Comparison of different chain n-fatty acids in modern plants on the Loess Plateau of China

Abstract

n-Fatty acids (n-FAs) are widely investigated in lake sediments, yet less attention has been given to soil sedimentary n-FAs primarily derived from terrestrial plants. In this study, we performed an analysis of n-FA distributions in modern plants on the Loess Plateau, China. It showed that n-FAs were generally dominated by n-C₁₆, and that short-chain (C₁₆–C₂₀), medium-chain (C₂₂–C₂₆) and long-chain (C₂₈–C₃₂) n-FAs accounted for 49.7%, 33.7% and 16.6%, respectively. The LTR (long-chain/total ratio), and medium-chain EOP (even/odd predominance) are likely to differentiate between dicots and monocots in modern plants. It is believed that this study will promote the paleo-application of soil sedimentary n-FAs on the Loess Plateau.

Keywords

n-fatty acids / terrestrial higher plants / Chinese Loess Plateau

Introduction

Lipid biomarkers, such as n-fatty acids (n-FAs), could serve as an important constituent of plant waxes biosynthesized by higher plants (e.g., ^{Eglinton and Eglinton, 2008}). n-FAs can be well-preserved in geologic archives (e.g., ^{Ficken et al., 1998}; ^{Naraoka and Ishiwatari, 2000}; ^{Ishiwatari et al., 2006}; ^{Freimuth et al., 2017}), and easily extracted by biochemical methods (^{Ishiwatari et al., 2005}; ^{Ardenghi et al., 2017}). Accordingly, n-FAs have been employed to reveal past environmental changes in various geochemical studies (e.g., ^{Ishiwatari et al., 2005}; ^{Diefendorf et al., 2011}; ^{He et al., 2016}; ^{Hou et al., 2017}).

Analyses of individual FAs were conducted to decode their origins in lacustrine deposits. For example, know-ledge of n-FA origins assumed that short chains (C₁₆–C₂₀) are mainly biosynthesized by hydrophytes, algae, and bacteria (e.g., ^{Cranwell et al., 1987}; ^{Fang et al., 2014}), medium chains (C₂₂–C₂₆) are primarily produced by aquatic plants (e.g., ^{Ficken et al., 2000}; ^{Wang and Liu, 2012}), and long chains (C₂₈–C₃₂) are typically contributed by terrestrial plants (e.g., ^{Douglas et al., 2012}; ^{Ouyang et al., 2015}). Other studies indicate that some medium- and long-chain n-FAs (e.g., n-C₂₆, n-C₂₈, and n-C₃₀) are also biosynthesized by bacteria and algae (^{Gong and Hollander, 1997}; ^{Naraoka and Ishiwatari, 1999}), and that relatively high abundances of C₂₂ and/or C₂₄ FAs occur in some terrestrial plants (^{Diefendorf et al., 2011}; ^{Wang and Liu, 2012}). Recent studies reported a general dominance of C₂₆–C₃₂ n-FAs in the majority of terrestrial plants, with a few exhibiting high levels of C₂₀–C₂₄ n-FAs (^{Gao et al., 2014}; ^{Feakins et al., 2016}). Collectively, these n-FA studies highlighted the relative contributions of terrigenous and aquatic plants to lake and oceanic sediments; however, less attention has been given to the relative contribution of intra-terrestrial plants (e.g., dicots versus monocots; woods versus grasses) to terrestrial soil sediments. Thus, it remains unclear if n-FA chain distributions can provide chemotaxonomic information for determining the relative contributions to sediments from different plant groups within terrestrial plants.

