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4-13-1993

Decomposition of Senescent Blades of the Seagrass wrightii in a Subtropical Lagoon

Stephen Opsahl University of at Austin

Ronald Benner University of South Carolina - Columbia, [email protected]

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Publication Info Marine Ecology Progress Series, ed. S. Y. Newell, Volume 94, 1993, pages 191-205. © Marine Ecology Progress Series 1993, Inter-Research.

This Article is brought to you by the Biological Sciences, Department of at Scholar Commons. It has been accepted for inclusion in Faculty Publications by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. MARINE ECOLOGY PROGRESS SERIES Published April 13 Mar. Ecol. Prog. Ser.

Decomposition of senescent blades of the seagrass Halodule wrightii in a subtropical lagoon

Stephen Opsahl, Ronald Benner

University of Texas at Austin, Marine Science Institute, Port Aransas, Texas 78373-1267,USA

ABSTRACT: Senescent blades from the seagrass Halodule wrightii Aschers were suspended in the water column of Laguna Madre (Texas, USA) for a period of 419 d, representing the longest seagrass decomposition study to date. The initial stage of decomposition was characterized by a rapid loss of organic matter (36 % in 24 d) attributed to leaching. A total of 76 % of the organic matter from seagrass tissues was lost by the end of the decomposition period. Of the major bulk constituents measured, neutral sugars were most abundant and accounted for 23 % of the ash-free dry wt of the initial senes- cent material. A complete and early loss of the cyclitol, myo-inositol, a reduction in glucose yields, and a relative enrichment in mannose were the most dynamic features of the neutral sugar fraction. An overall stability series: mannose > fucose > arabinose = rhamnose = galactose = xylose > glucose, re- flects patterns of selective degradation of the polymers from which these sugars are derived. Soluble and ester-bound phenolic acids were lost from tissues at relatively high rates. Susceptibility of individ- ual phenolic acids to removal from bulk tissue upon treatment with weak base correlated well to initial losses in the field. Early changes in total CuO oxidation phenol yields from H. wrightii were caused pnmarlly by the relatively rapid loss of soluble and ester-bound phenolic acids. Therefore, we took a new approach to quantlfy lignin-derived phenols by subtracting soluble and ester-bound phenols from total CuO oxldat~onylelds. When these losses were taken Into account, lignin-derived phenol yields were similar from freshly senescent and highly degraded detntus. Cutln, although initially selectively preserved relative to bulk tissue, was also found at near initial yields In the highly degraded detritus. Overall, the distribution of polymeric constituents (cellulose, hem~cellulose,lignin and cutin) was similar in freshly senescent and highly degraded tissues suggesting that the compounds which com- prise the ultrastructure of H,wrightij are degraded at similar rates Photobleached H. wriglitii blades were characterized by a much higher neutral sugar content, slmilar levels of cutin acids and a nearly complete absence of phenols con~paredto freshly senescent tissue. Photobleaching appears to be an important degradative mechanism which yields a polysaccharide-rich detritus that is devoid of the lignin signature characteristic of vascular tissues.

INTRODUCTION comprise a long-term reservoir of particulate organic matter which also contributes to higher trophic levels Seagrasses are major primary producers in many (Benner et al. 1984, Valiela et al. 1985, Benner et al. temperate and tropical coastal environments, and the 1988). Selective losses throughout decomposition alter fate of seagrass biomass is an integral component of the chemical composition of tissues and thus the avail- the complex flow of carbon and energy in these ability of individual constituents of the detritus to the ecosystems. Upon senescence, carbon losses due to microbial community. leaching contribute to labile pools of dissolved organic Carbohydrates are major components of vascular matter (Godshalk & Wetzel 1978, Rublee & Roman plant tissues. Within the total carbohydrate pool, 1982) leading to elevated levels of microbial produc- neutral sugars are important constituents since the tion (Benner & Hodson 1985, Benner et al. 198613, Blum majority of monosaccharides which form the structural & Mills 1991) which in turn support detrital food webs polymers cellulose, hemicellulose and pectin are (Robertson et al. 1982, Findlay et al. 1986, Peduzzi & included in this class. Cyclitols, also neutral sugars, are Herndl 1991). Structural constituents, such as lignin, important membrane constituents and intermediates cellulose and hemicellulose, degrade more slowly and in cellular metabolism (Loewus & Dickinson 1981), and

