Leaf wax n-alkane distribution and hydrogen isotopic fractionation in fen plant communities of two Mediterranean wetlands (Tenaghi Philippon, Nisí fen—Greece)

Many continental paleoclimate archives originate from wetland sedimentary sequences. While several studies have investigated biomarkers derived from peat-generating vegetation typical of temperate/boreal bogs (e.g., Sphagnum), only scant information is available on emergent plants predominant in temperate/subtropical coastal marshlands, peri-lacustrine and fen environments. Here, we address this gap, focusing on two wetlands in the Mediterranean (Nisí fen and Tenaghi Philippon, Greece). We examined the concentration, homologue distribution, and hydrogen stable isotopic composition (δ²H) of leaf wax n-alkanes in 13 fen plant species, their surrounding soil, and surface water during the wet growing season (spring) and the declining water table period (summer). Our findings indicate that local graminoid species primarily contribute to the soil n-alkane signal, with a lesser influence from forbs, likely owing to differences in morphology and vegetation structure. The δ²H values of surface and soil water align with local average annual precipitation δ²H, reflecting winter-spring precipitation. Consistently, the average δ²H of local surface, soil, and lower stem water showed negligible evaporative enrichment, confirming minimal ²H-fractionation during water uptake. We find that δ²H values of source water for wax compound synthesis in local fen plants accurately mirror local annual precipitation. Furthermore, despite differences between leaves and lower stems in n-alkane production rates, their δ²H values exhibit remarkable similarity, indicating a shared metabolic substrate, likely originating in leaves. Our net ²H-fractionation values (i.e., precipitation to leaf n-alkanes) align with those in Chinese highlands and other similar environments, suggesting consistency across diverse climatic zones. Notably, our data reveal a seasonal decrease in the carbon preference index (CPI) in plant samples, indicating wax lipid synthesis changes associated with increased aridity. Additionally, we introduce a new parity isotopic difference index (PID) based on the consistent δ²H difference between odd and even n-alkane homologues. The PID demonstrates a strong anticorrelation with plant CPI, suggesting a potential avenue to trace long-term aridity shifts through δ²H analysis of odd and even n-alkane homologues in sedimentary archives. While further development of the PID is necessary for broad application, these findings highlight the intricate interplay between plant physiology, environmental parameters, and sedimentary n-alkanes in unravelling past climatic conditions.

Highlights

• Soil n-alkane signal mainly determined from C₃ graminoids, particularly leaves;

• Alignment of δ²H values of surface, soil, lower stem water, and annual precipitation, confirm the potential of local plant communities to trace long-term seasonality shifts;

• Similar δ²H values of wax compounds in leaves and lower stems suggest a common metabolic substrate, likely originating in leaves;

• Net ²H-fractionation values align with studies in Chinese highlands, suggesting a consistent signal in peri-lacustrine/fen communities;

• Seasonal plant CPI shift indicates n-alkane synthesis changes linked to increased aridity;

• New odd/even-numbered n-alkanes δ²H difference index (PID) shows promise as a potential proxy for tracing long-term qualitative aridity shifts

1 Introduction

n-Alkanes are common components of epicuticular leaf waxes, which provide protection against mechanical damage and help regulating evapotranspiration (e.g., Eglinton and Hamilton, 1967; Koch and Ensikat, 2008). Owing to their covalent C-H bonds and absence of functional groups, n-alkanes exhibit remarkable resistance to degradation, allowing their preservation in sediments for millions of years (Eglinton and Logan, 1991). Their distribution of homologues can be source-specific, with different plant forms peaking at longer or shorter chain lengths. Additionally, their stable isotopic composition (δ²H and δ¹³C) can offer valuable insights into environmental conditions, such as precipitation δ²H, or differentiate between plant types (e.g., C₃ vs. C₄ plants) at the time of plant growth (e.g., Schefuß et al., 2003; Chikaraishi et al., 2004b; Sachse et al., 2004b; Feakins and Sessions, 2010; Freimuth et al., 2017). As a result, sedimentary plant waxes serve as valuable proxies for paleoclimate reconstructions, providing different information on past vegetation dynamics and hydroclimate (Jansen and Wiesenberg, 2017). However, plant n-alkane properties are highly responsive not only to changes in climatic-environmental parameters, but also to variations in plant types, morphology, and physiology. Consequently, regional studies on n-alkanes synthesis, occurrence, and deposition within contemporary plant communities play a pivotal role in refining the interpretation of sedimentary n-alkanes in downcore records.

1.1 Objectives

Here we focus on the (1) n-alkane abundance and distributions and (2) n-alkane and internal water ²H-composition of several species of helophytes (i.e., emergent plants such as reeds, sedges, etc.) that make up the dominant vegetation in two Greek wetland sites (Kalaitzidis, 2007).

In the last two decades, the qualitative interpretation of n-alkane distribution and sed-δ²H_wax has been frequently employed to infer past environmental shifts and develop paleo-hydrological reconstructions (e.g., Sachse et al., 2004a; Collins et al., 2013; Niedermeyer et al., 2016a; Tierney et al., 2017; Ardenghi et al., 2019; Butiseacă et al., 2022). In this context, several studies provided insight on the controls on n-alkane distribution and δ²H_wax of various plant communities in different environments and climates (Diefendorf and Freimuth, 2017; Liu and An, 2018 and refs therein). While a number of plant wax studies have investigated wetland vegetation such as at the Gannan Gahai lake or the Dajiuhu peatland in China (trees, grasses and aquatics; Duan et al., 2014; Zhao et al., 2018; Huang and Meyers, 2019), British coastal salt marshes (mainly halophytes; Eley et al., 2014, 2018) or high latitude bogs (Ficken et al., 1998; Pancost et al., 2002; Nichols et al., 2006, 2010; Brader et al., 2010), none to date focus specifically on the helophytic plant communities so common worldwide in temperate fens, marshes, and peri-lacustrine environments (e.g., Silliman and Schelske, 2003; Mead et al., 2005), from which derive a great number of continental paleo-archives (e.g., Fischer and Wilkes, 2003; Kaufman et al., 2020).

Especially in the Mediterranean region, where alkaline fens are characterised by the predominance of reeds such as sedges (Cyperaceae) and grasses (Poaceae) and absence of mosses (Sphagnum; Britton and Crivelli, 1993; Raeymaekers, 1998; Hajek et al., 2006; Tanneberger et al., 2017; Pontevedra-Pombal et al., 2019), these communities often produce sedimentary sequences (e.g., peat) that become valuable climate archives (e.g., Carrión and Van Geel, 1999; Mighall et al., 2006; Desprat et al., 2013; Pross et al., 2015).

In addition, studying the n-alkane properties of plants in these Mediterranean marshes has a particular paleo-climatic relevance due to the peculiar seasonality of Mediterranean geography and climate. Typically, due to changes between winter and summer atmospheric configuration (Xoplaki et al., 2003; Lionello et al., 2014) Mediterranean precipitation originates either locally (less ²H-depleted), mostly in summer, or from the Atlantic (more ²H-depleted) during the rest of the year (IAEA/WMO, 2017; Hellenic National Meteorological Service, 2018).

Factors like light availability, temperature, and the characteristic summer aridity (Vilà and Sardans, 1999; Peichl et al., 2018), roughly constrain the timing of the growing season between spring and summer. This transitional period is very sensitive to long term changes in seasonality, especially drought, which can greatly affect the δ²H of regional fresh water bodies (Dotsika et al., 2010) including, likely, the topogenous, karstic fed mires selected as our study sites. This means that major changes in atmospheric circulation in this region would likely translate into seasonality shifts, affecting the growing season of the local plant community in two main ways. First, changes in moisture levels would impact evapo-transpiration and drought-related stress mechanisms, which would likely be reflected in shifts in the production and distribution of plant n-alkanes as well as in their net ²H-fractionation. Second, the balance of moisture sources (Mediterranean vs. Atlantic) would be significantly altered, impacting the δ²H of source water available to plants, and thus, potentially, of their leaf waxes. Local fen plant communities can thus potentially serve as valuable recorders of long-term seasonality shifts, which could then be historically reconstructed from the resulting sedimentary archives (Ardenghi et al., 2019; Dixit et al., 2019).

Through this study we attempt to increase our understanding of the n-alkane environmental controls and ²H-fractionation mechanisms of these helophytic communities, as well as ascertaining the ²H-composition of their source water and its relation to the ²H-composition of precipitation. Our aim is to improve the interpretation of sedimentary n-alkane distribution and ²H-composition (sed-δ²H_wax) primarily in such archives, and, secondarily, in any environment where these types of plants provide an important share of sedimentary waxes.

1.2 n-Alkanes as biomarkers

1.2.1 Distribution

Various plant species and communities exhibit distinct patterns of n-alkane homologues, tipically characterised by a pronounced odd-over-even predominance, while most bacteria and few plant species preferentially produce even carbon numbered homologues (Dembicki et al., 1976 and refs. therein).

The shortest homologues (<C₂₁, typically C₁₇) are commonly associated with bacteria and algae (Han and Calvin, 1969; Arp et al., 1999; Sachse and Sachs, 2008; Chatterjee et al., 2023). Sphagnum spp. (C₂₃; Nott et al., 2000) and aquatic plant chain lengths tend to peak at C_21–25, in contrast to terrestrial plants, which often that peak at C_27–33 (Cranwell et al., 1987; Ficken et al., 2000; Gao et al., 2011; Bush and McInerney, 2013; Liu H. et al., 2019), although recent research challenges this dichotomy (e.g., Stefanescu et al., 2023).