Sedimentary n-FA δD values are increasingly employed as a robust proxy for revealing past changes in environment (e.g., ^{Hou et al., 2008}; ^{Seki et al., 2012}; ^{Aichner et al., 2015}; ^{Liu et al., 2018b}) due to higher concentrations of n-FAs than n-alkanes in lake sediments (Naraoka and Ishiwatari, 2000; ^{Pearson et al., 2007}). ^{Hou et al. (2008)} reported a significant correlation between δD values of C₂₈n-FA and precipitation δD (R²=0.76) along with precipitation and relative humidity gradients, indicating that sedimentary n-FA δD values can track variations in δD values of precipitation in the United States. ^{Seki et al. (2012)} found that sedimentary C₂₈n-FA δD values were likely to record temperature-induced precipitation δD values, suggesting that n-FA δD values could serve as an indicator to reconstruct paleo-temperatures in Lake Biwa, Japan. Based on paired measurements of n-FA δD and δ¹³C values from a sediment core in arid Central Asia, ^{Aichner et al. (2015)} showed remarkable episodes of cooler and wetter, as well as warmer and drier, climates over the late Holocene. ^{Liu et al. (2018b)} shed light on the effects of different photosynthetic pathways (C₃ vs. C₄) on intermolecular variation of n-FA δD values by investigating n-FA δD values from algae and submerged plants on the Tibetan Plateau of China. These isotopic paleo-applications of n-FAs in sediments are based on an in-depth understanding of plant-scale n-FA distributions and different plant groups. In terrestrial soil sediments, if n-FA chain distributions can be used to distinguish different plant groupings (e.g., dicots versus monocots; woods versus grasses), and if the end-member n-FA isotopic compositions of dicots and monocots (woods vs. grasses) can be measured, the relative contribution of dicots and monocots (woods vs. grasses) to terrestrial soil sediments could thus be derived by using a binary mixing model. Therefore, it is crucial to identify the relative contributions of different plant types into terrestrial soil sediments, which provide the basis for n-FA biomarkers to reconstruct paleoenvironments.

To address this question, we collected many plant material samples along sunny and shaded slopes of a catchment on the Loess Plateau, which contained various types of vegetation and enough loess-palaeosol sequences to potentially reconstruct paleoenvironments (^{An et al., 1991}; ^{Liu et al., 2017}). Note that our prior studies reported the distributions of long-chain n-alkanes among plant types (^{Liu et al., 2018a}; ^{Liu et al., 2019}) and n-alkane δD variations in modern plants in this catchment (^{Liu et al., 2017}). This study focused on n-FA distributions and concentrations in modern plants, with the objectives to assess discrepancy of n-FA distributions among different chains (short, medium and long), and analyze differences in n-FA contents among plant types (dicots vs. monocots).

Materials and methods

Sample collection

The site, Qiushui Valley, is situated in the center of the Loess Plateau (Fig. 1), with both semiarid and arid climates (^{An et al., 1991}). The mean annual precipitation (MAP) is approximately 540 mm, with an approximate mean annual temperature (MAT) of 9°C (^{Liu et al., 2017}). The descending trends of both the MAP and MAT exist across a southeast northwest transect (^{An et al., 1991}; ^{Wang et al., 2014}). The vegetation is dominated by grasses (e.g., Stipa bungeana, Bothriochloa ischemum) and shrubs (e.g., Artemisia sp.) (^{Liu et al., 2018a}). The intact leaves were sampled in May 2013, totaling 41 species. Approximately three to six individuals of each species were collected to guarantee a sufficient collection of plant materials. They were then stored in a cooler (approx. 4°C) for immediate transport to the laboratory.

n-FA extraction

n-FA extraction was conducted at the Institute of Earth Environment, Chinese Academy of Sciences (^{Wang and Liu, 2012}). Plant materials were freeze-dried for ultrasonic extraction using a mixture of methanol (MeOH) and dichloromethane (DCM) (1:9). The extracted lipids were dried with a gentle nitrogen stream, and directly methylated with 2 ml of 5% acetyl chloride in MeOH at 60°C for 12 h. When a NaCl solution (5%, 2 ml) was introduced, the methylated lipids were extracted with hexane three times. The FA methyl esters (FAMEs) were then segregated from the methylated mixtures using a silica gel column (100–200 mesh) by recycled elution with hexane and DCM, respectively. The FAMEs were further segregated into saturated and unsaturated lipids using an AgNO₃ (10 wt%) impregnated silica gel column with hexane/DCM (4:1) and DCM/ethyl acetate (4:1), respectively. The saturated FAMEs were analyzed using gas chromatography (GC) to determine the FA distributions.

