.3.3. Soils The average bulk d13C value of the two soils collected from nonvegetated terrestrial sites is –26.3‰; the mean value of two soils under moss is –24.7‰ (Table 2). Individual C19 to C23 n-alkanes from two soils of non-vegetated sites are about –27‰ (C17 is about –31‰), but longer chain n-alkane abundances were too low for carbon isotopic measurements (Fig. 4b). The n-alkanes from soil under moss appear to generally mimic the d13C trend of moss samples, showing a trend of carbon isotopic depletion with increasing carbon numbers (e.g., C19 and C20 is
–28‰, C20 to C23
–32‰; and longer chain n-alkanes
–38‰ to –35‰; Fig. 4b; Table 2; Supplementary Table S6). The n-alkanoic acids in two types of soils show more similar d13C values than n-alkanes (Fig. 5c; Table 2; Supplementary Table S5), with shorter chain n-alkanoic acids (C14–C18) at about –30‰, and increasing carbon isotopic depletion with increasing chain length to as low as –38‰. 3.3.4. Sediments The average bulk d13C values of Long, Daming, IIL3 and IIL9 Lake sediments are –17.9 ± 1.3‰, –10.7 ± 0.2‰, –13.4 ± 0.3‰ and –12.0 ± 0.2‰ (Table 2), respectively. Because short-chain n-alkanes co-eluted with large amounts of unknown compounds, the d13C values for C17–C23 n-alkanes were not obtained. For C24–C33 n-alkanes, the d13C values in Long Lake sediments are about –30‰, with relatively small differences among different carbon chain lengths. However, d13C values of n-alkanes at the other three lakes in colder regions and higher latitudes (Daming, IIL3 and IIL9 lakes) show much higher values and are more variable for different chain length homologues (Fig. 4c; Table 2; Supplementary Table S7). For example, C25–C27 n-alkanes have d13C values as high as –16‰ to –13‰. For C28 and C29 n-alkanes, d13C values were about –20‰. We were only able to obtain d13C data for C27 and C29 n-alkanes for IIL3, where the values were about –20‰ to –19‰. The d13C values of C24–C29 n-alkanes in Daming were similar to IIL9, C30 and C31 were about –24‰ (Fig. 4c; Table 2; Supplementary Table S7). The n-alkanoic acids show even more distinctive d13C distributions with chain length variations (Fig. 5d; Table 2; Supplementary Table S8). In contrast to various terrestrial biomass samples and soils, n-alkanoic acids from all lakes display increasing d13C values with increasing carbon chain length. The overall lowest d13C values were found in Long Lake, as is the case for n-alkanes. Daming Lake and IIL9 exhibit an overall 10‰ higher d13C values than Long Lake. The d13C values of C26–C28 long-chain n-alkanoic acids in Daming Lake and IIL9 are –15‰ to –14‰. IIL3 d13C values were in most

Table 2 The average carbon isotope values for diffferent chain length ranges of n-alkyl lipids and sample types. The relative 1r standard deviations of all samples are given in parentheses. ‘‘–” represents that the concentrations are too low or co-eluted with large amounts of unresolved compound mixtures for d13C measurements. TOC refers to d13C values of bulk organic matter. Sample type

n-alkanes

n-alkanoic acids

TOC

C17–C20

C21–C26

C27–C33

C14–C19

C20–C25

C26–C32

Environmental samples Lichen Moss Soil under moss Soil Microbial mat

–28.8(3.6) –27.3(2.5) –27.2(1.6) –28.5(2.1) –

–33.2(2.3) –34.5(1.0) –33.3(1.2) –26.3(0.4) –

–35.1(0.9) –37.6(2.2) –34.4(2.1) – –

–28.7(1.0) –31.5(1.5) –29.2(1.0) –28.5(1.8) –22.2(2.6)

–31.9(0.4) –34.1(1.5) –33.3(1.1) –33.0(1.3) –22.1(3.0)

–37.1(2.2) –36.5(1.0) –35.8(1.6) –34.0(0.7) –17.6(2.4)

–24.9(0.1) –26.3(2.2) –24.7(0.8) –26.3(0.3) –14.2(1.7)

Lake sediments IIL3 lake IIL9 lake Daming lake Long lake

– – – –

– –14.5(2.0) –15.7(0.6) –28.1(1.3)