Among terrestrial plants, trees/shrubs typically exhibit maximum abundances at C_27–29 while grasses show a predominance of C₃₁ (Cranwell, 1984; Meyers, 2003; Struck et al., 2020). While broad observations suggest that the average chain length (ACL) correlates with temperature/aridity-driven changes in production preferences along environmental gradients, different plant species may exhibit opposing behaviours (Hoffmann et al., 2013; Tipple and Pagani, 2013; Bush and McInerney, 2015; Teunissen van Manen et al., 2019).

Although ACL differences have been employed to discriminate among plant groups in particular environments (e.g., distinguishing gymnosperms/angiosperms and monocots/dicots on the Chinese Loess plateau; Liu et al., 2018), no definitive universally applicable correlation of ACL to plant groups has been established to date (e.g., Bush and McInerney, 2013). Recent studies emphasize the need to complement ACL and other indices (e.g., aquatic plant index) with additional other indicators (e.g., ¹³C-composition of n-alkanes) to effectively distinguish sources among submersed, floating, and emergent plants (e.g., Yu et al., 2021).

1.2.2 Stable isotopic composition

The hydrogen isotopic composition of leaf wax n-alkanes (δ²H_wax) from modern plants has been found to generally correlate with the δ²H value of the plant source water, which often reflects precipitation δ²H (δ²H_p; e.g., Sessions et al., 1999; Sauer et al., 2001; Sachse et al., 2004b). In fact, the δ²H_wax values from modern sedimentary wax compounds globally also robustly record local δ²H_p values (Ladd et al., 2018; Liu and An, 2019; McFarlin et al., 2019). On this basis, the δ²H of sedimentary n-alkanes, particularly of their C₂₉ and C₃₁ homologues, recovered both in marine and continental cores as well as in biologically reworked deposits (e.g., mammals’ middens; Chase et al., 2019), has been used as a tracer for paleo-precipitation isotopic composition (e.g., Sachse et al., 2004a; Niedermeyer et al., 2014, 2016b; Tierney et al., 2017; Ardenghi et al., 2019). The accuracy of δ²H_wax and chain length distribution as paleo-climate indicators relies on the understanding of n-alkane synthesis and of the influence and interaction of several internal (plant physiology) and external (environmental) variables.

As n-alkanes retain the δ²H values established at the time of biosynthesis (Sachse et al., 2012), the average δ²H_wax of single leaves can be biased toward growth water values available at the early stages of leaf growth (Sachse et al., 2010, 2015; Tipple et al., 2013; Tipple and Pagani, 2013; Gamarra and Kahmen, 2017). This may be caused by the timing of leaf synthesis (e.g., early growth often relies on synthates stored at the end of the previous year; Gao et al., 2015; Newberry et al., 2015; Freimuth et al., 2017) and, more generally, changes in the biosynthetic water pool linked to external factors such as temperature and relative humidity (Hou et al., 2008; Kahmen et al., 2008; Zhou et al., 2011; Douglas et al., 2012; Gao et al., 2014b; Bai et al., 2019; Jacob et al., 2021; Eensalu et al., 2023), light intensity (Yang et al., 2009), salinity (Ladd and Sachs, 2012; He et al., 2017; Ceccopieri et al., 2021; Wang et al., 2022), seasonality (Sessions, 2006; Pedentchouk et al., 2008; Kahmen et al., 2011; Newberry et al., 2015; Liu et al., 2017), elevation (e.g., Pérez-Angel et al., 2022) and precipitation δ²H (δ²H_p; Sachse et al., 2006, 2010, 2012; Liu and Yang, 2008; Rao et al., 2009; Feakins and Sessions, 2010).

Internal factors, linked to plant physiology (e.g., biosynthetic pathway, rooting depth) can also contribute to the variation in δ²H_wax (Smith and Freeman, 2006; Gao et al., 2015; Cormier et al., 2018, 2019; Eley et al., 2018) and modulate the correlation between δ²H_p and δ²H_wax, normally expressed as the net (or apparent) ²H-fractionation between n-alkanes and mean annual precipitation (e.g., ε_29/MAP) (Liu et al., 2006, 2016; Hou et al., 2007b; Sachse et al., 2012; Gao et al., 2014a; Gamarra et al., 2016; Eley et al., 2018). Despite the resulting high inter- and intra-specific variability characterising δ²H_wax (e.g., 40‰ between leaves of the same plant; Sachse et al., 2009; Newberry et al., 2015), larger datasets reveal a generally stable correlation between δ²H_wax and δ²H_p in plant communities and derived sediments (Chikaraishi and Naraoka, 2003; Sachse et al., 2006; Hou et al., 2008; Rao et al., 2009; Feakins and Sessions, 2010; Chen et al., 2022). Recent interpretive structural modelling has grouped and hierarchically classified multiple controlling factors such as δ²H_p, evapotranspiration, and plant types, according to their relative impact on δ²H_wax variability (Liu and An, 2018).

2 Methods

2.1 Approach

In order to investigate seasonal and species-specific differences characterising the plant communities building these sedimentary archives, we sampled several species of emergent plants from two climatically similar sites in northern Greece during the summer (dry season) of 2014 and the following spring (wet growing season). We analysed the abundance and distribution of n-alkanes in leaf, lower stem, and sub-surface soil samples, as well as the hydrogen isotopic composition of (1) water from soil, lower stems, and leaves, and of (2) n-alkanes extracted from leaves and lower stems.

2.1.1 Sampling sites

For this work we selected two sampling sites (Figure 1): one, the Tenaghi Philippon (TP) peatland in NE Greece (Figures 1, 2A), is the site of a historical permanent large wetland, recently (1930s) converted to farmland, as well as the site of important sedimentary paleoclimate archive (e.g., Wijmstra, 1969; Tzedakis et al., 2006; Pross et al., 2015; Schemmel et al., 2016; Ardenghi et al., 2019), and one, the Nisí fen, is a relatively undisturbed permanent-seasonal marshland at the Edessaios river’s source in NW Greece (Figures 1, 2B) and a good ecological analogue for the original TP environment (Kalaitzidis, 2007). Both the original TP marshland and Nisí fen are examples of topogenous mesotrophic mires (i.e., mires that are predominantly fed by local karstic aquifers) situated in the lowest areas of intra-mountainous basins. The two sites share similar basic climatic parameters (temperature, precipitation; Table 1) and the same Köppen-Geiger climate classification (Csa, i.e., warm temperate with arid, hot summers; Kottek et al., 2006) with localised humid conditions during most of the year, and occasional frost episodes, due to thermal inversion and cold bursts from the northern mountain chains, punctuating the relatively mild and wet winters (Christanis, 1994; Pross et al., 2015; Hellenic National Meteorological Service, 2018).

Figure 1

Figure 1. Map of northern Greece with the location of the two sampling sites and of the Thessaloniki GNIP station. Base map from Schlitzer, (2007); OpenStreetMap contributors, (2018).

Figure 2

Figure 2. Stylised maps of main vegetation communities and surface geology of (A) the Tenaghi Philippon site, and (B) the Nisí fen site (modified from Christanis, 1994), including our sampling stations (created with ArcGIS pro; Esri, 2023).

The TP peatland is the result of millions of years of accumulation of Cyperaceae and other peat-forming helophytes in what once was a fen environment characterised by alkaline conditions (Kalaitzidis, 2007). The original wetland was almost completely drained in the 1930s, resulting also in the alteration of its natural water regime. However, stable residual pockets of helophytic communities survive in areas along the draining canals. Circa 16% of the samples were collected from these areas, in an effort to provide a basis for direct data comparison between TP and the mostly undisturbed Nisí fen site.

2.1.2 Species

We selected six species of widespread perennial monocots: the C₃ graminoids Carex riparia (greater pond sedge), Cladium mariscus (saw-sedge), Phragmites australis (common reed), Typha angustifolia (narrowleaf cattail) and Scirpus lacustris (common club-rush), and the C₄ graminoid Cyperus longus (galingale). We focused on helophytes, firstly graminoids, as they have a preeminent role in the formation of peat deposits in these types of marshlands, which often produce important paleoclimatic archives (e.g., Miola et al., 2006; Borromei et al., 2010; Chawchai et al., 2015; Pross et al., 2015) and are not Sphagnum dominated such as acidic (e.g., Carex and Cladium are phylo-calcareous genera; Buczek, 2005; Theocharopoulos et al., 2006) peat-forming oligotrophic bogs (Anderson et al., 2013).

To cover a broader range of wetland species and plant forms, we sampled a group of five perennial eudicot species, the C₃ forbs Cirsium palustre (swamp thistle, biennial/perennial), Stachys palustris (marsh woundwort), Mentha aquatica (water mint), Lythrum salicaria (purple loosestrife), Galium uliginosum (fen bedstraw), as well as the C₃ pteridophyte Equisetum fluviatile (water horsetail) and the C₃ the perennial basal angiosperm Nymphaea alba (white water lily, a rooted hydrophyte with subaerial floating leaves). The latter four species have been sampled in smaller numbers (Supplementary Table S1) due to difficulties in locating/reaching them (L. salicaria, G. uliginosum, N. alba) and different morphology (E. fluviatile). All the plants listed above are common Eurasian wetland species, which are also widespread worldwide at the genus level (e.g., McNaughton, 1966; Mulligan et al., 1983; Theocharopoulos et al., 2006; Bryson and Carter, 2008; Trin et al., 2014).