GC quantification and data analysis

Quantification was determined using an Agilent 6890 Series instrument equipped with a split injector, Agilent HP1-ms column (60 m, 0.32 mm i.d., 0.25 mm film thickness), and a flame ionization detector (FID). The samples were injected in splitless mode, with a GC inlet temperature of 310°C and a flow rate of 1.2 mL/min. The oven temperature was initially elevated from 40°C to 150°C at 10°C/min, and then further elevated to 315°C at 6°C/min for 20 min. To calculate each n-FA concentration, peak areas were compared with a reference standard (i.e., n-C₁₈, n-C₂₀, n-C₂₄ and n-C₃₀) of known concentrations of individual n-FAs. All data were grouped by different plant types (dicots, monocots, others), and then analyzed by statistical analysis (e.g., mean, s.d., max., min., etc.) with Origin 2017. One-way ANOVA tests were performed to identify the significant differences between dicots and monocots at the significance of 0.01 by using SPSS 20 software. The sampling map was generated by using ArcGIS 10.2.

Results

Terrestrial plant n-FAs on the Loess Plateau

The n-FA concentrations and distributions in modern plants are provided in Table 1. The n-C₁₆ was the most abundant, followed by n-C₂₀, n-C₂₂ and n-C₂₄ in all plants (n = 41) (Fig. 2). Similar results were also observed for angiosperms (including dicots and monocots; Fig. 3(a)), dicots (e.g., Ziziphus jujuba Mill., Artemisia vestita, Armeniaca sibirica; Fig. 3(b)), and monocots (Bothriochloa ischemum, Stipa bungeana; Fig. 3(c)), respectively. Bryophyte n-FAs exhibited a bimodal pattern, with carbon maxima at n-C₁₆ and n-C₂₄ to n-C₃₀, whereas pteridophytes exhibited a unimodal pattern, with a maximum at n-C₂₈ (Fig. 3(d)). The n-FA distributions were grouped into short-chain (C₁₆–C₂₀), medium-chain (C₂₂–C₂₆), and long-chain n-FAs (C₂₈–C₃₂). The average proportions of short-, medium-, and long-chain n-FAs were estimated as 49.7%, 33.7% and 16.6%, respectively (Fig. 2).

Indicators (ACL and EOP) and concentrations

No significant discrepancy for ACL between dicots and monocots with regard to short-, medium-, and long-chain n-FAs in modern plants were found (Fig. 4(a)). The EOP values of both dicots and monocots were relatively higher for short-chain n-FAs than for medium- and long-chain. Dicots produced a higher EOP relative to monocots (Fig. 4(b)). Moreover, short-chain n-FA concentrations were 50.5±49.4 µg/g in dicots and 56.1±49.1 µg/g in monocots, medium-chain concentrations were 30.8± 27.2 µg/g in dicots and 15.8±12.0 µg/g in monocots, and long-chain concentrations were 7.2±6.3 µg/g in dicots and 12.3±10.6 µg/g in monocots, respectively (Fig. 4(c)). Based upon n-FA distributions, some new indices were examined to distinguish different plant types (Figs. 5 and 6). We found a significant difference between dicots and monocots (Fig. 5) in the LTR (p<0.01), but a relatively weak difference in the STR₁₆ (p = 0.035), suggesting that both the LTR and STR₁₆ may be helpful to differentiate dicots from monocots. Distributions of n-FA concentrations vs. EOP overlapped for short-chains (Fig. 6(a); p = 0.068) and long-chains (Fig. 6(c); p = 0.024) Similar results of n-FA concentrations vs. ACL for short-chains (Fig. 6(d); p = 0.066) and medium-chains (Fig. 6(e); p = 0.709) were observed. However, there was a significant difference in medium-chain EOP between dicots and monocots: 14.2 (n = 21, s.d. = 5.2) for dicots and 4.9 (n = 17, s.d. = 1.9) for monocots, respectively (p<0.01; Fig. 6(b)). Likewise, a one-way ANOVA test showed a significant difference in long-chain ACL between dicots and monocots (p<0.01; Fig. 6(f)).

