Isotopic and molecular distributions of biochemicals from fresh and buried Rhizophora mangle leaves†
© The Royal Society of Chemistry and the Division of Geochemistry of the American Chemical Society 2003
Received: 29 July 2003
Accepted: 24 November 2003
Published: 05 December 2003
Rhizophora mangle L. (red mangrove) is the dominant species of mangrove in the Americas. At Twin Cays, Belize (BZ) red mangroves are present in a variety of stand structures (tall >5 m in height, transition ~2–4 m and dwarf ~1–1.5 m). These height differences are coupled with very different stable carbon and nitrogen isotopic values (mean tall δ13C = -28.3‰, δ15N = 0‰; mean tall δ13C = -25.3‰, δ15N = -10‰). To determine the utility of using these distinct isotopic compositions as 'biomarkers' for paleoenvironmental reconstruction of mangrove ecosystems and nutrient availability, we investigated the distribution and isotopic (δ13C and δ15N) composition of different biochemical fractions (water soluble compounds, free lipids, acid hydrolysable compounds, individual amino acids, and the residual un-extractable compounds) in fresh and preserved red mangrove leaves from dwarf and tall trees. The distribution of biochemicals are similar in dwarf and tall red mangrove leaves, suggesting that, regardless of stand structure, red mangroves use nutrients for biosynthesis and metabolism in a similar manner. However, the δ13C and δ15N of the bulk leaf, the biochemical fractions, and seven amino acids can be used to distinguish dwarf and tall trees at Twin Cays, BZ. The data support the theory that the fractionation of carbon and nitrogen occurs prior to or during uptake in dwarf and tall red mangrove trees. Stable carbon and nitrogen isotopes could, therefore, be powerful tools for predicting levels of nutrient limitation at Twin Cays. The δ13C and δ15N of biochemical fractions within preserved leaves, reflect sedimentary cycling and nitrogen immobilization. The δ15N of the immobilized fraction reveals the overlying stand structure at the time of leaf deposition. The isotopic composition of preserved mangrove leaves could yield significant information about changes in ecosystem dynamics, nutrient limitation and past stand structure in mangrove paleoecosystems.
The mangrove environment that dominates the world's tropical and subtropical coasts is currently threatened by clear cutting and nitrification.[1, 2] Mangroves shield coasts from natural episodic events such as hurricanes; serve as nurseries for juvenile fish; are an important source of nutrients for offshore biological production; and are essential for keeping coastal waters free of sediment, which subsequently helps preserve coral reefs.
Rhizophora mangle L., the red mangrove, is one of the major species of mangrove in the Americas. It has a broad range of stand structures (< 1 m to > 60 m) for trees of the same age.[3, 4] These differences in height are due to variations in the balance and concentration of available nutrients (e.g., nitrogen vs. phosphorous vs. potassium), which can fluctuate over very small distances (i.e., meters).[3, 5–7] The red mangrove's adaptability to nutrient alterations and dynamic water levels has ensured survival of this species through sea-level change and anthropogenic nutrient release.
Twin Cays (16°50'N, 88°06'W), a peat-based, 92 ha archipelago of offshore (12 km) mangrove islands, is located inside the crest of the barrier reef of central Belize (BZ). The terrigenous input of organic matter is restricted to dust deposited by wind and rain. The shoreline gradient is intertidal and is interrupted by tidal creeks, open flats, and shallow interior ponds. The vegetation is dominated by the red mangrove with two other species, Avicennia germinans (L.) Stearn, (black mangrove) and Laguncuiaria racemosa (L.) Gaertn. f. (white mangrove), present in lower abundance. Mangrove forests at Twin Cays are characterized by a tree-height gradient that parallels other gradients, such as productivity and tidal flushing[3, 6–8] The tree-height gradient at the site can be subdivided into three zones: (1) A fringe of uniformly tall (5–6 m) red mangrove trees that dominate the seaward-most zone of the islands. (2) A transition zone (2–4 m in height), where all three mangrove species are present. (3) A zone of uniformly stunted (dwarf; ~1–1.5 m) red mangrove trees, with stands in the interior of the islands.