2.2 Sampling

Mediterranean spring is a relatively wet season, with the highest growth rate for plants. Conversely, low precipitation and high temperatures enhance local aridity in summer, lowering the water level of the mire. This enhances plant water stress, strongly slowing their growth rate (Vilà and Sardans, 1999; Peichl et al., 2018). With the intention to highlight potential resulting seasonal differences, we sampled at both sites in summer 2014 (5th to 7th July) and spring 2015 (26th to 30th May).

We analysed a total of 452 samples (Supplementary Table S1); of these, the majority (229) were samples of fully developed leaves, collected from the apical, Sun exposed sections of each plant. Similarly, 161 samples were collected from the base of the main stem (except for N. alba). All leaf and lower stem samples come from different mature individuals of a species, sampled in close proximity, and complemented by a total of 47 soil samples collected at ca. 20 cm depth within each sampling station, from patches not directly exposed to sunlight to avoid potential ²H-fractionation bias due to intense soil water evaporation. Additionally, 10 surface water samples were collected from swamp areas in proximity of the sampling stations at both sites.

All samples were collected during mid-daytime (ca 10:00 to 17:00), with mean air temperatures of 24°C–28°C (Nisí) and 28°C–29°C (TP) in summer, and 21°C–24°C (Nisí) and 26°C (TP) in spring. The selected leaves and the lower stem sections were severed with a clean metal knife. Additionally, we quickly and carefully de-veined eudicot leaves with a clean metal blade to avoid mixing the water contained in leaf cells with that present in the leaf vascular system. All samples (folded leaves, stem sections and subsurface soil) were then inserted into glass gastight vials (Labco Exetainer^©), placed in a field refrigerator maintained at 5°C and subsequently frozen at −20°C in the laboratory. Surface water samples were collected in LDPE plastic bottles (60 mL), completely filled, tightly sealed, and kept refrigerated at max 5°C until analysis.

2.3 Water extraction and isotopic analysis

δ²H values of the 10 surface water samples were obtained using a Los Gatos Research liquid water isotope analyser–cavity ringdown laser spectroscopy (LGR LWIA-24d) at the Senckenberg BiK-F laboratories, Frankfurt. Delta values are given as permil deviation from VSMOW (±1‰).

Leaf, stem, and soil water was extracted through cryogenic vacuum distillation and its isotopic composition was measured using a Thermo Electron high temperature conversion elemental analyser coupled to a Delta^PlusV mass spectrometer (TC/EA-MS) via a Conflo IV interface, at the Botanical Institute laboratories of the University of Basel, following the procedure described in Newberry et al. (2017). The hydrogen and oxygen isotopic compositions are expressed as permil deviation from VSMOW and the long-term external precision was found to be ±0.8‰ (standard deviation).

Table 1

Table 1. Main topographical and environmental parameters of the two sampling sites.

2.4 n-Alkane quantification and isotopic analysis

We obtained and measured n-alkanes in all 47 soil samples. Due to the high number of leaf and stem samples, we carefully selected a subset of 137 samples (105 leaf and 32 stem, ca. 3 leaf and 1-2 stem samples per each species per sampling station) from the original population to facilitate n-alkane analysis. The selection process ensured that each species was adequately represented and that the subset population reflected the water isotopic characteristics (δ²H) of the original population (Table 3B).

The n-alkanes of leaf, stem and soil samples were extracted via accelerated solvent extraction, following the procedure described in Ardenghi et al. (2017), analysed via a ThermoScientific Trace GC Ultra–DSQII (GC-MS), and quantified using an external standard (Alk C₇-C₄₀ – Supelco 49452-U, 1,000 ng/μL) at the Senckenberg BiK-F laboratories in Frankfurt a.M., Germany. The n-alkane δ²H values of leaf and stem samples were determined using a ThermoScientific GC-Isolink II gas chromatograph coupled to a Delta^PlusV isotope ratio mass spectrometer via a ConFlo IV interface (GC-irMS), following the same procedure described in Ardenghi et al. (2019), at the physiological plant ecology laboratory at the Institute of Botany of the University of Basel, Switzerland. The overall analytical uncertainty (pooled sample size weighted standard deviation) was found to be ±1.7‰ (Polissar and D’Andrea, 2014). All n-alkane homologues (odd- and even-numbered) analysed for δ²H with peak areas over 2 Vs were evaluated. However, as the linear response was tested only above 15 Vs, a correction for peak areas lower than 40 Vs was applied, based on a non-linear regression curve derived by an internal standard (Squalane; see Supplementary Material S1, Supplementary Figure S1).

The distribution of n-alkanes is summarised by two indices: the carbon preference index (CPI; Eq. 1; Marzi et al., 1993), and the average chain length index (ACL; Eq. 2; Poynter, 1989). Both indices are calculated on the C_14-37 homologue range.

$CPI=\frac{\left({\displaystyle {\sum }_{i=n}^m}{C}_{2i+1}\right)+\left({\displaystyle {\sum }_{i=n+1}^{m+1}}{C}_{2i+1}\right)}{2\left({\displaystyle {\sum }_{i=n+1}^{m+1}}{C}_{2i}\right)}(1)$

$ACL=\frac{{\displaystyle {\sum }_i^m}\left[C_{2i+1}\times \left(2i+1\right)\right]}{\left({\displaystyle {\sum }_i^m}{C}_{2i+1}\right)}(2)$

To obtain an isotopic value comparable between samples with different homologue distribution, we calculated the hydrogen isotopic composition of the concentration weighted mean n-alkane (δ²H_CWMA; Eq. 3; Newberry et al., 2015). Where k is the carbon number of the considered homologues, n the number of individual homologues recovered per sample (in the C₁₅–C₃₇ range), and total indicates the sum of n homologues:

${\delta }^2{H}_{CWMA}={\displaystyle \sum _{k=n}}\frac{{\delta D}_k\times {Conc}_k}{{Conc}_{total}}(3)$

In our data, we also detected a consistent pattern in the relative difference between the δ²H of odd and even homologues. To express numerically this difference and allow its comparison between samples, we devised the “parity isotopic difference index” (PID, Eq. 4).

$\text{PID}=\frac{\left[{\displaystyle {\sum }_{\mathrm{i}=\mathrm{n}}^{\mathrm{m}}}\left({\mathrm{C}}_{2\mathrm{i}+2}-{\mathrm{C}}_{2\mathrm{i}+1}\right)⟺{\mathrm{C}}_{2\mathrm{i}+2}\wedge {\mathrm{C}}_{2\mathrm{i}+1}\ne \varnothing \right]+\left[{\displaystyle {\sum }_{\mathrm{j}=\mathrm{q}}^{\mathrm{p}}}\left({\mathrm{C}}_{2\mathrm{j}}-{\mathrm{C}}_{2\mathrm{j}+1}\right)⟺{\mathrm{C}}_{2\mathrm{j}}\wedge {\mathrm{C}}_{2\mathrm{j}+1}\ne \varnothing \right]}{{\displaystyle {\sum }_{\mathrm{k}=\mathrm{s}}^{\mathrm{r}}}{\mathrm{C}}_{\mathrm{k}}/{\mathrm{C}}_{\mathrm{k}}⟺{\mathrm{C}}_{\mathrm{k}}\wedge {\mathrm{C}}_{\mathrm{k}+1}\ne \varnothing }(4)$

The formula indicates the average δ²H difference between adjacent odd-even homologues (double headed arrow means “if, and only if,” “C” is the δ²H value of the respective homologue), and it is designed to buffer against missing data (Ø) and remove from the calculation any pair of non-adjacent homologues (to reduce the skewing influence due to potential drift in different areas of the analytical range).

Positive PID values indicate that odd homologues are generally more ²H-depleted than even homologues, and vice versa; high absolute values indicate strong odd/even ²H-difference and vice versa, while zero indicates no overall difference.

Isotopic ²H-fractionation between the different hydrogen pools of surface water (δ²H_surfacew), soil water (δ²H_soilw), lower stem water (δ² H_stw), leaf water (δ²H_lw) and n-alkanes (δ²H_stwax for stems and δ²H_lwax for leaves) was calculated according to Eq. 5 (Coplen, 2011); both δ and ε are then expressed as permil units (‰; Cohen et al., 2007).Where a and b represent the different hydrogen pools:

${\epsilon }_{a-b}=\left[\frac{{\delta }^2{H}_a+1}{{\delta }^2{H}_b+1}\right]-1(5)$

Here we define ε_stw-lw as the ²H-fractionation between each species mean seasonal leaf water δ²H and the respective mean lower stem water δ²H. ²H-fractionations between plant water and wax n-alkanes are indicated as ε_stw-stwax (stem/stem), ε_stw-lwax (stem/leaf) and ε_lw-lwax (leaf/leaf).

3 Results

3.1 n-Alkanes

3.1.1 n-Alkane concentrations

The concentration of n-alkanes (n-C_14–37) in our plant samples (Supplementary Figure S2; Table 2) exhibited a wide range, spanning from 1.3 to 2,147.3 μg/g. In contrast, soil samples displayed relatively lower and less variable concentrations, ranging from 0.4 to 45.5 μg/g).