Discussion

Differentiating dicots from monocots using the n-FA ratios

It is well-recognized that significant discrepancies in n-alkane δD values across plant groups (grasses/woods; dicots/monocots) existed in modern plants (^{Hou et al., 2007}; ^{Sachse et al., 2012}; ^{Liu et al., 2016}; ^{Liu and An, 2018}; ^{Wang et al., 2018}). However, it has not yet to be determined if plant types affect n-FAs in modern plants, because the biosynthesis of n-FAs and n-alkanes originated from the same precursor (i.e., acyl-ACP), in conjunction with approximate fractionation mechanisms. For example, C₃₁ n-alkane and C₃₂ n-FA were biosynthesized from the same precursor C₃₂ n-alkyl acyl-ACP (^{Chikaraishi and Naraoka, 2007}). Based on n-FA studies, short chains (<C₂₂) were predominantly produced by bacteria (^{Cranwell et al., 1987}), algae, and phytoplankton (^{Fang et al., 2014}), but long chains (C₂₈–C₃₂) were primarily generated by terrestrial higher plants (^{Ouyang et al., 2015}). Thus, it is plausible to group n-FA distributions into short, medium, and long chains.

The C₁₆–C₃₂ n-FA distributions were characterized by even/odd predominance (EOP), similar to the CPI patterns of n-alkanes in terrestrial plants (e.g., ^{Bush and McInerney, 2013}; ^{Badewien et al., 2015}; ^{Huang et al., 2016}). By analyzing C₁₆–C₃₂ n-FA distributions, we observed that the C₁₆ n-FA is commonly predominant and that long-chain n-FAs have low abundances relative to short- and medium-chain n-FAs in most plants, except for pteridophytes (Figs. 2 and 3). The dominance of C₁₆ n-FA in terrestrial plants is most likely associated with different biosynthesized compartments within a leaf: n-C₁₆ FA is generally biosynthesized in the chloroplast while above C₂₀ chain lengths are biosynthesized in the cytoplasm (^{Kunst and Samuels, 2003}; ^{Samuels et al., 2008}). We surmise that the utility of the different-chain n-FA ratios, such as the LTR (long-chain/total) and STR₁₆ (n-C₁₆/total), could distinguish the differences between plant types (dicots vs. monocots). The biochemical difference is their leaf veinal structures, with monocots having parallel veins whereas dicots primarily being reticulate veins (^{Helliker and Ehleringer, 2000}). We know that C₁₆ n-FA is widespread in both aquatic and terrigenous plants (^{Bush and McInerney, 2013}), but long-chain n-FAs are primarily derived from terrigenous plants (^{Chikaraishi and Naraoka, 2007}; ^{Douglas et al., 2012}). The source of C₁₆ n-FA represents a mixture of terrigenous and aquatic plants, but long-chain n-FAs typically indicate terrigenous plants; thus, the LTR, rather than the STR₁₆, is able to distinguish different plant types. Several indices based on n-FA distributions were introduced to discern vegetation and climate information, such as the EOP, ACL, and n-FA concentration. We analyzed the relationships between EOP/ACL and n-FA concentration across short-chains, medium-chains, and long-chains by using log-log plots (Fig. 6). The medium-chain EOP and long-chain ACL are likely to be used to differentiate dicots from monocots, whereas the difference in medium-chain EOP and long-chain ACL is likely associated with the biochemical characteristics and/or differences in the in situ microclimates. Overall, the indices (LTR, medium-chain EOP and long-chain ACL) provide good potential to distinguish between dicots and monocots in modern plants in a valley of the Loess Plateau, China.

Here we summarize the published results of n-FAs in terrestrial plants (e.g., ^{Diefendorf et al., 2011}; ^{Gao et al., 2011}; ^{Douglas et al., 2012}; ^{Wang and Liu, 2012}; ^{Feakins et al., 2016}). The average values of long-chain ACL, medium-chain EOP, and LTR for dicots were 29.80±0.89 (n = 279), 5.58±2.94 (n = 23), 0.51±0.23 (n = 287), respectively. We could not obtain the STR₁₆ for dicots because the previous studies reported the n-FA carbon ranges that were concentrated in medium- and long-chain n-FAs. In contrast, the average values of long-chain ACL and medium-chain EOP, LTR, and STR₁₆ for monocots were 29.50±0.13, 3.59±0.35, 0.24±0.03, and 0.16±0.03 (n = 14), respectively. Based on our compiled data set, there were significant discrepancies in medium-chain EOP and LTR were observed between dicots and monocots (p<0.01), yet there was no significant difference in long-chain ACL (p = 0.153). It is thus possible for medium-chain EOP and LTR to distinguish between dicots and monocots in terrestrial plants at large scales, instead of long-chain ACL. In this study, however, we also examined STR₁₆ (C₁₆/total n-FAs) because the C₁₆ n-FA was typically predominant in most terrestrial plants on the Chinese Loess Plateau (Figs. 2 and 3). However, prior studies did not keep an eye on n-C₁₆ FA. It is important to report the relative abundance of n-C₁₆ FA in future studies.