–18.7(0.2) –16.9(3.9) –20.7(5.8) –29.2(0.9)

–22.7(1.8) –23.3(1.9) –19.8(1.3) –30.0(1.3)

–20.6(1.4) –19.4(3.2) –14.9(2.6) –24.5(3.2)

–18.3(2.0) –14.5(2.1) –13.3(1.4) –21.7(1.3)

–13.4(0.3) –12.0(0.2) –10.7(0.2) –17.9(1.3)

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X. Chen et al. / Organic Geochemistry 138 (2019) 103909

Fig. 4. The d13C values of n-alkanes from environmental samples and sediments. (a): the average d13C values of n-alkanes in terrestrial biomass samples (moss and lichen). (b): the average d13C values of n-alkanes in two soil samples and two soilunder-moss samples. (c) the average d13C values of n-alkanes in lake sediments. Error bars represent 1r standard deviation of all measured data. The unconnected lines and no dots are samples that were not measured for carbon isotope ratios.

cases between the Daming Lake/IIL9 and Long Lake n-alkanoic acids. 4. Discussion 4.1. Comparison of distribution and d13C of n-alkyl lipids in lichen and moss samples between Antarctica and mid-latitude regions In our study, the chain-length maxima of n-alkanes in moss is at C27 and C29, and is longer than those reported in Sphagnum moss in lower latitude regions, which generally maximize at C23 (and occasionally C25) n-alkane (Nichols et al., 2009; Ortiz et al., 2011; Bush and McInerney, 2013; Huang et al., 2012a, 2012b). Among the long-chain n-alkanoic acids, C24 and C28 n-alkanoic acids are the

Fig. 5. The d13C values of n-alkanoic acids from environmental samples and sediments. (a): the d13C values of n-alkanoic acids in three microbial mat samples. (b): the average d13C values of n-alkanoic acids in terrestrail biomass samples (lichen and moss). (c) the average d13C values of n-alkanoic acids in two soil samples and two soil-under-moss samples. (d) the average d13C values of n-alkanoic acids in lake sediments. Error bars represent 1r standard deviation of all measured data. The unconnected lines and no dots were not measured for carbon isotope ratios.

X. Chen et al. / Organic Geochemistry 138 (2019) 103909

dominant component in the Antarctic moss samples we collected. These features also differ from previous studies that found C22 and C24 n-alkanoic acids were most abundant in temperate moss samples (Nichols et al., 2009; Huang et al., 2012a, 2012b). Notably, the species of moss in this study including Sanionia uncinata, Syntrichum magellanica and Bryum argenteum are distinct from Sphagnum in lower latitude regions. Our results suggest that the relative abundance of long-chain n-alkyl lipids are higher in Antarctic moss than those from lower latitude regions, which may result from the influence of different species or biosynthetic variations in colder and dryer environments. For example, there have been many reports of plants synthesizing longer chain n-alkyl lipids to prevent water loss at dryer conditions (Dodd and Afzal-Rafii, 2000; Nichols et al., 2006). The distributions of n-alkyl lipids in Antarctic lichen are also different from those reported in low latitude lichen. C29 and C23 n-alkanes were most abundant with a strong odd-over-even carbon preference (CPI23-33 ranges from 3.5 to 8.2) in five Coticolous lichen samples collected from western Hubei Province (Huang et al., 2012a, 2012b). In our study of Antarctic lichen, C22, C27 and C29 n-alkanes are the most abundant with little odd-over-even carbon preference (Fig. 2c; Supplementary Fig. S3). The distributions of n-alkanoic acids in Antarctic lichen are nevertheless similar to those of fifteen lichen species collected from a wide range of temperate and tropical climates, all of which show that short-chain nalkanoic acids (C16 and C18) are the most abundant, but long-chain n-alkanoic acids (>C24) are relatively low in abundance (Vu et al., 2016). The species of lichen in this study is Usnea aurantiacoatra which differs from previous studies in lower latitude regions. Higher proportions of long-chain n-alkyl lipids in Antarctic lichen than those reported for lower latitude samples may also originate from similar mechanisms as discussed above for mosses. The d13C values of n-alkanes in lichen and moss are broadly similar and both display a trend of lower d13C values (
–40‰ to –29‰ in moss and
–36‰ to –30‰ in lichen) with increasing carbon chain-length (Fig. 4a), which falls into the range of C3 plants (Chikaraishi and Naraoka, 2007). The d13C values of long-chain nalkanes (C23 to C31) in lichen sampled from the western Hubei Province vary between –38.3‰ to –31.4‰ (Huang et al., 2012a, 2012b) and hence show similar values to our study. Moreover, the d13C values of long-chain n-alkane (C23 to C31) in Antarctic moss are similar to those of four moss species in southern and southwest China reported by Huang et al. (2010, 2012a, 2012b) and three Sphagnum species in Europe (Brader et al., 2010; van Winden et al., 2010). Our results suggest that Antarctic lichen (Usnea aurantiacoatra) and moss (Sanionia uncinata, Syntrichum magellanica and Bryum argenteum) have in general similar d13C values as their lower latitude counterparts. 4.2. Comparison of distribution characteristics and d13C of n-alkyl lipids from microbial mats in Antarctica lakes and those from lower latitude regions The distributions of n-alkanes and n-alkanoic acids in our microbial mat samples (Fig. 2a and b) show maxima at shortchain lengths, which is similar to those reported from three meltwater ponds from the McMurdo Ice Shelf, where cyanobacteria were the primary producers in microbial mats (Matsumoto et al., 1993; Jungblut et al., 2009). However, there is no previous report of individual d13C values of individual n-alkanoic acids from microbial mats in Antarctic lakes. The bulk d13C values of organic matter in microbial mats collected from IIL2, IIL5 and IIL9 ponds vary from –16‰ to –12.6‰, similar to other shallow lake microbial mats collected from the McMurdo Dry Valleys and South Shetland Islands (Lawson et al., 2004). The values are
5–10‰ higher than microbial mats and