Table 2

Table 2. n-alkanes absolute concentrations (µg/g), with relative standard deviation (σ), carbon preference index (CPI) and average chain length (ACL) for all leaf, lower stem, and soil samples (n = 187) analysed in this study.

Concentrations were higher in eudicots/forbs and lower in monocots/graminoids. Leaves consistently showed approximately the same to ∼80 times more n-alkanes than their corresponding lower stem samples (Table 2). This distinction between leaves and stems was notably pronounced among forbs in spring, while graminoids, such as C. mariscus and S. lacustris, exhibited less overall leaf/stem differentiation. Our data revealed no consistent difference in n-alkane concentrations between spring and summer at either site. Nevertheless, the leaves of four species (S. Palustris, C. longus, S. lacustris in Nisí, and P. australis in TP) exhibited a spring-to-summer increase in mean concentration surpassing their respective standard deviation (Table 2). Similarly, while no distinct difference emerged between samples of the same species from Nisí and TP (only four species in common; Supplementary Table S1), leaves of C. longus and P. australis showed higher n-alkane concentration in TP, albeit often close to the respective standard deviation values; Table 2).

3.1.2 n-Alkane distribution

The carbon preference index (CPI) exhibited a wide range across samples: from 0.4 to 86.2 in leaves (with a mean of specific averages at 16.1 ± 7.6), 1.8 to 54.4 in lower stems (mean of specific averages at 14.4 ± 7.1), and 0.9 to 10.4 in soil samples (mean of specific averages at 5.2 ± 1.7). Overall, CPI displayed considerable inter- and intra-specific variability, with all samples recording values well above 1, except for three C. longus leaf samples, characterised by low n-alkane concentrations (Table 2). This suggests a prevalent odd-over-even predominance, notably stronger in plant samples (with a mean of 15.2 ± 7.3) than in soil samples. The sole discernible pattern was seasonal, with CPI generally decreasing from spring to summer, especially in graminoids (Supplementary Table S3) in both leaf and lower stem samples.

The average chain length index (ACL) exhibited a diverse range across samples: from 25.5 to 32.0 in leaves (with a mean of specific averages at 29.0 ± 0.5), 21.4 to 31.7 in stems (mean of specific averages at 27.7 ± 0.6), and 26.4 to 29.4 in soil samples (mean of specific averages at 28.0 ± 0.7). Most samples fell within the typical ACL range for terrestrial plants (27–31), including the floating hydrophyte N. alba. The exception was E. fluviatile, the only pteridophyte (i.e., seedless/flowerless vascular plants, mostly ferns) analysed in this study, with an ACL range of 25–26. Eudicots/forbs and leaves showed ACL higher than, respectively, monocots/graminoids and stems (Supplementary Table S3), as well as soil samples (ca. 27–29). ACL also showed a general increase from spring to summer in eudicot/forb leaves (average increase of 0.4–2.2), although stability was observed in monocot/graminoid leaves. Most stem samples showed an average increase of 0.6–5.8 during this seasonal transition.

3.1.3 n-Alkane hydrogen stable isotopic composition

Plant wax n-alkane δ²H values in the C_20–35 range exhibited a broad spectrum, ranging from −281‰ to −48‰ (mean ≈ −180‰, σ ≈ 41‰; detailed descriptive statistics for each homologue are reported in Supplementary Table S4). Notably, even chain lengths displayed generally less depleted values (mean ≈ −140‰) compared to odd chain lengths (mean ≈ −171‰; Figure 3). For both odd and even homologues, the most depleted values were observed in the C_27–31 range (odd ≈ −179‰, even ≈ −147‰), while progressively less depleted values characterised shorter (odd ≈ −171‰, even ≈ −154‰) and longer (odd ≈ −176‰, even ≈ −129‰) homologues.

Figure 3

Figure 3. Average δ²H values of individual n-alkane homologues from leaf samples divided by species, season and sites: blue (spring/Nisí), red (summer/Nisí), green (spring/TP), orange (summer/TP). Error bars indicate the standard error of the mean.

The δ²H weighted mean (δ²H_CWMA) strongly reflected the values of C₂₇-C₂₉ and C₃₁, the three most abundant homologues in all leaf samples and most lower stem samples (Supplementary Table S4). In leaf samples, δ²H_CWMA ranged from −273‰ to −115‰ (average −198‰ ± 28‰), while in stem samples, it ranged from −253‰ to −152‰ (average −196‰ ± 27‰). Among leaf samples, species with the least depleted δ²H values were G. uliginosum among forbs, C. longus among graminoids, E. fluviatile, and N. alba, while M. aquatica and C. mariscus exhibited the most depleted values. Lower stem samples displayed higher interspecific variability.

Summer δ²H values of concentration-weighted mean n-alkanes from leaves (CWMA δ²H_lwax) were generally less depleted than spring values (Supplementary Table S3; Figure 4), with exception noted for S. lacustris and E. fluviatile (likely influenced by sampling bias; see Section 4.2.3), as well as S. palustris and C. riparia. In Nisí, focusing on C₃ species, graminoids exhibited the most depleted δ²H_lwax (−214‰, −215‰), while generally less depleted values (Supplementary Table S3) characterised forbs (−205‰, −183‰) and E. fluviatile (−171‰).

Figure 4

Figure 4. Mean δ²H values of leaf and lower stem water (lw; stw) and leaf and lower stem wax n-alkanes (lwax; stwax), by species, season and site. Vertical bars indicate SEM.

3.2 Hydrogen stable isotopic composition of water

3.2.1 Surface and soil water ²H composition

Surface and soil water δ²H values (Figure 5; Supplementary Table S2) varied between −72‰ and +37‰. Across both sites and seasons, the δ²H values for surface and soil water were well clustered around their means, with a maximum standard deviation of 1.8‰. Notably, surface water maintained a consistent mean δ²H value (ca −54‰) in both seasons at both sites. In contrast, soil water δ²H values consistently trended lower in Nisí compared to TP, demonstrating an increase from spring (−49.1‰ TP; −60.2‰ Nisí) to summer (−46.5‰ TP; −49.9‰ Nisí; Supplementary Table S2). Both surface water and soil water showed more ²H-depleted values compared to water from leaves and lower stems.

Figure 5

Figure 5. (A) Boxplots comparing δ²H values of surface water, soil water, lower stem water and leaf water (n = 452), divided by site and season. Red crosses mark simple means, black dots individual values, empty dots outliers, black line the median; bottom numbers indicate sample size. The subset for P. australis (B) shows how the negative spring to summer δ²H shift for plant water at the TP site, apparent in (A), is due to missing summer data-points for the other collected species.

3.2.2 Lower stem water and leaf water δ²H values

Leaf water δ²H values for ranged from 37‰ to −41‰, with two outliers at −71‰ (mean ≈ −8‰, median ≈ −8‰, σ ≈ 14‰). In contrast, lower stem water δ²H values varied between −18‰ and −65‰ (mean ≈ −49‰, median ≈ −49‰, σ ≈ 10‰).

Seasonal, species-specific mean δ²H values for lower stem water ranged from −62‰ to −24‰, while leaf water values ranged from −25‰ to +29‰. Except for those of M. aquatica, S. palustris, and S. lacustris, spring values typically exhibit a greater ²H-depletion compared to summer values (approximately 5‰–15‰). However, the variability closely aligned with the standard deviation for each species, as outlined in Table 3B and Figure 5. Similarly, for both lower stem and leaf water, forbs displayed higher δ²H values than graminoids (ca 5‰–10‰ on average) and TP showed higher values than Nisí (Table 3B; Supplementary Table S3). Stem to leaf water ²H-fractionation (ε_stw-lw) ranged between +13‰ and +59‰ (Table 3B).

Table 3

Table 3. Hydrogen stable isotopic composition of (A) soil water and surface water, and (B) internal water and n-alkanes of lower stem and leaf samples, divided by species and season.

4 Discussion

4.1 Variability of n-alkane concentration/distribution in plants and soil

4.1.1 Variability between plant forms and between leaves and stems

Leaf samples show consistently higher n-alkane concentrations than stem samples (up to 80 times and ca. 15 times on average; 3.1.1, Table 2). This can be attributed to the necessity for a thicker cuticle to mitigate desiccation in the upper organs, more exposed to sunlight (i.e., leaves and flowers; Gamarra and Kahmen, 2015; Speckert et al., 2023). More in detail, while forbs show on average higher leaf n-alkane concentrations than graminoids, they also show greater difference between leaves and stems (40 times on average) than graminoids (7 times on average). This is common (e.g., He et al., 2020; Corcoran et al., 2022) and likely due to morphological differences between these two groups.

The CPI (Table 2) shows no consistent differences between species nor between leaves and stems, with several species (e.g., P. australis, C. palustris) exhibiting contrasting leaf-to-stem CPI ratio values in different sampling instances. The CPI values decrease from leaf/stem samples to soil, likely due to preferential degradation of longer homologues (Yan et al., 2021) and the production of microbial derived shorter/even homologues (Thomas et al., 2021; Corcoran et al., 2022 and refs therein), making it impossible to use CPI as a proxy to trace specific plant sources in sediments (Chen et al., 2022).