Q Inter-Research 1993 Mar. Ecol. Prog.Ser. 94: 191-205, 1993

have been shown to be abundant in seagrasses (Drew thesis that seagrass-derived carbon is transferred to 1980). Taken together, these neutral sugars are likely higher trophic levels in this and other systems (Fry & to be the predominant form of carbohydrate in sea- Parker 1979, Stevenson 1988) via a microbial food web grasses and therefore would comprise the bulk of (Chin-Leo & Benner 1991). To evaluate this transfer of carbon flux from decomposing seagrass tissue. Little carbon more closely, we measured changes in the information regarding the neutral sugar content of aldose, cyclitol, soluble and ester-bound phenols, and seagrasses or the reactivity of specific carbohydrates lignin- and cutin-derived cupric oxide oxidation prod- during decomposition is presently available. ucts during the decomposition of H. wrightii. The Phenolic compounds are common constituents of duration of this experiment (419 d) and the analytical vascular and may be separated into 3 opera- techniques used to measure chemical changes make tionally-defined classes: soluble, ester-bound and this study unique in the area of seagrass decomposi- lignin-derived (Wilson et al. 1986, this study). Soluble tion. Trends are discussed in terms of the chem- phenolic acids have been shown to be abundant in a ical composition of seagrass detritus and biomarker variety of seagrasses (Zapata & McMillan 1979, Buchs- applications. baum et al. 1991) and seagrass extracts rich in soluble phenols have been found to inhibit the growth of bacteria and algae (Harrison & Chan 1980). Ester- MATERIALS AND METHODS bound phenols may be linked to lignin (Higuchi et al. 1967) or cell-wall carbohydrate (Hartley 1973), and are Site description. Seagrass decomposition experi- degraded at considerably lower rates than soluble ments were conducted in the Laguna Madre, which phenols (Wilson et al. 1986). Lignin is a structural spans approximately 200 km from Corpus Chnsti, polymer of phenylpropane subunits which are linked Texas, to the Mexican border. The lagoon covers a by carbon-carbon and ether bonds. Lignin is a unique total area of over 1100 km2 and is separated from the constituent of vascular plants (Sarkanen & Ludwig Gulf of Mexico by Padre Island. This system is unusual 1971) that is typically found to be resistant to microbial in that high levels of sunlight and evaporation, low degradation (Benner et al. 1986b). Therefore, lignin rainfall, minimal freshwater inflow and distant tidal can be useful as a biomarker for -derived passes all contribute to hypersaline conditions charac- organic matter in heterogeneous samples such as teristic of this lagoon throughout much of the year. The sediments, and dissolved organic matter (Hedges average water depth in the Laguna Madre is about 1 m & Parker 1976, Meyers-Schulte & Hedges 1986, and there is a very limited tidal range, however, occa- Hamilton & Hedges 1988). By making these distinc- sional inundations such as hurricanes, completely flush tions between individual pools of phenolic compounds, the system with freshwater. associated relative reactivities and degradation rates The vast majority of the lagoon bottom is covered by can be investigated. seagrasses with Halodule wrightii being the predomi- Cutin is an important component of the protective nant species. With the exception of ducks which feed outer covering (cuticle) of vascular plant herbaceous on H. wrightii roots and during the winter, tissues (Martin & Juniper 1970). Taxonomic distinc- grazers have a minimal impact on the seagrass bio- tions based on characteristic distributions of individual mass. Typical throughout the duration of this cutin-derived hydroxy acids have been established study ranged from 33 to 55%" and water temperatures (Holloway 1982, Goni & Hedges 1990b) so that these ranged from 0 to 30°C. compounds may serve as specific geochemical indi- Decomposition experiment. Samples of green and cators of particular plant types. The degradation of senescent seagrass material were collected during cutin acids in conifer needles has been studied (Goni & September 1988. Attached blades of Halodule wnghtii Hedges 1990c), and as a group, cutin acids were found which were entirely green in color were collected for to be more reactive than either the lignin or carbo- a representation of fresh, actlvely growing tissue. hydrate fractions. The reactivity of individual cutin Attached blades which were green with some areas of acids was also shown to vary. In order to determine the yellowish-brown color were picked to represent senes- relative stability of seagrass-derived cutin, chemical cent tissue. This senescent tissue was chosen as the changes which take place during the decomposition of initial starting material in the litter bag experiment. seagrass detritus need to be evaluated. Large quantities of decaying seagrass are pushed In the present study, we investigated the short- and onto the shoreline during periods of strong wind and long-term decomposition of senescent above-ground remain out of the water for long periods of time. This Halodule wrightii Aschers tissues in the Laguna portion of the shoreline is typically dry, although high Madre, Texas, USA. Increasing evidence, such as and rain will occasionally saturate this area with stable carbon isotope analyses, supports the hypo- water. A sample of dried seagrass blades which was Opsahl & Benner:Decomposition of senescent Halodule wrightii 193