Fertilization experiments at Twin Cays have shown that trees in the fringe zone are nitrogen limited; dwarf trees are phosphorous limited; and trees in the transition zone are limited by both nitrogen and phosphorous. Previous studies at Twin Cays have described the bulk carbon and nitrogen isotopic composition of red mangrove leaves from experimental plots that traverse all three zones of nutrient limitation. A wide range of carbon and nitrogen isotope values has been described for red mangrove, with dwarf trees having the highest carbon (mean δ13C = -25.3‰) and lowest nitrogen isotopic composition (mean δ15N = -10‰), compared with tall trees of the same species (mean δ13C = -28.3‰ and mean δ15N = 0.0‰). This contrasts to relatively uniform carbon and nitrogen isotopic compositions of the peat matrix throughout the differing nutrient gradients at Twin Cays, previously reported.[9, 10]
These isotopic distinctions within the same species of red mangrove may permit inference about past stand structure if (1) it is established which chemical fractions hold the isotopic signal and (2) despite diagenesis, this isotopic signal is retained by the preserved leaves. This paper addresses both of these requirements by measuring the carbon and nitrogen isotopic composition of a variety of biochemical fractions (water soluble compounds, free lipids, acid hydrolysable compounds, individual amino acids and the insoluble residual compounds) in fresh and preserved red mangrove leaves sampled at Twin Cays, BZ. Although previous work has shown that amino acids are labile, they are thought to degrade relatively slowly after burial. [12–15] Recent research has highlighted the immobilization of nitrogen by tannins in mangrove ecosystems; nitrogen from amino acids is believed to be incorporated into complex, recalcitrant pools of organic matter and preserved in peat sediments. These factors provided a focus for the research presented in this paper.
Fresh leaves were sampled from red mangrove trees growing in areas of apparent phosphorous limitation and flooding (dwarf trees) and in areas of nitrogen limitation (tall trees). Preserved leaves were analyzed from two peat cores (~50 cm long) under two mangrove stands at Twin Cays, one from under a dwarf stand and one from under a tall stand. Wooller et al., provided information on the taphonomy of dwarf and tall trees in terms of their bulk isotopic characteristics, and this paper is essentially a continuation of that study.
Field collection of samples from Belize
A fully expanded canopy leaf was taken from three tall and three dwarf Red mangrove trees during October and November 2000 from Twin Cays, BZ. Leaf samples were dried at 50°C under N2 at the Smithsonian Marine Station, Carrie Bow Cay, BZ. Samples were then packaged and transported to the Geophysical Laboratory, Carnegie Institution of Washington for stable isotope analyses.
Leaf samples from mangrove peat
A sediment core was taken to a depth of 50 cm from peat underlying both a dwarf and a tall red mangrove stand. The two sediment cores were sectioned into one cm intervals, placed into Petri dishes and kept at 4°C. Sections of the sediment core were sampled for the remains of red mangrove leaves. Leaf remains were located at ~30 cm in both cores and picked from the surrounding peat. The leaf remains were frozen and freeze dried in a manner similar to the fresh leaf samples and analyzed as reported below. Preserved leaf remains from under the modern dwarf and tall red mangrove stands will be described as 'PL dwarf' and 'PL tall', respectively.
The freeze-dried fresh red mangrove leaves from three dwarf trees and three tall trees and the preserved leaves from the peat cores, were sequentially extracted for free lipids and bound amino acids. Free lipids were extracted by sonication (3 × 5 min) of the dry leaf (20 mg) with dichloromethane and methanol (9 : 1, 10 ml) and separated from the residual leaf via centrifugation. Bound amino acids were extracted from the remaining leaf material, by hydrolysis (1 ml, 6N HCl constant boiling, Pierce Chemicals, 110°C, 20 h), as previously described.[17, 18] Sugars, carbohydrates and free amino acids were extracted from a separate portion of the same leaf, by sonication with water (ultra pure, 3 × 10 ml × 5 min). The water extracts were separated by centrifugation from the residual leaf and freeze-dried for isotopic analysis. The bulk lipid extract, the water soluble extract, the bound amino acid extract, and a portion of residual leaf at each stage of extraction were analyzed for total percent organic carbon (TOC), total percent nitrogen (TN) and stable carbon and nitrogen isotopic composition, as described below.
Bulk carbon and isitrogesi stable isotope analyses
An aliquot (~700 to 800 μg) of each extract was weighed into a tin capsule, which was sealed and introduced via the EA carousel into the autosampler (A2100) of a CE Instruments, NA 2500 series elemental analyzer (EA). Isotope ratios of the combustion gases were analyzed using continuous-flow, stable isotope ratio mass spectrometry (Finnigan MAT, Delta plus XL[10, 17]).