In terms of ACL, leaves show similar, although generally higher values then the corresponding stem samples (Table 2). Relative to leaves, stem samples often display a broader distribution of odd chain lengths centred around the same C_max (Supplementary Figure S2), along with analogous but generally lower ACL values, with a few exceptions (e.g., C. riparia in Nisí in spring; Table 2). These observations align with previously reported differences between leaves and roots in C₄ grasses such as Enneapogon avenaceus and Astrebla pectinata (Kuhn et al., 2010), but are in contrast with the findings of He et al. (2020) for similar Poaceae and Cyperaceae emergent species, reporting much lower ACL values in roots relative to leaves. The similarity in ACL and the higher concentrations of n-alkanes in leaves compared to stems indicate that, as observed in alpine and temperate grassland species (Gamarra and Kahmen, 2015), also in these fen environments the distinctive signal deposited in sediments is predominantly characterized by the chain length distribution of the leaves (and not the stems) of helophytic plants.

In terms of plant forms, ACL values in our data generally differ between forbs and graminoids. Graminoids leaves exhibit lower ACL (27–29) than forbs leaves (29–32), with the exception of the C₄ graminoid C. longus (29–31), as highlighted by the PCA in Figure 6; stem samples show an analogous pattern. Very slight, although opposite, differences between forbs and graminoids ACL have been detected before on plants from a similar fen environment on the Chinese Loess Plateau (Liu et al., 2018). Overall, graminoids show higher values on the Chinese Loess Plateau (∼30.0) than in Nisí and TP (∼28.6), while forbs have a similar/lower ACL range than graminoids (Supplementary Table S3). On one hand, this opposite behaviour of ACL could be a response to the differences between the climates of the Chinese Loess Plateau (Köppen BS) and of the Mediterranean borderlands. In fact, even if both are characterised by seasonal aridity, the timing is different between these two regions: on the Chinese Loess Plateau winter (and not summer) is the most arid season (Kong et al., 2018). Alternatively, the different behaviour of ACL in our study could simply be the result of different species associations (Diefendorf et al., 2021).

Figure 6

Figure 6. Principal component analysis scores for proportional abundances (0-1) of C_21-33 n-alkane concentrations (odd homologues only), for soil samples and leaf samples, divided by growth form and photosynthetic pathway. Nymphaea alba (being a floating hydrophyte) and Equisetum fluviatile (the only pteridophyte) have been assigned to two separate classes. The distribution of the sample groups describes more in detail what already suggested by ACL values. C₃ forbs, mainly characterised by C₃₁, C₃₃ and C₂₉ predominance, are separated by the majority of C₃ graminoids, characterised by a strong C₂₇ and C₂₉ components (except for a few S. lacustris and C. riparia samples), as is the aquatic N. alba (probably reflecting the subaerial condition of its leaves). The C₄ graminoid C. longus plots between these two groups, while the fern E. fluviatile constitutes a separate cluster down the C₂₃ and C₂₅ directions. Soil samples clustering between C₃ and C₄ graminoids, (with several points in the C₂₃ direction), seems to indicate an origin strongly controlled by these two groups. Produced with the R statistical software, using the FactoMiner package (Lê et al., 2008).

Also, the other two plant forms present in our data show ACL values lower than forbs/eudicots. The floating hydrophyte N. alba shows an ACL (∼29) more characteristic of emerged helophytes (probably indicating the subaerial condition of its leaf blades; Yu et al., 2021). Interestingly, the only pteridophyta (E. fluviatile) shows an ACL (25–26, the lowest in our results) closer to submerged hydrophytes than to terrestrial plants (Figure 6; Yu et al., 2021).

In our soil samples, ACL is relatively stable (27.3–28.7; Table 2) and mean values tend to be similar to mean ACL values of monocots/graminoids (leaves and stems) in both locations and seasons, likely reflecting a major contribution of this plant group to sediment/peat deposition (Liu et al., 2018), as also highlighted in the PCA (Figure 6).

We are aware that caution must be applied to the interpretation of ACL environmental significance, as (1) it has already been shown that relative humidity gradients can have opposite effects on the ACL of different species (Hoffmann et al., 2013; Chen et al., 2022) and (2) plant contributions to sediments in any environment is subject to multiple taphonomic variables (e.g., Corcoran et al., 2022 and refs therein). However, our results indicate ACL as a potential tool to differentiate between plant form (forbs vs. graminoids) relative contribution in this particular environment. Based on our data, the n-alkane signal stored in the sediments of this fen environment with this type of helophytic plant community seems to be mostly representative of local graminoids, and particularly of their leaves.

4.1.2 Seasonality

Our samples show no consistent change in n-alkane concentration between seasons, at both sites (Table 2). While S. palustris, C. longus, and S. lacustris leaves show clear increases from spring to summer, the opposite is true for M. aquatica, C. mariscus, and E. fluviatile, while values for all the other species remain within a standard deviation and thus substantially unvaried. Concentrations are expected to positively correlate to the summer temperature increase, to enhance the cuticular transpiration barrier, as shown, for example, in Juniperus monosperma leaves, (Diefendorf et al., 2021; Shi et al., 2021 and refs therein). The reason behind this inconsistency is unclear, but it likely results from the effect of other factors controlling n-alkane production and preservation, such as, for example, leaf abrasion, aridity, and variability in the leaf sampling procedure (Eglinton and Eglinton, 2008).

Similarly, ACL values do not show a clear seasonal pattern. Forbs such as C. palustris, M. aquatica, S. palustris, as well as S. lacustris and E. fluviatile show increasing ACL values from spring to summer, but all other species show no clear seasonal change. While seasonal ACL increases have been detected before (e.g., from April to September in Salix viminalis leaves, Newberry et al., 2015, in other eudicot/monocot species, Cui et al., 2008, and even in a 15 years study of a subalpine meadow, Shi et al., 2021), Diefendorf et al. (2021) found ACL to be unrelated to temperature increases and to be instead relatively constant within a species. Based on our data, neither n-alkane concentration nor ACL do appear to provide valuable information on seasonality in these type of fen vegetational communities.

In contrast, CPI shows a consistent seasonal decrease from spring to summer. The decrease in CPI seems generally related to an increase of even homologues, while odd homologues concentrations appear relatively stable (Supplementary Figure S2). An analogous CPI seasonal pattern has been detected in maple leaves (Chikaraishi and Naraoka, 2006) and other eudicot/monocot species (Cui et al., 2008) and linked to leaf senescence. However, as none of our leaves/stems showed signs of fading when collected in July, this is an unlikely explanation for the seasonal CPI shift in our samples. Instead, the CPI increase could be related to the enhanced summer aridity. Drought conditions have been shown to speed up lipid metabolism while affecting enzymatic chain elongation, leading to an increased production of mid-short n-alkanes homologues and their relative degradation by-products, including even homologues (Post-Beittenmiller, 1996; Shepherd and Griffiths, 2006; Srivastava and Wiesenberg, 2018; Speckert et al., 2023).

In general, our data indicate that CPI values in plant samples differ between the growing (higher values) and the dry (lower values) seasons. However, due to preferential degradation and microbial activity (Thomas et al., 2021; Corcoran et al., 2022 and refs therein; see Section 4.1.1), soil/sediment CPI is likely an unreliable proxy for tracing seasonality in sediments, at least in this kind of Mediterranean fen environments.

4.2 Water ²H signal from source to leaf water: precipitation and evapotranspiration

4.2.1 Mean annual precipitation as plant source water pool

Surface water and soil water at a depth of 20–30 cm exhibited very similar hydrogen isotopic values (Figure 5), pointing to a clear link between the two water pools. δ²H values for surface and soil water show very similar ranges at both sites in each season (except for 3 unusually high values in TP spring soil water). This reinforces the hypothesis that the Nisí site can be considered as an analogue for the ancient TP site in terms of δ²H of source water based on the opinion of researchers that have described the environmental, climatic, and geological characteristics of both sites (e.g., Christanis, 1994; Kalaitzidis, 2007; see Table 1) as well as on the data of spring/source water δ²H reported here and throughout Greece (Dotsika et al., 2010).

The sole contemporary dataset for annual precipitation δ²H (δ²H_p) in northern Greece is derived from the Thessaloniki GNIP station, covering a period of 2.5 years from 2001 to 2003). The station is situated approximately 105 km W-SW of the Tenaghi Philippon peatland and 90 km E-SE of Nisí fen (IAEA/WMO, 2017; Figure 1).

The δ²H_p values range from −99‰ to −3‰ (with an annual precipitation weighted average of −48‰) and show a distinct seasonal pattern. The most negative values characterise the abundant precipitation during the wet winter-spring period reflecting a weighted mean of approximately −52‰. Conversely, the least negative are observed during the dry Mediterranean summer, hovering around −31‰. As a result, surface and soil water δ²H at the end of spring serve as a reliable approximation of the δ²H_p annual weighted mean (ca. −48‰).

Our data affirms this pattern, as soil (and surface) water consistently exhibit δ²H values similar to this annual mean not only in spring (−49.1‰ in TP; −60.2‰ in Nisí), but also in summer (−46.5‰ in TP; −49.9‰ in Nisí). This alignment is likely attributed to the elevated amounts of winter-spring precipitation and, most probably, to the smearing effect of the karstic aquifer feeding the mire(s) (Kalaitzidis, 2007; Dotsika et al., 2010). Remarkably, the OIPC provides comparable annual δ²H values for precipitation (−51‰ ± 2‰ in Nisí; −48‰ ± 1‰ in TP); similarly, local sampling in NW (−60.4‰ ± 0.8‰) and NE (−50.6‰ ± 0.7‰) Greece, where Nisí fen and TP are located, respectively, supports similar values for spring water (Dotsika et al., 2010). This suggests that these helophytic plant communities in these Mediterranean regions do indeed utilise water that faithfully represents local annual δ²H_p values.