entirely white in color (photobleached) was collected chemical analyses could be evaluated on an ash-free from the supralittoral portion of the shoreline adjacent dry wt (AFDW) basis. to the study site. This material is subjected to high Neutral sugar determinations. Neutral sugars were levels of sunlight which is not attenuated by the water measured by the method of Cowie & Hedges (1984a). column. An additional sample of brown seagrass A 20 mg sample was pre-treated with 12 M H2S04for detritus was collected directly underneath the photo- 2 h, diluted to 1.2 M and hydrolyzed 3 h at 100°C. After bleached layer. Photobleached material is present hydrolysis, adonitol was added to the mixture as an along virtually the entire perimeter of the Laguna internal standard. The sample was neutralized with Madre. Ba(OH)2, deionized in a mixed bed of cation/anion Immediately after collection, all seagrass blades exchange resins, dried in a Savant Speed-Vac centrifu- were rinsed in distilled water to remove salt. Separa- gal evaporating system and resuspended in pyridine. tion of epiphytic algae from associated seagrass tissue Sorbitol was added to the sample as an absolute was not a concern in this study since the high salinities recovery standard. Next, an equal volun~eof 0.4 % restrict growth of epiphytes. The effects of drying leaf (w/v) LiC104 in pyridine was added and the mixture litter before experimental n~anipulations has been a then held at 60°C for a 48 h equilibration period. The topic of concern in decomposition studies. In the mixture was derivatized by adding Sylon BFT Laguna Madre, large wracks of seagrass detritus are (Supelco) and held at 60°C for an additional 10 min. frequently pushed out of the water during which time The resulting mixture of trimethylsilyl derivatives was they undergo considerable drying at temperatures in separated on a Hewellet Packard 5890 gas chromato- excess of 40°C. The extent to which seagrass detritus graph using simultaneous analyses on non-polar and undergoes submergence and drying in its natural polar capillary columns (DB-1 and DB-1701, J&W setting has not yet been determined. In this study, a Scientific). The column temperature program began drying temperature of 45°C was chosen to minimize at 14OoC, held for 4 min, increased to 270°C at potential changes in chemical composition yet permit 6°C min-', and then held for an additional 4 min. The accurate mass loss measurements, although we do DB-1 column was used for most quantifications but recognize that rates of decomposition, primarily during occasional CO-elutions were detected by comparison the leaching phase, may be enhanced as a result of with the DB-1701 column and these sugars were drying (Harrison & Mann 1975). quantified using DB-1701 data. At equilibrium, each Litter bags. Litter bags measuring 43 X 33 cm were sugar may have from 1 to 5 detectable isomeric peaks constructed out of 202 pm Nitex mesh (Tetko, Inc.). and the largest of these which was clearly resolved Senescent seagrass blades (10 g) were placed in each was used for quantification. Lyxose and ribose were litter bag and the bags were sewn shut with nylon nearly absent in all tissues, producing small peaks on thread. Bags were spaced 30 cm apart and suspended the gas chromatograph whch could not be identified in the water column from nylon rope which was or quantified reliably, and therefore were considered secured to PVC pipes. Bags were attached to the nylon trace constituents. rope using cable ties. The design was such that the Individual sugar monomers produced during the litter bags would be suspended in the water column hydrolysis of polymeric carbohydrate, become hy- above the sediment but would remain submerged drated at the glycosidic linkage during cleavage. For even during the lowest tides. Bags were placed in the final calculation of neutral sugar yields, this weight field on October 28, 1988 and 2 bags were harvested at gain is subtracted from total yields, reducing them by approximately monthly intervals. ca 10% depending on the molecular weight of the After collection, bags were transported to the labora- sugar monomer. Neutral sugar yields have not been tory where they were rinsed in distilled water. The adjusted to account for incomplete hydrolysis of poly- contents of the bags were emptied into a clean glass mers. For example, under optimal conditions, hydroly- dish filled with distilled water, which facilitated careful sis of purified cellulose yielded an 80% recovery of separation of recognizable seagrass detritus from other glucose (Cowie & Hedges 1984a). material. Most of the extraneous material consisted Sample mean deviation is reported to be 5 to 10 % of small bivalves and molluscs whch likely entered for individual sugars but typically falls below 5 % for during larval or juvenile stages and matured inside the more abundant sugars (Cowie & Hedges 1984a). In bags. Once rinsed and separated, the seagrass detritus this study, we analyzed each bag twice for neutral was placed in a drying oven at 45°C for 48 h and the sugar content. We then calculated sample mean devi- contents were then weighed. The contents of the bags ations for replicate litter bags harvested at each time were not combined and all chemical analyses were point. Average percent mean deviations for each made on individual bags. Duplicate sub-samples from sugar calculated from the time series ranged from 4 to each bag were combusted at 550°C for 6 h so that all 13 %. 194 Mar. Ecol. Prog. Ser. 94: 191-205, 1993