Carbon and nitrogen stable isotope analyses of individual amino acids and free lipids
The bound amino acids (extracted from mangrove leaves) were derivatized with 2-propanol and trifluoracetic anhydride (N-TFA) to form N-TFA amino acid isopropyl esters, similar to previously reported methods.[17, 18, 20] The carbon and nitrogen isotopic composition of each derivatized amino acid was determined by isotope ratio monitoring gas chromatography mass spectrometry (irmGC-MS). irmGC-MS analyses were performed using a Varian gas chromatograph coupled to a Finnigan MAT, Delta plus XL via a combustion interface. The carbon isotopic composition of the individual compounds was measured relative to gaseous CO2 or N2 standards that were introduced as a series of pulses, directly into the ion source at times that did not coincide with the sample peaks. Amino acids were separated via a 50 m HP Ultra-1 analytical GC column that operated under a set temperature program (held at 75°C for 2 min, ramped to 90°C at 4°C per min, held for 4 min, ramped to 280°C at 30°C per min and held for 15 min, the injector temperature was 220°C). The derivatization agents add carbon by way of the addition of isopropyl and N-trifluoroacetyl groups to the amino acid. It was therefore, necessary to calculate δ13C values against known standards that were derivatized and extracted along with each batch of samples. The equation used to determine the final δ13C value for each amino acid was as follows:
where dsa and dst refer to the derivatized sample and amino acid standard, respectively, and Pstd is equal to the proportion of carbon in the derivative from the amino acid. Each sample was run a total of three times and each group (dwarf or tall) had three fresh leaves, making nine runs per group of dwarf or tall trees. The maximum standard deviation for amino acids from plant leaves was determined to be ± 0.86‰. Reproducibility for the δ13C of amino acid standard mixtures was ± 0.59‰. Glycine (gly) co-eluted with an unknown compound and was not used in this study. Derivitization did not disrupt the nitrogen molecules of the amino acids, so nitrogen isotopes were determined directly from the Finnigan Isodat software. Standards were analyzed once and only one of each of the dwarf and tall fresh and preserved leaves were analyzed (four in total).
Distribution of chemical groups in red mangrove leaves
Dwarf red mangrove fresh leaves: Biochemical and isotopic characteristics of separated chemical fractions
Tall red mangrove fresh leaves: Biochemical and isotopic characteristics of separated fractions
Preserved leaves: Biochemical and isotopic characteristics of separated chemical fractions
Carbon and nitrogen isotopic composition of chemical fractions in red mangrove leaves
The carbon and nitrogen isotopic composition of each chemically extracted compound group for dwarf and tall leaves are shown in Tables 1 and 2. Each compound group is isotopically distinct from the bulk leaf, but without exception each has isotopic signatures (δ13C and δ15N) indicative of the parent leaf (Tables 1 and 2). The mean δ13C composition of free lipids is ~2.5‰ more positive in leaves from dwarf trees compared with those from tall trees (n = 3). In contrast to the lipids the carbon isotopic composition of the acid hydrolysable fraction (bound amino acids) in all fresh leaves is ~2‰ heavier than the corresponding parent leaf (Tables 1 and 2). The remainder of the leaf, which does not contain a measurable amount of nitrogen, follows a similar trend with respect to the δ13C composition, being ~2.5%‰ heavier in leaves from dwarf trees compared with those from tall trees. The only measurable nitrogen-containing fraction of the leaf is that within the bound amino acids. The carbon isotopic signature follows the same trend as above (~2.5‰ more positive in leaves from dwarf trees compared with those from tall trees). The nitrogen isotopic compositions are also indicative of the parent leaves being ~9.5‰ lighter in the leaves from dwarf trees compared with those from tall trees (Tables 1 and 2).
A 1-2‰ difference between the δ13C values was evident between the two leaves in each biochemical fraction, in a consistent direction (dwarf > tall). δ15N values were similar in both of the preserved leaves in each fraction of biochemicals and were distinctly different in leaves from dwarf trees compared with those from tall trees (Tables 123). The water-soluble fraction (Table 3), in contrast to the fresh leaves, contained nitrogen and each leaf had a similar nitrogen isotopic composition (-4.94 and -4.69‰ for 'PL dwarf' and 'PL tall', respectively; Table 3). The lipid fractions of each dwarf and tall red mangrove leaf analyzed as part of this study are relatively light (~3‰) in carbon isotopic composition compared with the parent (bulk) leaf (Tables 1 and 2). The δ13C values of the residual leaf fractions of 'PL dwarf' and 'PL tall' were heavier by ~1.5‰ than δ13C values for bulk 'PL dwarf' and 'PL tall'. δ15N values of the residual biochemical fraction were distinctive for each preserved leaf. 'PL dwarf' had a δ15N values similar to that of fresh leaves from dwarf red mangrove (-14.61‰ vs. -9.99‰; Tables 1 and 3) whereas 'PL tall' had a δ15N value similar to that of fresh leaves from tall red mangrove (0.57 vs. 0.07‰, Tables 2 and 3).