4.2.2 Soil water to lower stem water

Most species showed less depleted stem water δ²H (δ²H_stw) values in the summer, and, within the same species, δ²H_stw values were less depleted in TP than in Nisí (Table 3B). The ²H-fractionation between the soil water and the lower stem water (ε_soilw-stw) of each species (Table 4) was 1) consistently positive (except for P. australis), 2) often within the respective standard deviations, and 3) lacking a clear seasonal pattern. Additionally, as ²H-fractionation during water absorption in roots is considered unlikely (Ehleringer and Dawson, 1992; White et al., 1994; Jacob et al., 2021), the tendency to less depleted δ²H_stw values appears to reflect an occasional initial minor evapotranspiration effect occurring at the crown root and/or at the base of the stem (Eensalu et al., 2023). This difference is more pronounced for forbs (δ²H_stw up to approximately 22‰ less depleted than δ²H_soilw) than for graminids (generally in the 0‰–5‰ range) and mirrors the δ¹⁸O differences between the soil and stem water of two grasses (Dactylis glomerata, Lolium perenne) and a forb (Trifolium pratense; Barnard et al., 2006). We speculate that the overall low/absent enrichment of stem water relative to soil water in monocots/graminoids might be attributed to a protective outer sheath present at the base of most grasses (which was removed during sampling; Barnard et al., 2006). The occasional negative difference between the δ²H_stw and the δ²H_soilw values for P. australis (down to ca. −10‰ in summer), could be explained by its greater rooting depth (Burdick et al., 2001; Moore et al., 2012; Huang et al., 2018; Zhao et al., 2018; Huang and Meyers, 2019), a phenomenon already documented for some species growing in arid environments (Feakins and Sessions, 2010; Corcoran et al., 2022).

Table 4

Table 4. Values of ²H-fractionation between soil water, plant internal water, and plant n-alkanes (CMWA; see Table 3B).

Overall, the data indicates an absence of evaporative enrichment between soil and stem water or at the very least, a very limited occurrence of it. Despite specific variations in rooting depth, leading to a decoupling between δ²H_stw and δ²H_soilw, when considering the plant community as a whole, the average δ²H_stw is representative of the δ²H_soilw for both seasons and sites (Figure 5). Moreover, δ²H_soilw has been previously linked to the local mean annual δ²H_p (see Section 4.2.1). This demonstrates that the initial source of biosynthetic hydrogen within the plants (δ²H_stw) at both sites isotopically reflects mean annual precipitation, with an at least annual integration or smoothening of the seasonal differences of the δ²H_p signal, a conclusion comparable to findings reported in similar studies (e.g., Eensalu et al., 2023). Consequently, any intra- and inter-specific δ²H_wax variability will primarily result from differences in physiology (e.g., growth form) and/or environmental conditions (e.g., aridity) during the growing season, affecting the internal processes of the plants (e.g., Griepentrog et al., 2019).

4.2.3 Lower stem water to leaf water

Generally, δ²H_lw values exhibited greater enrichment in summer compared to spring (Table 3). In most species, the difference between spring and summer δ²H_lw closely mirrored the seasonal variation in δ²H_stw (ca. 5‰–10‰). M. aquatica also conformed to this pattern when a single clear outlier (−72‰) was excluded from the summer mean. The seemingly distinct behaviour of S. lacustris (ca 10‰ more depleted in summer) can be attributed to a sampling bias: its morphology posed challenges in clearly separating stem and leaf sections in the summer specimens. Similarly, the significant seasonal difference in E. fluviatile δ²H_lw (ca 36‰ more negative in summer) is likely linked to the different physiology of this genus, which produces green photosynthesising stems and leaves in spring while new, low photosynthesising stems, crowned by a spore-bearing cone, emerge in summer (the latter were, in fact, sampled as “whole”).

The ²H-fractionation between lower stem and leaf water (ε_stw-lw; +27‰ to +60‰; Table 4) tends to be greater in species with greater height, such as P. australis, T. angustifolia, and C. mariscus (notably, Cladium jamaicense in the Florida Everglades also showed very depleted δ²H_lwax values, −231‰; He et al., 2020), and relatively broader, more exposed leaves (as in C. palustris), potentially indicating increased evapo-transpiration.

It is important to note that our dataset offers a discrete rather than continuous representation of internal water δ²H, a parameter that has been found to exhibit considerable variability, with seasonal to hourly fluctuations (Sachse et al., 2009, 2010). Our sampling approach captures only selected “snapshots” of stem/leaf water δ²H distributed over a few days in spring and summer. Nevertheless, our ε_stw-lw values demonstrate consistency between spring (+44.7‰ ± 12‰) and summer (+45.2‰ ± 10‰) species-specific averages. This stability aligns with findings in studies involving a limited number of tundra vascular plant species in western Greenland (σ=12‰; Berke et al., 2019) and suggests a levelling out of the considerable variability in stem/leaf evapo-transpiration over the extended time frame of the entire vegetative season.

4.3 Water to n-alkanes ²H-fractionation

4.3.1 Leaf water to leaf n-alkanes

The ²H-fractionation from leaf water to leaf n-alkanes (ε_lw-lwax) is known to present high specific variability. While interspecific variability is predominantly associated with differences in plant physiology, intraspecific variability (up to ca. 70‰) is attributed to diurnal/seasonal variability of leaf water δ²H values (Sachse et al., 2012). Given these considerations, the study of ε_lw-lwax (as well as ε_stw-stwax) tipically requires high frequency discrete sampling, ideally approximating continuous sampling, of leaf/stem water and wax n-alkanes throughout the entire vegetative period (e.g., Kahmen et al., 2013; Tipple et al., 2013; Newberry et al., 2015; Sachse et al., 2015).

As previously mentioned (see Section 4.2.3), our study does not rely on such continuous sampling, but rather adopts a more sporadic approach. Nevertheless, our sampling occurred during the main arc of the day, over an average of 2-3 consecutive days per season and site (see Section 2.2). To mitigate the uncertainty inherent in the sporadic sampling of individual specimens, we aggregated the leaf and lower stem water δ²H values of all samples (395), without regard to species, computing season and site averages for δ²H_lw and δ²H_stw (Table 3). We regard these averages as robust estimates of community-wide δ²H_lw and δ²H_stw during spring and summer (Feakins and Sessions, 2010; Sachse et al., 2010). Utilising these consolidated values (Table 3B) and species-specific averages of δ²H_lwax and δ²H_stwax (CWMA), we derived species-specific ²H-fractionation values from lower stem water to leaf water (ε_stw-lwax), leaf water to leaf wax (ε_lw-lwax), stem water to stem wax (ε_stw-stwax), and leaf water to stem wax (ε_lw-stwax), during growth and maturity seasons (Table 4).

At Nisí, especially in summer (Supplementary Table S3), C₃ graminoids showed significantly greater ε_lw-lwax (average of species-specific mean values: spring −202‰ ± 11‰, summer −213‰ ± 10‰) than forbs (spr. −195‰ ± 8‰, sum. −176‰ ± 11‰; Table 4). The resulting ε_lw-lwax difference between C₃ graminoids and forbs in summer (i.e., at the end of the growing season; 36‰ ± 15‰) closely mirrors the reported difference in net fractionation between the same groups (37‰ ± 42‰; Sachse et al., 2012). The larger associated error with net fractionation (±42‰) suggests that, while biosynthetic fractionation may tend to a stable mean value within plant groups, much of uncertainty in net fractionation likely stems from variations in environmental conditions affecting the water pool (δ²H_p/δ²H_soilw/δ²H_stw/δ²H_lw).

4.3.2 Leaf and lower stem water to lower stem n-alkanes

The δ²H values of n-alkanes in stem samples did not exhibit a discernible seasonal pattern and our δ²H_stwax values were relatively similar to the δ²H_lwax values of their leaf counterpart (although often slightly less depleted; Figure 4). ε_stw-stwax values tended to be lower than ε_lw-lwax and displayed significant variability (Figure 7; Table 4). However, when δ²H_lw is used instead of δ²H_stw, the resulting ε_lw-stwax values are much closer to ε_lw-lwax than ε_stw-stwax (Figure 7).

Figure 7

Figure 7. Mean ²H-fractionation between 1) leaf water and n-alkanes (green), 2) lower stem water and n-alkanes (purple), 3) leaf water and lower stem n-alkanes (orange), for all sampled species at both sites, in spring and summer. The values of lower stem and leaf water δ²H are a pooled mean for all species per each season and site (Table 3; Table 4). Vertical bars indicate propagated SEM.

Our proposed explanation for the higher similarity between ε_lw-stwax and ε_lw-lwax (than between ε_stw-stwax and ε_lw-lwax) involves a shared substrate for the n-alkanes synthesis between lower stems and leaves. Due the low photosynthetic activity at the base of the stem (Brazel and Ó’Maoileídigh, 2019 and refs. therein), n-alkanes produced there must derive their hydrogen (at least partially) from a substrate previously synthetised in leaves (Sessions, 2006; Shepherd and Griffiths, 2006). The δ²H of this substrate would be marked by δ²H_lw, used locally and then transported down to other parts of the plant, influencing the δ²H value of locally produced n-alkanes, especially in organs less exposed to light (as, in our case, the base of the stem). This would explain both the more stable relationship of δ²H_stwax to δ²H_lw (ε_lw-stwax) compared to δ²H_stw (ε_stw-stwax), as well as δ²H_stwax slightly less depleted values compared to δ²H_lwax, as the substrate delivered to the stem would be ²H-enriched after part of ¹H was preferentially removed by biosynthetic ²H-fractionation during the synthesis of leaf wax compounds.