9

terrestrial algae from deeper lakes in McMurdo Dry Valleys (Wharton et al., 1993; Lee et al., 2009). The high d13C values of microbial mats in our samples may be related to a CO2 diffusionlimited environment caused by insufficient CO2 supply and high photosynthetic efficiency in our shallow lakes (Bird et al., 1991; Wei et al., 2016). Interestingly, n-alkanoic acids with different chain lengths have different d13C values with longer chain nalkanoic acids having higher d13C values in our samples (Fig. 5a). The carbon isotopic fractionation of n-alkanoic acids during biosynthetic chain elongation processes generally lead to more 13 C-depletion with increasing chain length for acetogenic lipids (Monson and Hayes, 1980). Therefore, increasing d13C values with increasing n-alkanoic acid chain length observed in our samples are unlikely due to biosynthetic fraction during chain elongation processes. We propose that long-chain n-alkanoic acids may be derived from heterotrophic microbes, as we will discuss below in more detail for lake sediment samples. 4.3. Comparison of distribution and carbon isotopic characteristics of n-alkyl lipids from soils in Antarctica Long-chain n-alkanes (>C23) and n-alkanoic acids (>C24) in soil under moss exhibit similar trends as those in moss and consistent with their similar origins (Fig. 2e–h). Previous studies also reported that lichen and/or vascular plant debris in some lake sediments and soils from the Dry Valleys of Victoria Land and McMurdo contain amounts of long-chain n-alkanes (>C23) with no obvious oddover-even carbon preference (CPI15-35
1–2) and n-alkanoic acids (>C20) with no obvious even-over-odd carbon predominance (CPI10-30
1–2) (Matsumoto et al., 1981, 1990a, 1990b, 2010). However, soils from our non-vegetated sites contain much lower relative abundances of long-chain n-alkanes (>C23) and nalkanoic acids (>C24) (Fig. 2I and j), compared to soils directly under moss, and they may have more complex origins as discussed below based on their carbon isotopic values. Note that due to the extremely low concentrations of long-chain even carbon nalkanes, and long-chain odd carbon n-alkanoic acids, we did not calculate CPI values for soils from non-vegetated sites, The d13C values of individual C19 to C23 n-alkanes from two soils at non-vegetated sites were about –27‰ (C17
–31‰; Fig. 4b; Table 2; Supplementary Fig. S6), similar to soils from McMurdo Dry Valleys (Matsumoto et al., 2010). These short-chain nalkanes in soils may partially originate from debris of microalgae and cyanobacteria in soils and/or dried up lake beds. The longchain n-alkanoic acids in our soil samples show similar d13C values to those of the moss (Fig. 5), but
4‰ lower d13C values than those previously reported for soil samples from the McMurdo Dry Valleys by Matsumoto et al. (2010). The authors hypothesized that long-chain n-alkanoic acids in Dry Valley soils originated from plant debris deposited in the past warm periods such as the Miocene and Pliocene. Our results suggest a more complex origin for these long-chain n-alkanoic acids in soils from coastal Antarctica: i.e., an origin from modern moss, either from local sources or from aeolian transport of moss from elsewhere in Antarctica. 4.4. Microbial origin of long-chain n-alkanes in lake sediments Abundant long-chain n-alkanes (>C23) with high d13C values (up to –12‰) found in IIL9, IIL3 and Daming Lake sediments (Fig. 6a; Table 2; Supplementary Table S7) are in sharp contrast to those in moss, lichen and soil samples discussed above. There are four probable sources for long-chain n-alkanes in these lakes: (1) long-range aeolian transportation of dust from adjacent vegetated land masses; (2) terrestrial moss and lichen on or around Antarctica; (3) algae living in microbial mats or in the lake; (4) hetero-