Distributions of individual amino acids in red mangrove leaves
However, the relative abundance of identifiable individual amino acids within the leaves from dwarf and tall trees is similar (Fig. 1a). The only noticeable difference between leaves from dwarf and tall trees is observed with the largest amino acid component, which is alanine in the tall leaves, whereas in the dwarf leaves it is glutamic acid. However, the two groups of leaves cannot be statistically distinguished on the basis of their amino acid composition (T <Tcrit at α = 0.05).
Preserved leaves are depleted, in their absolute abundance of amino acids when compared to the fresh leaves (Fig. 1b) and the composition of amino acids is also clearly different. Glycine, tyrosine and arginine are all undetectable in the preserved leaf and ten of the twelve remaining amino acids are present at a lower relative concentration to those in the fresh leaves. Proline and hydroxy-proline are the only amino acids that are present at concentrations within one standard deviation of the modern leaves (proline 4.69, 3.95 vs. 3.34, 5.60 ng mg-1; 'PL dwarf', 'PL tall' vs. fresh dwarf, tall, respectively), or greater (hydroxy-proline 2.12, 1.98 vs. 0.49, 1.23 ng mg-1; 'PLdwarf', 'PL tall' vs. fresh dwarf, tall, respectively) quantities in the preserved leaves versus the fresh leaves (Fig. 1b).
Compound specific carbon and nitrogen isotopic composition of amino acids in red mangrove leaves
δ13C values for individual amino acids in fresh dwarf and tall red mangrove leaves
Dwarf red mangrove leaves
Tall red mangrove leaves
δ13C values for individual amino acids in preserved leaves below dwarf and tall red mangrove stands (n = 3)
Fresh dwarf and tall red mangrove leaves
The similarity in distribution of the majority of biochemical fractions in dwarf and tall red mangrove trees suggests that each of the stands has similar requirements for growth, and each are utilizing the available nutrients in a similar manner. The only exception is the relative depletion of bound amino acids in dwarf versus tall leaves. Amino acid synthesis requires relatively more phosphate molecules than lipid synthesis, and this could explain why the phosphate-limited (i.e. stressed) dwarf trees have a smaller proportion of bound amino acids within their leaves.
Although the stable carbon isotopic composition of each biochemical fraction in the fresh leaves are different compared to that of the bulk leaf, the tall and dwarf leaves can be distinguished on this basis. Tall leaves have a stable carbon isotopic composition that is ~3‰ lighter than dwarf leaves for each biochemical fraction. Free lipids synthesis preferentially utilizes 12C over 13C, [22–24] whereas amino acid synthesis retains 13C and has a relatively heavy stable isotopic composition relative to the bulk leaf.
The unextractable leaf fraction does not contain nitrogen in any of the fresh leaves, and we assume that this fraction consists largely of tannins and lignins. Total polyphenolic compounds, including condensed tannins and residual phenolics (e.g., lignin) have been reported to comprise over 40% by weight of red mangrove leaves (using previously described methods; dwarf leaf = 41.7%, tall leaf = 43.3%) with condensed tannins significantly more abundant in tall red mangrove leaves (20.4%) than dwarf red mangrove leaves (17.7%). There was no significant difference in percentage of the leaf remains after all extractions, between dwarf and tall red mangrove leaves analyzed in this study.
In summary, the carbon isotopic signature of the whole leaf, free lipids, water-soluble extract, and the residual leaf can be used to distinguish dwarf and tall red mangrove leaves. In addition the nitrogen isotopic composition of the whole leaf and the acid hydrolysed fraction of the leaf could be used in a similar manner.
Individual amino acids
The distributions of individual amino acids in dwarf and tall red mangrove leaves were similar (Fig. 1a.), implying that phosphorous limitation, excess nitrogen, water and/or salinity stress experienced by the dwarf red mangrove stands does not alter amino acid biosynthesis and metabolism. The range of δ13C values for all amino acids is wide (Table 4) as recognized in other plants. There is however, a general trend of δ13C values; the majority of amino acids (7 of 12) from tall leaves are relatively depleted in C compared with the dwarf leaves. We do not suggest that the analysis of one individual amino acid would distinguish dwarf and tall red mangrove stands. However, if the total data set of all amino acids is used, the carbon isotopic signature of the leaf is reflected and can be used to discern stand structure.