Soluble phenolic acids. Numerous monomeric and to the sample after bombing as an internal standard. polymeric aromatic constituents are released upon The sample was rinsed out of the mini-bomb with extraction in 80 % methanol (Van Sumere 1989). This 20 m1 of 1 N NaOH, centrifuged and decanted. This extraction releases free phenolics but leaves those rinsing procedure was repeated 2 times and the com- bound by ester-linkages intact (Van Sumere 1989). In bined extracts were acidified to pH 1 with 6N HCl. The the present study, several important monomeric mixture was extracted 3 times with freshly distilled phenols released by methanol extraction were identi- ether and dried under nitrogen. The dried sample was fied and quantified. Vanillic, syringic, p-coumaric and resuspended in pyridine from which a subsample was ferulic are among the most abundant phenolic acids transferred to another vial. This subsample was mixed found in seagrasses, although others have been identi- with an equal volume of Sylon BFT (Supelco Inc.) and fied (Zapata & McMillan 1979). We have focused on heated at 60°C in a heating block for 10 min. The these phenolic acids in order to make comparisons resulting trimethylsilyl derivatives were separated with these same compounds which are also found in, using the same gas chromatograph and capillary ester-bound form and are produced from lignin during columns used for neutral sugars. CuO oxidation (see below). Concentrations for most compounds agreed very To measure soluble phenolic acids, a 200 mg sample well between columns (+ 10 %) with the exception of p- of dried plant material was extracted 3 times in 10 m1 hydroxybenzaldehyde and ferulic acid which have of 80% methanol at 80°C for 10 min. Ethylvanillin been previously reported to have CO-eluting com- was added as an internal standard prior to the initial pounds on the DB-l column (Hamilton & Hedges methanol extraction. The extracts were combined and 1988). Each litter bag was analyzed for lignin-derived dried by evaporation under nitrogen. Extracts were phenols. Sample mean deviations were calculated then resuspended in water and acidified to pH 1 with from replicate litter bags harvested at each time point. 6 N HCl. Subsequent ether extraction and analysis for The average percent mean deviation for individual individual phenols proceeded as described below for phenols throughout the time series ranged from 10 to lignin-derived phenols. 20 %. Peak identification for some of the phenols was Ester-bound phenols. A relatively mild hydrolysis sometimes difficult in the more highly degraded with weak base was used to release soluble and ester- samples because they occurred in low concentrations. bound phenols (Whitehead et al. 1981). Ester-bound To verify identification of these peaks, some samples phenols are defined herein as those phenols which are were CO-injectedwith a small amount of the standard released upon mild base hydrolysis minus the soluble solution. Elevated peak areas for perfectly CO-eluting phenols which are extracted in 80% methanol. peaks on both columns was accepted as a means of Recovery and determination of ester-bound phenols positive identification for CO-injectedsamples. For pos- proceeded as follows: a 200 mg sample of dried plant itive identification of the lignin-derived phenols (which material was placed in a 50 m1 screw-cap tube with were low in concentration relative to most other 16 m1 of 1 N NaOH. This mixture was then spiked with vascular plants), the initial senescent starting material the internal standard ethylvanillin. The sample was was analyzed by gas chromatography/mass spectro- reacted for 20 h at room temperature with a magnetic metry (GUMS), which verified the presence of all 1l stir bar for mixing. At the end of the hydrolysis, the index phenols produced during the CuO oxidation of samples were filtered through pre-combusted What- lignin. In order to quantify those phenols derived ex- man GF/F filters. The hydrolysate was acidified to clusively from lignin, they must be distinguished from pH 1 with 6 N HCl. Subsequent ether extraction and soluble and ester-bound phenols. We herein define analysis for individual phenols proceeded as described lignin-derived phenols as those phenols produced below for lignin-derived phenols. The yields of soluble from the CuO oxidation of whole tissue minus phenols phenols were always a small (