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X. Chen et al. / Organic Geochemistry 138 (2019) 103909

trophic microbes decomposing dead primary producers living in the lakes. Long-chain n-alkanes in remote sediments (
55–65°) of the southern Pacific Ocean are considered biomarkers for terrestrial vascular plant waxes transported by aeolian dust to remote sediments (Lamy et al., 2014; Jaeschke et al., 2017). Long-range aeolian transportation of dust from adjacent vegetated land masses and petrogenic sources could thus be a possible source for long-chain n-alkanes in Antarctica. However, Bendle et al. (2007) found that the long-chain n-alkanes (C29 and C31) in the southernmost Southern Ocean aerosol samples are characterized by low d13C values (–37‰ to –30.8‰) consistent with the dominance of C3 plants in high latitude regions. Fossil fuel combustion has been found to be an important source of long-chain n-alkanes in aerosols during winter in northern Switzerland (Nelson et al., 2018). However, the exceptionally high d13C values of long-chain n-alkanes in our study lakes and ponds suggest that any input from petrogenic sources or adjacent vegetated land masses is likely to be negligible. Similarly, the d13C values of long-chain n-alkanes in IIL9, IIL3 and Daming Lake sediments were about 15–20‰ higher than in terrestrial biomass (lichen and moss) and soil under moss measured in our study (Fig. 4). Thus, moss and lichen are likely not the main source for long-chain n-alkanes in IIL9, IIL3 and Daming Lake. Moreover, the long-chain n-alkanes in microbial mats are minor components and can also be neglected (Fig. 2a). The exception here is for Long Lake, where the sediment long-chain nalkanes display lower d13C values that are similar to moss and lichen growing around the lake (Fig. 4). Carrizo et al. (2019) also found abundant long-chain n-alkanes with relative low d13C values (
–30‰ to –28‰) in two large lake surface sediments from Fildes Peninsula, Antarctica, indicating the dominant source of long-chain n-alkanes derived from biogenic materials such as moss and lichen