Implications for paleoenvironmental reconstruction on the Loess Plateau

We revealed some indicators for differentiating dicots from monocots, i.e., the LTR and medium-chain EOP, based upon an investigation of n-FA distributions in modern plants and a compared analysis of n-FAs from published studies. These indicators provide an alternative way to qualitatively interpret the sources of soil sediments from different plant types in a terrestrial ecosystem, such as the Loess Plateau of China. Combined with these indicators and n-FA δD values in terrestrial soil sediments, we could possibly evaluate the quantitative sources from different plant types into soil sediments. With regard to the n-FA sources in soil sediments, plant wax n-FA transportation also plays an important role in determining n-FA composition in sediments. Sediments enrich plant waxes at an ecosystem scale using aerial particulate waxes, soil erosion, and in situ leaf fall (^{Diefendorf and Freimuth, 2017}). It is thus possible that multiple factors (e.g., wind transmission, dust deposition, and in situ plant biomass) could complicate the input sources of soil sedimentary n-FAs. Moreover, straight chain fatty acids act as major lipid components of living plants and their occurrence in sediments is well-documented (^{Ishiwatari et al., 2006}) due to the excellent preservation of n-FAs in geologic archives (e.g., ^{Freimuth et al., 2017}), despite the likely effect of lipid degradation on sedimentary CPI values in several studies (^{Buggle et al., 2010}; ^{Zech et al., 2013}). Even though a simple analysis of in situ plant wax n-FA distributions on the Loess Plateau was performed in this study, a systematic investigation on these distributions across large spatial-temporal scales is essential in future studies. The effect of wind transmission and dust deposition on soil sediments must be determined when interpreting n-FA sources in soil sediments. Consequently, it is quite complex to qualitatively and quantitatively resolve the input sources of n-FAs in terrestrial soil sediments. This study will improve the terrestrial biomarker (i.e., n-FAs) as a proxy for paleoenvironmental reconstruction in a terrestrial ecosystem.

Analysis of n-FA chain length distributions in sediments could possibly be used to decipher leaf wax plant sources, yet more investigation of taxonomic differences in chain-length distributions would be required (^{Douglas et al., 2012}). Our analysis of n-FA distributions provides an approach to distinguish different plant types in modern plants, but the uncertainty needs to be constrained and additional factors must be considered when interpreting sedimentary n-FA δD values. Concerted efforts must be made to conduct more lipid experiments across various climates and biomes, and an examination should be performed to better understand the mechanism underlying lipid biosynthesis. Multi-proxy research can assist in the development and strengthen the validity of paleoenvironmental studies (^{Horton et al., 2016}). For example, pollen records could help to constrain vegetation-related shifts given pollen provides a taxonomically detailed indication of vegetation change. The interpretation in a terrestrial ecosystem could be strengthened with an increased knowledge of the lipid chemotaxonomy of modern plants, and the n-FA distributions and isotopic values (e.g., δD) would be combined to analyze on the Loess Plateau, China.

Conclusions

An investigation of n-FA distributions from terrestrial plants was performed on the Loess Plateau of China. These distributions were generally dominated by n-C₁₆, with a high abundance of n-C₂₀, n-C₂₂ and n-C₂₄. The indicators (i.e., LTR, medium-chain EOP) discussed herein are likely to differentiate between dicots and monocots in a terrestrial ecosystem. Our results suggest that a systematic investigation of n-FA distributions across large spatial-temporal scales is crucial for future studies.

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