acids was a problem on the DB-1701 column, therefore been shown to release more soluble constituents from all measurements of these conlpounds were made the particulate detritus of aquatic macrophytes (God- using the DB-1 column. Commercial standards are not shalk & Wetzel 1978, Benner & Hodson 1985, Valiela et available for most of these compounds. Therefore, rel- al. 1985). A total of 76 % of the organic mass was lost ative retention times were related to a series of CI3to after 419 d providing a decay rate of 0.18 'Xodd', which saturated fatty acids as described by Goni & falls within the lower range of decay rates based on Hedges (1990a). All concentrations for cutin-derived linear models reported for a variety of seagrasses compounds for which we had no standards were calcu- summarized in a review by Harrison (1989) However, lated using the response factor of ethylvanillin. this survey includes decomposition data from studies Additionally, the initial starting material was analyzed which vary in length, seagrass species, initlal starting by GUMS to confirm the identity of the 4 cutin acids material, litter bag mesh size and field conditions measured. The abundance of individual isomers of 2 of making any direct comparisons of decay rates difficult. the CI6 hydroxylated fatty acids [(8, 9 or 10)- dihydroxyhexadecanoic acid (X, 0-CI6)and (7 or 8)- hydroxyhexadecane-l,16-dioicacid (X-C,,DA)] were Neutral sugars not determined for the time series, therefore only the sum total which elutes as a single peak during gas Yields of the individual neutral sugars from all chromatography was reported for each of these com- samples are presented in Table l. Glucose was pounds. Average percent mean deviations were the most abundant neutral sugar accounting for calculated as described for lignin-derived phenols and 176.4 mg g-I AFDW and 74 % of the total neutral sugar ranged from 12 to 16 % for the individual cutin-derived yield in freshly senescent tissues. Corresponding hydroxy acids. yields of other neutral sugars ranged from 3.3 mg g.' in the case of myo-inositol to 17.3 mg g-' for xylose. Myo-inositol was the only cyclitol recovered from RESULTS AND DISCUSSION Halodule wrightii which agrees with results from a study on the soluble carbohydrate composition of Weight loss seagrasses by Drew (1980). Total neutral sugar yields accounted for 24 % of the total organic mass of freshly Decomposition, in this study, refers to the loss of dis- senescent H. wrightii blades. solved and particulate organic matter from the original Large changes in distributional patterns of indi- starting material which was placed in the litter bags. vidual sugars were evident during the first 24 d of Losses include complete mlneralization, leaching of decomposition. Like conifer needles (Hedges & Weliky dissolved organic constituents and loss of small par- 1989) and mangrove leaves (Benner et al. 1990a), ticulate matter (< 202 pm) Halodule wrightii lost Halodule wrightii lost all myo-inositol during the 35.5% of its initial weight (AFDW) after only 24 d in earliest stages of decomposition, and this cyclitol was the field (Fig. l), primarily due to leaching which has the most labile constituent measured. Losses of indi- vidual sugars during the first 24 d of decomposition ranged from 21 % in the case of glucose to 54 O/ufor mannose. Approximately 26% of the total initial neutral sugar pool was lost during this time. Abundance patterns for neutral sugars during the latter portion of the litter-bag study included relative enrichment in mannose, arabinose and fucose, which accounted for 8.6, 6.8 and 3.0 mg g-' AFDW of senescent tissue and 17.0, 8.1 and 4.3 mg g-' AFDW after 419 d of decomposition. Rhamnose and xylose yields demonstrated little net change during the study. t,,,,[ l.., l,...l, .,, Glucose underwent a major reduction in yields from 0 r 0 100 200 300 400 500 176.4 to 111.2 mg AFDW. Xylose was the only sugar which maintained a fairly constant proportion of Days in Field the total neutral sugar content (8.0 +0.4 %). Yields of galactose varied starting with 12.2 mg g-' AFDW in Fig. 1. Halodule wrightjj. Weight loss expressed as a percent- senescent tissue, which decreased rapidly to 7.5 mg g-' age of ash-free dry weight (AFDW) remaining. Each symbol represents the average weight loss for replicate litter bags. AFDW after 49 d and then consistently increased to Error bars depict sample mean deviations 12.1 mg g-' AFDW by the end of the study. Total neu- 196 Mar. Ecol. Prog. Ser. 94: 191-205, 1993