living around lakes. This suggests that moss and lichen may become important sources for long-chain n-alkanes in Long Lake – our warmest and wettest settings. We note, however, that long-chain n-alkanes in Long Lake sediments display little oddover-even predominance (Fig. 3a). While modern moss longchain n-alkanes show strong odd-over-even predominance (Fig. 2e), the maximal chain length in moss and lichen is longer (Fig. 2c and e). It has been widely observed in aquatic sediments (from middle and low latitudes), that the CPI values of longchain n-alkanes (1.4–6.6; Sachse et al., 2004; Duan et al., 2011) are significantly lower than those found in living plants (4.3–40.3; Collister et al., 1994). However, due to the relatively wide range of variability of CPI values in plants, the reason for such discrepancy between plants and sediments has never been satisfactorily addressed. From our data here, it is likely that such discrepancy originates from microbial inputs during the transport and sedimentation process of these long-chain n-alkyl lipids to aquatic sediments. Our results suggest sources other than modern lichen and moss could also contribute to Long Lake long-chain nalkanes. The most plausible source for long-chain n-alkanes in our study lakes in Antarctica is heterotrophic microbes decomposing dead cyanobacteria and algal biomass in the lakes. It is expected that these decomposers would have d13C values
1‰ higher than the d13C values of their food sources (Hullar et al., 1996; Sun et al., 2005). The bulk biomass d13C values of microbial mats we collected from our study lakes are in the range of –16.0‰ to –12.6‰ (Table 2). These values readily reconcile with the extraordinarily high d13C values for long-chain n-alkanes in our study lakes (with the exception of Long Lake). Our proposal is consistent with recent results based on hydrogen isotopic values of soil long-chain nalkanes (Zech et al., 2011; Li et al., 2018). We also note the relatively low CPI values in our lake sediment samples (Fig. 3), which also support a microbial origin of these compounds. We can further quantify the relative input sources of long-chain n-alkanes in our lake sediments, using a binary isotope mass balance model.

d13 C s ¼ f  d13 C a þ ð1  f Þ  d13 C t

Fig. 6. The d13C values of n-alkanes from sediments and the percentage of n-alkanes from aquatic sources calculated using a carbon isotope binary mixing model. (a): the average d13C values of n-alkanes in lake sediments. (b): the percent of n-alkanes from aquatic sources (%). Error bars in (a) represent 1r standard deviation of all measured samples. Error bars in (b) represent uncertainty in the resulting estimates of aquatic source of long-chain n-alkanes, based on error propagation calculations. The unconnected lines and no dots were not measured for carbon isotope ratios.

ð1Þ

where d13Cs is the d13C values of n-alkanes from lake sediments, d13Ca is the d13C values of n-alkanes from the heterotrophic microbes, d13Ct is average d13C values in the n-alkanes from terrestrial biomass (lichen and moss), and f is the proportion of n-alkanes from microbial decomposers. It is impossible to know the exact end-member d13C values of heterotrophic microbes and those from terrestrial sources. Therefore, end-members are estimated based on our measured carbon isotopic values of microbial mats in lake water and various (albeit sparse) plants on the adjacent land, assuming common isotopic fractionation values. For examples, previous studies have shown that the d13C values of fatty acids produced by heterotrophic microbes (Escherichia coli and Shewanella putrefaciens) are depleted by
3‰ relative to the biomass they consumed (Monson and Hayes, 1982; Blair et al., 1985; Teece et al., 1999). Bouillon and Boschker (2006) also showed that the d13C values of n-alkyl lipids from heterotrophic microbes are about 4‰ more negative than bulk organic matter, this value is within the range of –3‰ expected from fatty acid synthesis in general (Hayes, 2001). Therefore, we assumed the d13C values of long-chain nalkyl lipids from heterotrophic microbes are depleted by 3‰ to total organic matter. The organic matter in surface sediments is mainly derived from microbial mats, with an average bulk d13C value of – 14.2 ± 1.7‰ (n = 3; Table 2). Thus, the carbon isotopic values of long-chain n-alkyl lipids from heterotrophic microbes are estimated to be –17.2 ± 1.7‰. The d13C values of long-chain n-alkanes produced by different species of terrestrial plants (moss and lichen) we collected and analyzed in this study from different regions show rela-