This proposed mechanism closely parallels to the one employed to explain less depleted δ²H_lwax at the base of some monocot and eudicot leaves (Gao et al., 2015). It potentially aligns with other findings indicating that the δ²H values of n-alkanes in leaves are more positive than in stems and more negative than in roots (Gamarra and Kahmen, 2015; He et al., 2020). The δ²H_wax of those stems, exposed to light, would be influenced by the more negative δ²H_stw, unlike our lower stem samples (confined to the section close to the roots), relying more on leaf-synthesised substrate. However, another study (Liu J. et al., 2019) found δ²H_wax to be more negative in roots than leaves in two C₃ species (Artemisia vestita–monocot, and Stipa bungeana–eudicot), and vice versa in Bothriochloa ischaemum, a C₄ monocot species. This underscores the necessity of gathering additional data on plant organs other than leaves to elucidate the variability in δ²H_wax between different plant sections.

4.4 Net ²H-fractionation

Studies on the leaf n-alkane ²H-composition of graminoids and forbs in wetland helophytic communities are limited, with most focused on the highlands of China (Duan et al., 2014; Zhao et al., 2018; Huang and Meyers, 2019; Liu et al., 2023). It is important to note that the degree to which our net fractionation ranges (ε_C29/MAP; C₃ graminoids, summer, −207‰ to −168‰, average −182‰; C₃ forbs, summer, −156‰ to −92‰, average −145‰; Table 5) overlap to the one from other locations might have limited significance. For example, the net-fractionation values for four Chinese macroregions (Liu et al., 2023) show inconsistent overlapping to our monocot and dicot ε_C29/MAP ranges, across different environments and climatic regions. This could derive from variations in sampled species and climatic/geographical conditions. Nevertheless, interesting similarities exist in the ²H data between some Chinese sites and our Greek locations. For instance, the CMWA δ²H_lwax values range at lake Gahai (−205‰ to −192‰) for two emergent C₃ forbs in summer (Knorringia sibirica and Hippuris vulgaris; Duan et al., 2014) falls within the interval observed in our summer forb data (−205‰ to −141‰, species specific; Table 4). Interestingly, the range for all plants from aquatics to trees (−246‰ to −130‰; Duan et al., 2014) is the same we have for just emergent plants (−251‰ to −138‰, graminoids and forbs, summer), probably due to an under sampling of helophytes at Gahai. The net fractionation also aligns, with P. sibirica and H. vulgaris showing a ε_C29/MAP-ε_C27/MAP range (−163‰ to −141‰) substantially matching our ε_C29/MAP range for forbs (−171‰ to −147‰, excluding G. uliginosum −92‰) in summer. However, correlations between more enriched δ²H_lwax and greater leaf surface area or higher ACL, suggested in previous studies, do not hold for our data, neither for forbs (e.g., G. uliginosum shows the most enriched δ²H_lwax while having much smaller leaves than other sampled forb species), nor graminoids.

Table 5

Table 5. Net ²H-fractionation from OIPC 3.1 δ²H_p values of mean annual precipitation (MAP) to leaf wax n-alkane δ²H values, calculated for just n-C₂₉ (ε_C29/MAP) and for the concentration weighted mean of n-alkanes (ε_CMWA/MAP).

More interesting is the comparison with Dajiuhu peatland data (Zhao et al., 2018) which is facilitated by the fact that the analysed emergent C₃ graminoid species belong to the same families as ours (Poaceae, Typhaceae, Cyperaceae, and, in one case, the same genus Carex). In Dajiuhu, the δ²H_lwax range for emergent C₃ graminoids (−253‰ to −165‰) substantially matches ours (−256‰ to −170‰; summer) when all n-C_23-33 odd homologues are considered; less overlap occurs when only n-C₂₉ values are considered (−240‰ to −213‰ in Dajiuhu, −252‰ to −176‰ in Nisí-TP). Even as the OIPC δ²H_MAP for Dajiuhu (−70‰) and Nisí-TP (−51‰) show a ca. 20‰ difference, the average ε_C29/MAP values for C₃ graminoids coincide (Dajiuhu −171‰, Nisí-TP -175‰; Table 5). Dajiuhu’s climate is monsoonal (and not Mediterranean), with heavy, ²H-depleted precipitation during late June and early July, followed by hot and dry summers (Zhao et al., 2018). However, the Dajiuhu and Nisí-TP sites are characterised by similar wetland environments as well as a similar relationship between vegetative period and seasonality, even if in Dajiuhu this is shifted later into summer.

These data show that, while fractionation values may vary greatly between individuals of similar species and sampling instances, a certain integration occurs when the plant communities are considered in their entirety. In particular, they suggest that net fractionation may be relatively constant (Liu et al., 2023) in similar C₃ graminoids helophytic communities, regardless of absolute differences in precipitation δ²H and temperature, as long as (1) the relationship between growing season and precipitation/aridity periods is similar (as between Nisí-TP and Dajiuhu), and (2) the source water δ²H selected to calculate net fractionation actually reflects the real source water δ²H in the local environment (Yu et al., 2021).

However, it should be noted that other studies incidentally show very similar ε_C29/MAP values for C₃ graminoid helophytes (P. australis, −166‰, Qiushui valley, Liu et al., 2017; Typha latifolia, −150‰, Blood Pond, Hou et al., 2007a) or even just C₃ graminoids (Smith and Freeman, 2006; Hou et al., 2007a; Oakes and Hren, 2015; Daniels et al., 2017; Freimuth et al., 2019), without sharing similar climatic conditions and/or species families.

Unfortunately, the same level of comparison between Dajiuhu and Nisí-TP can not be achieved with C₃ forbs species belonging to different families (ε_C29/MAP Dajiuhu −110‰, Nisí-TP -145‰ summer; Table 5). However, both studies report net fractionation to decrease (from more negative to less negative) from graminoids to forbs (Zhao et al., 2018; Liu et al., 2023). Other studies involving vascular plants in wetland environments generally report lower (less negative) ε_C29/MAP values. Both graminoids (n=2) and forbs (n=2) from the Northern Norwegian Hollabåttjønnen bog show extremely low values (−53‰ if δ²H_MAP or −93‰ if July δ²H for graminoids, −71‰ if δ²H_MAP, −110‰ if July δ²H for forbs; Balascio et al., 2018), as well as P. australis from the East Anglian Stiffkey saltmarshes (−144‰; Eley et al., 2014). The same P. australis in Stockholm, Sweden shows lower ε_C29/MAP values (−132‰, only 1 data-point; Yang et al., 2011) and substantially lower values (−95‰) from three high altitude sites along a latitudinal gradient in inner China (Duan and He, 2011). This is a reminder that, although wetland helophytes (particularly C₃ graminoids) appear to tend toward a stable net fractionation value, other factors such as, for example, higher latitude effects on seasonality and growing season (Yang et al., 2011; Daniels et al., 2017; Balascio et al., 2018; Corcoran et al., 2022), salinity effects (Eley et al., 2014; Ceccopieri et al., 2021) and elevation (Duan and He, 2011), must be taken into account when attempting any kind of δ²H_p reconstruction from sedimentary δ²H_wax from wetlands.

4.5 Seasonal differences in the δ²H values of odd/even homologues

Odd numbered n-alkanes consistently exhibited more ²H-depletion than their adjacent even carbon numbered counterparts, with few exceptions (Figure 3). This pattern does not appear to be an analytical artefact, as (1) we assessed only peaks over 2 Vs and corrected for peak area values lower than 40 Vs (see Supplementary Material S1, Supplementary Figure S1), (2) it is evident also in samples where even homologues concentrations occasionally match or exceed odd homologues concentrations, and (3) it aligns with the findings of previous studies on leaf wax n-alkanes, n-alkanols, n-aldehydes and n-fatty acids in higher plants (e.g., Collister et al., 1994; Yang and Huang, 2003; Chikaraishi et al., 2004a; Sachse et al., 2006; Hou et al., 2007b).

To quantify and characterise this odd/even ²H-composition pattern, we devised a Parity Isotopic Difference index (PID; see Section 2.4). PID values (Table 6), ranging from −20 to 51, are almost always positive (except for C. riparia, C. palustre and T. angustifolia in spring) indicating consistently lower δ²H values for n-alkane even homologues. Interestingly, PID values are also consistently higher in summer samples (an increase between +12 and +70 relative to spring values), with the only exception of M. aquatica and S. palustris. The PID average (all species) is significantly (ca 6 times; Supplementary Table S3) higher in summer than in spring, indicating a spring to summer increase in the difference between odd and even δ²H_lwax values.

Table 6

Table 6. Values of PID (Parity Isotopic Difference index) and CPI (Carbon Preference Index) from leaf wax n-alkanes per each species, divided by site and season.