Table 1. Halodule wrightii. Neutral sugar yields (mg carbohydrate g-' AFDW) for green, senescent, decomposing and photobleached seagrass blades. Deviation: average percent mean deviation for decornposlng samples. GLU: glucose; GAL: galactose; MAN: mannose; RHA: rhamnose; FUC: fucose; XYL:xylose; ARA: arabinose; MYO:myo-inos~tol; Total: total yield of neutral sugars

Sample GLU GAL MAN RH A FUC XYL ARA MY0 Total

Green 169.9 11.1 6.7 7.5 2.5 17.1 6.1 7.0 Senescent 176.4 12.2 8.6 9.6 3.0 17.3 6.8 3.3 Decomposing (d) 24 213.7 9.0 6.1 10.5 2.8 20.9 7 0 0.0 4 9 184.7 7.5 7.9 10.7 2.7 18.3 6.5 0.0 136 180.4 8.6 7.2 10.5 2.4 19.2 7.8 0.0 166 159.7 8.5 7.9 11.6 2.9 16.2 8.0 0.0 195 138.8 8.7 8.0 12.1 2.9 16.6 8.0 0.0 227 141.6 8.0 8.1 11.9 2.7 16.0 8.2 0.0 269 161.6 9.5 9.3 12.7 3.4 19.1 8.8 0.0 305 142.4 10.0 14.5 11.6 3.8 16.4 8.1 0.0 339 117.6 10.4 18.3 10.5 4.5 14.4 7.6 0.0 367 136.2 9.8 13.2 11.1 3.7 16.3 7.9 0.0 395 130.9 10.8 15.3 11.2 4.5 17.1 8.2 0.0 419 111.2 12.1 17.0 10.1 4.3 15.2 8.1 0.0 Deviation: (4 %) (6%) (13 %) (5 %) (7 %) (5%) (4 %) Photobleached 321.5 8.3 8.3 14.8 3.3 30.2 6.6 0.0 tral sugar yields varied from 237.2 mg g-' AFDW in conversion of commercially prepared cellulose to senescent tissues, to 178.0 mg g-I AFDW by the end of glucose without the H2S04 pretreatment. Therefore the study, and this trend in total neutral sugar yield we estimate that non-cellulose glucose accounts for was essentially determined by changes in glucose ca 10% of the total glucose pool in senescent H. abundance. wrightii and cellulose comprises the remaining 90 %. The neutral sugar profile from photobleached Notably, 82 % of the total mannose pool also required detritus varied considerably from senescent and highly pretreatment for recovery. Therefore, mannose, degraded submerged tissues with respect to individual which is a C, epimer of glucose, may be formed pri- sugars and total recoveries (Table 1).The yield of glu- marily as a hydrolysis product from the relatively cose (321.5 mg g-' AFDW) was 1.8 times that of senes- large amount of glucose present in the hydrolysate cent tissue and 2.9 times that of highly decomposed mixtures. submerged material. Glucose accounted for 82% of the total neutral sugar content and 32% of the total organic mass of this detritus. The xylose yield was Soluble phenolic acids 30.2 mg g-' AFDW, 1.8 times that of senescent tissue. This detritus was also slightly enriched in rhamnose, Three soluble phenolic acids were detected in low slightly depleted in galactose and completely depleted concentrations in the methanol extract of senescent in myo-inositol relative to undegraded material. tissue (Table 2). Vanillic acid was more abundant Overall, 39% of the total organic mass of bleached I0.18 mg (10 g AFDW)-'1 than p-coumaric I0.09 mg detritus could be accounted for as neutral sugars (10 g AFDW)-'1 and ferulic acid I0.13 mg (10 g compared to 18 to 27 O/ofor the submerged blades in AFDW)-l].These soluble phenolic acids made up ca the litter bag study. 