X. Chen et al. / Organic Geochemistry 138 (2019) 103909

tively similar d13C values. Therefore, we choose the average d13C values of all terrestrial plant (moss and lichen) and 1r standard deviation to represent our terreatrial isotope end-member (C24 = –35.3 ± 1.5‰, C25 = –34.9 ± 1.4‰, C26 = –35.5 ± 2.1‰, C27 = –36.2 ± 2.5‰, C28 = –34.7 ± 2.1‰, C29 = –35.4 ± 2.0‰, C30 = –35.3 ± 2.4‰, C31 = –36.6 ± 2.3‰, C32 = –36.1 ± 3.5‰, C33 = –36.4 ± 4.6‰). Calculations based on this binary carbon isotope model indicate that virtually all C24 to C29 n-alkanes in IIL9, IIL3 and Daming Lake sediments originate from heterotrophic microbial decomposers that reside in the lake water column and sediments. Interestingly, the relative microbial contributions to C31 and C32 n-alkanes in Daming Lake is lower, suggesting heterotrophic microbes in Daming Lake produce lower amounts of these longer chain n-alkanes and terrestrial plant sources increase in relative importance. In our warmest and wettest site, Long Lake, our isotope model suggests less than 50% of long-chain n-alkanes originate from heterotrophic microbes (Fig. 6b). This indicates that terrestrial vascular plants can quickly rise in their importance as sources for longchain n-alkanes as they start to be present around our study sites. We note that Matsumoto et al. (1993) reported long-chain n-alkanes and n-alkanoic acids (>C20) in some lake sediments from the McMurdo Dry Valleys and suggested that microorganisms might be an important source (Matsumoto et al., 1993). However, the relatively low CPI values of the long-chain n-alkanes and n-alkanoic acids in these soils may signify partial origin from microbial sources, because several studies have shown longchain n-alkanes from microbial sources do not display high CPI values (Clark and Blumer, 1967; Ladygina et al., 2006). Our compound-specific carbon isotope data provide a solid support for the microbial origin of long-chain n-alkanes. More accurate measurement of d13C end-members in and around our study lakes will further constrain the microbial contributions. For example, the end-member d13C value of microbial source is calculated based on the averaged bulk d13C value of three microbial mat samples (–17.2 ± 1.7‰). Average d13C values of all terrestrial plants (moss and lichen) and 1r standard deviation are used to represent our terrestrial isotope end-members. We estimate
10% uncertainty in the resulting estimates of heterotrophic microbial source of long-chain n-alkanes (Fig. 6b), based on error propagation calculations (since the binary model is a simple linear mixing, any end-member uncertainties directly translate into result uncertainties; Chave et al., 2004). Such level of uncertainties, however, does not affect our conclusion that long-chain n-alkanes in the study lakes are predominantly derived from microbial sources, particularly heterotrophic microbes. 4.5. Microbial origin of long-chain (>C24) n-alkanoic acids in lake sediments Unlike the relatively low d13C values (
–30‰) of long-chain nalkanes in Long Lake sediments, long-chain n-alkanoic acids display relatively high d13C values of
–22‰. These carbon isotopic values also support a primary origin of these compounds from heterotrophic microbial decomposers. Carbon isotope values of longchain n-alkanoic acids in all terrestrial plant samples we measured (Fig. 5b; Table 2) range from about –38‰ to –30‰, and hence are unlikely to be the main sources of these compounds in our study lakes. Volkman et al. (1980) reported that diatoms (e.g., Melosira and Biddulphia) may have contributed from 30% to 80% of C24 to C28 n-alkanoic acids in an intertidal sandy sediment. The proposal of production of long-chain n-alkanoic acids by diatoms is based on an enrichment culture inoculated with fresh sediments (Volkman et al., 1980). However, long-chain n-alkanoic acids have never been found in isolated diatom cultures and the proposed diatom origin of these long-chain n-alkyl lipids remains tentative (Volkman et al., 1998). The abundance of long-chain n-alkanoic acids is extre-