Our preliminary nitrogen isotopic data suggest that the isotopic composition of all but one amino acid (valine) reflects the nitrogen isotopic composition of the bulk leaves. Leaves from dwarf trees are isotopically lighter than compared to leaves from tall trees (Fig. 3). The distribution of amino acids within tall and dwarf trees is similar and the carbon and nitrogen isotopic composition of each leaf appear to have a similar fractionation during synthesis. This suggests that, whatever the physiological differences between the two stands of red mangrove trees, they are not detectable through differences in amino acid fractionation. Differences between carbon and nitrogen incorporation into the plants are the most important processes for isotopic fractionation of these two nutrients. This finding implies that stable carbon and nitrogen isotopes could be used to assess gradients of nutrient limitation in this complex ecosystem.
Red mangrove leaves preserved in peat
The bulk isotopic signatures of preserved leaves from the peat do not reflect the isotopic compositions of the leaves from the living trees above them. These differences may suggest that the leaves have been diagenetically transformed since deposition; that the isotopic composition of red mangrove at each of the sites has changed since the time of deposition; or that the preserved leaves were transported from a different stand structure prior to deposition. Wooller et al., ascertained that the δ13C of senescent leaves from dwarf and tall red mangrove from Twin Cays retained the isotopic signature of the fresh leaves, so it is assumed that the differences in TOC and δ13C of the lipids in preserved versus fresh leaves must have occurred after deposition to the peat sediments. We believe that diagenetic degradation has selectively degraded 12C versus C in the lipid fraction of the preserved leaves, which would then assume a more positive δ13C signature. The relative depletion in TOC within lipid fractions of the leaves could therefore be explained by sedimentary diagenesis.
The low C/N in 'PL dwarf' and 'PL tall' (5.04 and 5.90, respectively) is indicative of a microbial signature.[9, 10] A relative accumulation of TN would suggest that there has been little or no degradation of nitrogen containing compounds, or that during microbial re-working a different pool of nitrogen was incorporated into the acid hydrolysable traction of the preserved leaf. Based on the C/N of the acid hydrolysed extract and the CSIA of amino acids in the preserved leaves we suggest that the acid hydrolysable fraction of PL dwarf and PL tall is reflecting a microbial signature.
The residual, un-extractable fraction of the preserved leaves contains nitrogen. Moreover, the δ15N of PL dwarf is very light (-14.61‰) whereas, the δ15N value of PL tall is similar to that of the δ15N of fresh tall leaves (+0.57‰). The matrix surrounding the preserved leaves showed little variation in δ15 N (-2‰ to 1.75‰), indeed the only source of nitrogen to the peat sediments that has been shown to have such a wide variation of δ15N values is the difference between fresh red mangrove leaves (leaves from dwarf versus tall red mangrove stands; Tables 1 and 2[7, 10]). Nitrogen immobilization is known to occur in mangrove peat sediments, where it has been reported that nitrogen-containing compounds become bound/absorbed by tannins and are thus effectively un-digestible by bacteria. For preserved leaves to retain a δ15N signature in the residual chemical fraction that is different by ~15 ‰, immobilization of nitrogen probably occurs rapidly, does not discriminate between N and N, and therefore, possibly reflects the δ15N composition of the original nitrogen in the leaf. We conclude that the residual chemical fractions of 'PL dwarf' and 'PL tall' have δ15N signatures that reflect fallen leaves from dwarf and tall mangrove stands, respectively, and that this is the fraction that holds the greatest promise for determining stand structure from isotopic patterns in mangrove peat cores.
Compound specific isotopic analysis of amino acids shows that (1) labeling of all amino acids in both carbon and nitrogen is derived from the original plant carbon and nitrogen, but (2) microbes have completely altered the specific δ13C values. Carbon isotopic differences from the source leaf are apparent. The nitrogen isotopic composition of individual amino acids in the preserved leaves, where differences between the same amino acid in the leaves are up to 10‰ (Fig. 3b), continue to hold the signature of modern dwarf verses tall red mangrove leaves.
This research has shown how the stable isotopes of carbon and nitrogen might be used to predict past mangrove stand structure and alterations in nutrient limitation. The nature of the δ15N of insoluble organic matter in preserved leaves holds promise and is the subject of our future work. Modern tree height gradients are indicative of nitrogen versus phosphorus limitation and height of the water table. Hence, the stable isotopes of carbon and nitrogen will be used to discern the response of red mangrove to environmental change.
† Presented at the ACS Division of Geochemistry Symposium 'Stable isotope signatures for establishing paleoenvironmental change', Orlando, April 2002.
We would like to thank the Station Managers, Dan Miller and Claudette DeCourley, for assistance at the Carrie Bow Marine Station, Belize. This work was funded by NSF Biocomplexity awards to Myrna Jacobson and Marilyn Fogel (Grant numbers 99–81357 and 99–81535). With thanks to Diane O'Brien, Christian Ostertag-Henning, Maureen Reap and an anonymous reviewer whose constructive comments improved our paper.
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