4 % of the total phenol y~eld(Table 2) from CuO oxida- The H2SO4 pretreatment is necessary in order to tion of senescent Halodule wrightii. Soluble phenolic efficiently hydrolyze cellulose. An additional analysis, acids were virtually absent from all litter bag samples in which the 12 M H2S04pretreatment was excluded, (with the exception of low levels of ferulic acid after was made on senescent Halodule wrightii. By exclud- 24 d) which indicates that these compounds are rapidly ing the pretreatment, a measurement of non-cellulose leached from H. wrightii detritus. glucose is possible and cellulose can then be calcu- In contrast to these relatively low concentrations of lated by difference from a sample in which the pre- specific phenolic acids, Buchsbaum et al. (1991)report treatment has been included. About 14% of the that fresh blades from the seagrass Zostera marina glucose was recovered without the H2S04 pretreat- were comprised of ca 10 % total soluble phenolics ment. However, Cowie & Hedges (1984b) found a 4 % as measured using the Folin-Denis procedure after Opsahl & Benner: Decomposition of senescent Halodule wrightii 197

Table 2. Halodule wrightii. Phenol yields [mg phenol (10 g AFDW)-'1 for green, senescent, decomposing and photo- bleached seagrass blades. Deviation: average percent mean deviation for decomposing samples (lignin-derived phenols only). VAL: vanillin; VON: acetovanillone; VAD: vanillic acid; SAL: syringaldehyde; SON: acetosyringone; SAD: syringic acid; CAD: p-coumaric acid; FAD: ferulic acid; C8: sum of all 8 phenols

Sample VAL VON VA D SAL SON SAD CAD FAD X8

Soluble phenols Green 0.15 0.00 0.00 Senescent 0.18 0.00 0.00 Decomposing (d) 24 49 136 305 3 67 419

Ester-bound phenols Green 2.38 0.00 0.00 Senescent 2.55 0.00 0.00 Decomposing (d) 24 49 136 305 367 419

Green Senescent Decomposing (d) 24 4 9 136 166 195 227 269 305 339 367 395 419 Deviation: Photobleached

"Green, senescent and 24 to 136 d tissue calculated as total phenols produced upon CuO oxidation minus soluble and ester- bound phenols. Beyond 136 d, total CuO oxidation yields are presented because soluble and ester-bound phenols are virtually absent

extraction in 80 % methanol. This colorimetric determi- Ester-bound phenols nation of the total soluble phenolic pool includes mea- surement of a heterogeneous mixture of monomeric Four phenols, vanillic, syringic, p-coumaric and and polymeric phenolic compounds. If these 2 different ferulic acid, were identified in the base hydrolysates species of seagrass are similar in their total phenolic (Table 2). Ester-bound vanillic, p-coumanc and ferulic composition, then the phenolic acids that we identified acid accounted for 93, 92 and 71 % respectively of their comprise only a small fraction of the total phenols in totals (soluble + ester-bound phenols) released from the methanol soluble extract. senescent tissue upon hydrolysis in base indicating Mar. Ecol. Prog. Ser. 94: 191-205, 1993