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mely low in our microbial mat samples (Fig. 2b), which are dominantly composed of cyanobacteria and algae (Fumanti et al., 1997; Jungblut et al., 2009). We thus propose that the contribution of long-chain n-alkanoic acids from diatoms or other microalgae is insignificant in our study lakes and ponds. Using a similar binary isotope mass balance model, we can assess the percentage of terrestrial and aquatic (microbial) sources of longchain n-alkanoic acids. Here we apply the same carbon isotope endmember values as used for long-chain n-alkanes (i.e., heterotrophic microbes: –17.2 ± 1.7‰; terrestrial plants: C24 = –34.2 ± 2.5‰, C25 = –35.6 ± 0.5‰, C26 = –35.9 ± 2.4‰, C27 = –36.9 ± 0.6‰, C28 = 38.2 ± 2.3‰, C29 = –37.5 ± 1.0‰, C30 = –36.1 ± 1.2‰, C31 = –35‰). Our calculated results indicate that the percentage of long-chain n-alkanoic acids (>C24) derived from aquatic sources in our four study lakes is > 50%, even for Long Lake, our warmest and wettest site (Fig. 7b). In particular, the aquatic microbial contribution of long-chain n-alkanoic acids in IIL9, IIL3 and Daming Lakes is approximately 80%. Moreover, long-chain n-alkanoic acids in Long Lake from aquatic sources (>60%) are much higher in concentration than long-chain n-alkanes (<40%; Fig. 6b and 7b), suggesting long-chain n-alkanoic acids are more likely to originate from aquatic microbial (most likely heterotrophic microbes) sources than long-chain nalkanes. As with the n-alkanes, our estimated microbial contributions for n-alkanoic acids have approximately 10% uncertainties based on end-member standard deviations. Analyses of more endmember samples will help reduce uncertainties. Tentative evidence suggesting microbial production of longchain n-alkyl lipids has previously been found in low and mid latitude ocean samples (Nishimura and Baker, 1986; Gong and Hollander, 1997; Naraoka and Ishiwatari, 2000) and lake sediments (Bovee and Pearson, 2014; van Bree et al., 2018; Makou et al, 2018). For example, the carbon isotopic values of long-chain nalkanoic acids (>C24) in open marine sediments of the western North Pacific are enriched in 13C by
6‰ relative to those in the bay and riverine sediments, suggesting marine microalgae and/or heterotrophic microorganisms may be the dominant source of long-chain n-alkanoic acids in the open marine sediments (Naraoka and Ishiwatari, 2000). Makou et al. (2018) also found abundant long-chain n-alkanoic acids (>C24) with relatively old 14 C ages (
2600–6500 years) in meromictic lake sediments, suggesting a predominant microbial origin. Our results, therefore, have broad implications for deciphering the origin and subsequent paleoclimate applications of long-chain n-alkyl lipids in aquatic sediments from low to high latitude regions. 4.6. Sources of short-chain n-alkanes and n-alkanoic acids in lake sediments Short-chain n-alkyl lipids are also abundant in sediments of our four study lakes. The n-alkanes in soil and microbial mats show maxima at C17 and C20, respectively, whereas these short-chain n-alkanes are minor in abundance in terrestrial biomass (lichen and moss). Thus, short-chain n-alkanes in our lake sediments should mainly derive from microbes living in the lakes, and potentially small amount from soil microbes. This is consistent with the proposal of Matsumoto et al. (1993, 2010) that short-chain nalkanes originate from microalgae and cyanobacterial debris in lake sediments and soils from the McMurdo Dry Valleys and Vestfold Hills of Antarctica (Volkman et al., 1986, 1988; Matsumoto et al., 1993, 2010). Short-chain n-alkanes in microbial mats collected from three melt-water ponds from the McMurdo Ice Shelf were thought to mainly originate from cyanobacteria (Jungblut et al., 2009). The average d13C values of C16 and C18 n-alkanoic acids from microbial mats were about –21‰ and –26‰ (Fig. 5a), whereas d13C values of terrestrial biomass (lichen and moss) and soils were

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waxes can rapidly dominate the sediment pool of long-chain nalkanes in warmer and wetter settings where vascular plants are present. However, long-chain n-alkanoic acids have an even greater tendency to originate from aquatic sources than longchain n-alkanes, as demonstrated by the calculated relatively high percentage of aquatic sources (>60%) in Long Lake. Therefore, caution must be taken in using long-chain n-alkyl lipids (and especially long-chain n-alkanoic acids) for paleoclimate and paleoenvironmental reconstructions in sites with little terrestrial plant presence (e.g., Antarctic cold deserts, or other arid regions lacking leaf waxes of aeolian origin). In lower latitude sites, it is also important to take into consideration the possibility of microbially derived long-chain n-alkyl lipids when interpreting their distribution and isotopic values for paleoclimate and paleoenvironmental applications. For example, our results suggest that the lower carbon preference index values for long-chain n-alkanes in low and middle latitude ocean and lake sediments may mostly originate from microbial contributions. The chain-length of long-chain n-alkyl lipids in Antarctic lichen and moss are 2 to 3 carbon numbers higher than temperate lichen and moss. Such chain-length difference may result from a greater need to protect the plants from desiccation in the dry and cold environment of Antarctica. The d13C values of long-chain nalkanes (C23–C33) and n-alkanoic acids (C22–C32) for lichen and moss samples are, however, similar for Antarctic and temperate regions. Fig. 7. The d13C values of n-alkanoic acids from sediments and the percentage of nalkanoic acids from aquatic sources calculated using a carbon isotope binary mixing model. (a): the average d13C values of n-alkanoic acids in lake sediments. (b): the percent of n-alkanoic acids from aquatic sources (%). Error bars in (a) represent 1r standard deviation of all measured samples. Error bars in (b) represent uncertainty in the resulting estimates of aquatic source of long-chain n-alkanoic acids, based on error propagation calculations.