The cause of this behaviour is currently unknown. Drawing from prior investigations (Zhou et al., 2010; Eley et al., 2018), we posit that either environmental factors (e.g., aridity) or physiological influences may have affected the a) ²H-composition of pyruvate, the precursor to both odd and even n-alkane homologues, and/or b) the availability of pyruvate from different sources (e.g., NADPH, carbohydrates; Schmidt et al., 2003; Ladd and Sachs, 2012). The production of n-alkanes is predominantly driven by odd-numbered homologues through the pyruvate-acetate pathway, and primarily utilises strongly ²H-depleted NADPH derived pyruvate (Schmidt et al., 2003). However, a drought related increase in lipid metabolism, which would also lead to an uptick in even-numbered n-alkanes production (which would result also in strongly decreasing CPI values; see Section 4.1.2), could potentially enhance the use of the pyruvate-propionate pathway for the synthesis of even-homologues (Zhou et al., 2010), tapping into less ²H-depleted sources of hydrogen (e.g., pyruvate from carbohydrates or leaf water; Eley et al., 2018), creating a discernible differential fractionating effect on these distinct biosynthetic pathways, and thus be also recorded as a shift in PID.

Interestingly, our data revealed a notable monotonic anti-correlation of PID with plant CPI (Spearman ρ = −0.625, p = 0.001; Kendall τ = −0.492, p = 0.001), which also varies seasonally (see Section 4.1.2). This inverse correlation is evident (Figure 8) across all species, apart from M. aquatica and S. palustris, where seasonal differences in PID and CPI are minimal and can be considered substantially unaltered. The strong correlation with CPI suggests that the PID records a genuine signal, likely arising from seasonal difference in the production of even-homologues.

Figure 8

Figure 8. PID (Parity Isotopic Difference index) and CPI (Carbon Preference Index) cross-plot. Spring and summer values (when both available) for the same species and site are connected by a straight line. All paired values show a PID increase and a CPI decrease from spring to summer (except Phragmites australis in TP and Stachys palustris). Interestingly, PID (and often CPI too) of the few species with available data show virtually the same values in Nisí and TP during spring (Scirpus lacustris, Phragmites australis, Typha angustifolia.

However, due to the low temporal resolution of our data (only spring/summer, each represented by one data-point) and the absence of comparable data from other studies, we lack sufficient data for a conclusive outcome on this matter. Nevertheless, we can speculate on a new hypothetical proxy mechanism involving both CPI and PID. Seasonal variations in plant CPI suggests that shifts in aridity during the vegetative season consistently modify the production of n-alkane even and odd homologues. Despite the lack of a reliable correlation between plant CPI and soil/sediment CPI, as discussed above (4.1.2), due to preferential degradation of longer homologues and microbial activity (Thomas et al., 2021; Corcoran et al., 2022 and refs therein), assuming that the same degradation processes do not modify the ²H-composition of n-alkanes in soil/sediment, the correlation between PID and aridity (and thus also CPI) could potentially be reliable.

The PID index might become a valuable proxy for qualitatively tracing aridity shifts during the growing season of the plant community that contributed to the sedimentary archive. Should these seasonal relationships between CPI and PID, along with the absence of additional ²H-fractionation in sediment, be substantiated through specific studies, it may introduce innovative approaches to qualitatively discern long-term aridity variations within sedimentary paleo-archives. Nevertheless, further research is essential to uncover its origin and assess its applicability for paleo-applications.

5 Conclusion

In order to better understand the environmental and plant physiological controls of n-alkanes as biomarkers in sedimentary archives originated from wetlands, we analysed specific and seasonal variations of (1) n-alkane concentration, distribution, and hydrogen isotopic composition, and (2) ²H-fractionation steps from source water to wax n-alkanes, in leaves/stems of several species from a Mediterranean helophytic plant community.

Based on our concentration data and resulting average chain length index values, it appears that local graminoid species are the predominant source of the local soil n-alkane signal, with a lesser contribution from forbs. This might be attributed to differences in morphology and vegetation structure between these two plant groups.

Our data reveal that the δ²H values of surface and soil water collected between spring and summer align with the local average annual precipitation δ²H signal, which, in turn, closely reflects the weighted average δ²H of winter-spring precipitation derived from available stations data. Furthermore, we observe a consistent agreement in the average δ²H of local surface water, soil water, and stem water, indicating little to no evaporative enrichment of soil water relative to winter-spring precipitation, and confirming negligible ²H-fractionation in the root-stem during water uptake, as previously reported in the literature. Overall, these findings confirm that the δ²H values of the source water accessible to local fen plant communities for wax compounds synthesis indeed reflect the δ²H of annual precipitation, underscoring the potential of δ²H of local plant derived n-alkanes as tracer of long-term seasonality shifts.

Specifically, our concentration and distribution data indicate that, consistently with similar studies in other environments, the primary source of the local sedimentary n-alkane signal derives mainly from the leaves of local fen plants. However, this leaf-stem difference appears to have minimal impact in terms of the δ²H signal. In fact, as anticipated, while leaf water exhibits higher ²H-enrichment compared to stem water, indicative of transpirative enrichment in the leaf water pool, we observe comparable n-alkane δ²H values between lower stems and leaves. This similarity implies that the production of wax compounds in both organs relies on a shared metabolic substrate, likely originating in leaves and synthesised from leaf water.

While we detect significant inter- and intra-specific variability in leaf wax δ²H and related net ²H-fractionation, when integrated at a community level, we noticed a general tendency to stabilise toward known average values from literature, particularly when it comes to plant forms. Compared to studies conducted in similar environments worldwide, our average net ²H-fractionation values (between precipitation and leaf n-alkanes) still rank among the most negative, but this is likely due to a combination of plant typology (grasses) and random differences in the selection of species sampled. However, the C₃ forbs δ²H values for n-C₂₉ (−152‰ ± 27‰) align with those reported for comparable wetland environments in the Chinese highlands, as do C₃ graminoids values (−170‰ ± 26‰), which mirror findings from other studies in similar settings. This similarity suggests relatively consistent net fractionation values for these peri-lacustrine/fen communities not only in the Mediterranean but also in other climatic zones, provided similar seasonality and absence of particular skewing factors (e.g., higher latitude seasonality, salinity). Overall, this means that if information on the main constraining factors is known, these helophytic communities could potentially provide an accurate estimate (at least qualitative) of net ²H-fractionation between δ²H_p to δ²H_wax, particularly when this signal is then buried into the sediment and integrated over longer time scales.

In the Mediterranean context, any future attempt to use δ²H_wax from lacustrine/palustrine sedimentary archives as a paleo-precipitation proxy should consider factors, such as the long-term stability of graminoid dominance and of their net ²H-fractionation value to trace any change in precipitation patterns that would result from the local climate shifting away from typical Mediterranean conditions. In particular, the interpretation of the δ²H n-alkane patterns of the TP sedimentary record (Ardenghi et al., 2019) rely on the assumptions that (1) the long term shift in n-alkane δ²H does not derive from a shift in the composition of the local plant community, and that (2) plant derived n-alkane δ²H is a reliable proxy for the source water of local plants. Our data seem to confirm both points, as (1) local graminoids (more common during colder periods; Pross et al., 2015) present greater net fractionation than forbs, and (2) leaf n-alkane δ²H values show to be a reliable proxy for source plant water δ²H in the local community.

Our data also highlights a consistent decrease from spring to summer of the carbon preference index (CPI) in plant samples, pointing toward seasonal changes in wax lipid synthesis, likely linked to increased drought. Unfortunately, generally lower soil CPI values, likely due to non-homogeneous microbial production and degradation of n-alkane homologues, show that this seasonal signal seems can not be traced through the analysis of soil/sediment CPI.

However, another seasonal pattern emerged from our data: we observed a distinct and consistent ²H-difference between odd- and even-numbered n-alkanes (less and more depleted, respectively) in our plant samples. To quantify this, we introduced a new parity isotopic difference index (PID). The PID consistently increases from spring to summer, indicating a seasonal environmental or physiological influence associated with increased production of even homologues, implying a possible differential ²H-fractionation between the biosynthetic pathways of odd and even n-alkane homologues. This PID seasonal pattern also exhibits a clear inverse correlation to CPI. While we lack sufficient data for a conclusive outcome on this matter, if these seasonal relationships of CPI and PID are validated in specific studies, it could potentially open new avenues to trace long term qualitative aridity shifts through the analysis of the δ²H in odd and even n-alkane homologues in sedimentary archives.

Data availability statement

All raw data will be available on the Pangaea database (https://www.pangaea.de/). The data will also be made available upon request.

Author contributions

NA: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing–original draft, Writing–review and editing, Software. AM: Funding acquisition, Resources, Supervision, Writing–review and editing. JM: Software, Writing–review and editing. DS: Writing–review and editing. AK: Methodology, Resources, Writing–review and editing. EN: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing–review and editing.

Funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. Financial support by the Senckenberg Biodiversity and Climate Research Centre (SBiK-F) is gratefully acknowledged.

Acknowledgments

We thank Dr. Elissavet Dotsika and Dr. Stavros Kalaitzidis for sharing with us their knowledge on Greek peatlands and isotopic hydrology, and Patrick Nowara and Dr. Paraskevi Chantzi for their invaluable help during the field campaign. We thank Ulrich Treffert for his invaluable laboratory support, as well as Dr. Daniel Nelson, Dr. Sarah Newberry, and Dr. Victor Evrard for their fundamental help to processing and analysing our samples at the Basel University botanical laboratories.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor MR-G declared a past Authorship with one of the authors DS.

The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/feart.2024.1359157/full#supplementary-material

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