about –30‰ (Fig. 5band c; Table 2). The d13C values of C16 and C18 n-alkanoic acids in IIL3, IIL9 and Daming Lake were about –26‰ to –20‰, i.e., between values for the terrestrial biomass and microbial mat end-members. Thus, short-chain (C16 and C18) nalkanoic acids in our study lake sediments may have mixed terrestrial microbial and aquatic microbial sources.

5. Conclusions Long-chain n-alkyl lipids (n-alkanes and n-alkanoic acids) in aquatic sediments have traditionally been ascribed to an origin from terrestrial vascular plant leaf waxes. Here we show for the first time that long-chain n-alkyl lipids in lake sediments can be predominantly or partially derived from microbial sources, particularly heterotrophic microbes. The general absence of terrestrial vascular plants in three of our study sites, except for small amount of mosses and lichens, provided ideal samples for assessing potential aquatic sources of long-chain n-alkyl lipids in lake sediments. Our results suggest a more widespread occurrence of microbially derived long-chain n-alkanes and long-chain n-alkanoic acids in sediments and soils. Applying a binary carbon isotope mass balance model to the four study lakes and ponds in Antarctica allows us to quantitatively assess the relative terrestrial and aquatic inputs of long-chain nalkyl lipids in lake sediments. Our analyses show that aquatic microbial sources are the dominant producers of long-chain nalkyl lipids inventory in our three higher latitudes (69°S to 74°S) Antarctic lake sediments. When vascular plants and moss are present, as is the case of Long Lake, a relatively lower latitude (
62°S) lake in the Antarctic Peninsula, long-chain n-alkanes from microbial sources decrease dramatically, indicating vascular plant leaf

Acknowledgements This work was jointly supported by a National Natural Science Foundation of China (grant numbers: 41576183 and 41776188), and United States National Science Foundation (grant numbers: OPP-1503846; EAR-1762431). We would like to thank the Chinese Arctic and Antarctic Administration of the State Oceanic Administration for project support. We also thank the United States Antarctic Program (USAP), Antarctic Support Contract and Italian Mario Zucchelli Station for logistical support. We also thank Prof. Steven D. Emslie who made great efforts improving our preliminary manuscript and Dr. Jiaju Zhao for his kind help in experimental analysis and Libin Wu, Jing Jin and Xueying Wang for preparing samples. Prof. Pengcheng Wu, Dr. Jian Yang and Haiying Wang are acknowledged for their help in moss and lichen species identification. Steven D. Emslie, R. Murray, and A. McKenzie provided valuable field assistance. Dr. Tao Huang and Fubin Liu are also thanked for collecting sediment samples in the field. We are grateful to Dr. Elizabeth Canuel, Dr. Phil Meyers and three anonymous reviewers whose comments significantly improved the quality of the manuscript. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.orggeochem.2019.103909. Associate Editor—Philip A. Meyers References Aichner, B., Hilt, S., Périllon, C., Gillefalk, M., Sachse, D., 2017. Biosynthetic hydrogen isotopic fractionation factors during lipid synthesis in submerged aquatic macrophytes: effect of groundwater discharge and salinity. Organic Geochemistry 113, 10–16. Alberdi, M., Bravo, L.A., Gutiérrez, A., Gidekel, M., Corcuera, L.J., 2002. Ecophysiology of Antarctic vascular plants. Physiologia Plantarum 115, 479–486. Baroni, C., Orombelli, G., 1994. Holocene glacier variations in the Terra Nova Bay area (Victoria Land, Antarctica). Antarctic Science 6, 497–505. Bendle, J., Kawamura, K., Yamazaki, K., Niwai, T., 2007. Latitudinal distribution of terrestrial lipid biomarkers and n-alkane compound-specific stable carbon

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