that these ester-bound phenols are approximately an CuO oxidat~oninto 3 categories: soluble, ester-bound order of magnitude more abundant than soluble phe- and lignin-derived phenols. We have defined lignin- nolic acids in Halodule wrightii. These phenolic acids derived phenols as those phenols produced from the were the dominant components of the total soluble and CuO oxidation of whole tissue minus phenols released ester-bound phenols in H. wrightii. Vanillin, aceto- after mild base hydrolysis. Using this definition, we are vanillone, syringaldehyde, and acetosyringone were now able to exclude soluble and ester-bound phenols undetectable in the basic hydrolysate (Table 2), from those which are derived exclusively from lignin. although vanillin has been reported in base extracts Other studies have questioned whether or not lignin of soils and plant roots (Whitehead et al. 1981). is present in seagrasses (see Buchsbaum et al. 1991 However, their inherently low concentrations in sea- and references therein). Part of this discrepancy lies in grass tissues make detection and quantification diffi- the methodologies used to measure lignin. Acid deter- cult and trace amounts of some of these compounds gent or Klason lignin (Klumpp & van der Valk 1984, may in fact be released during mild base hydrolysis. Buchsbaum et al. 1991), acetyl bromide digestion (Wil- Over 75 % of the ester-bound phenols were lost from son et al. 1986) and conjugated and non-conjugated litter bags after only 24 d in the field (Table 2), indi- phenols (Baydoun & Brett 1985) have all been used to cating that ester-bound phenols are labile relative to measure lignin in seagrasses. These methods rely on bulk tissue and susceptible to leaching, microbial gravimetric or colorirnetric measurements for final de- decomposition, or both. Between 24 and 136 d, the per- termination of lignin and are subject to contamination centage of initial ester-bound phenol remaining had by non-lignin compounds, such as tannins and cutin decreased to <5 % and was comprised solely of p- (Benner et al. 1990b). Alternatively, molecular-level coumaric acid. As seen from the decomposition series, analyses of CuO oxidation products (Hedges & Mann the large pool of relatively labile soluble and ester- 1979, this study) or pyrolysis mass spectrometry (Boon bound phenols, initially present in these tissues, was & Haverkamp 1982) provide positive identification of lost during the first few months of decomposition. lignin through recognition of characteristic compounds or mass fragments. As seen in Table 2, all 8 characteristic lignin-derived Lignin-derived phenols phenols were present in all litter-bag samples. Based on the presence of lignin-derived CuO oxidation Lignin is a heteropolymer of phenylpropane subunits products (which were verified by GUMS), we provide bound by ether and carbon-carbon linkages and is unambiguous evidence for the presence of lignin in unique to vascular plants (Sarkanen & Ludwig 1971). Halodule wrightii. For comparison, Haddad & Martens Rigorous conditions, such as alkaline CuO oxidation, (1987) also detected vanillyl, syringyl and cinnamyl are required for cleavage of the carbon-carbon and phenols in the seagrasses H. wrightii and Ruppia sp. ether bonds in lignin. The CuO oxidation of lignin pro- These authors also analyzed Zostera manna, another duces 11 predominant phenolic monomers, and these species of seagrass, and found vanillyl but no syringyl are separated into 4 families: the p-hydroxy, vanillyl, phenols. Likewise, Wilson et al. (1985) also analyzed syringyl and cinnamyl phenols (P, V, S, C respectively). Z. marina and found vanillyl but no syringyl phenols, The p-hydroxy, vanillyl and syringyl families each while Hedges & Mann (1979) did not find any vanillyl have 3 members, an acid, aldehyde and ketone, and or syringyl phenols in Z. manna. Based on these the 2 cinnamyl phenols (p-coumaric and ferulic studies, the lignin content of Z. marina is questionable acid) are both acids. p-Hydroxybenzaldehyde and p- because it lacks the charactenstic syringyl phenols. hydroxybenzoic acid may have both lignin (Sarkanen Furthermore, Boon & Haverkamp (1982) analyzed & Ludwig 1971) and non-lignin sources (Hedges & Z. manna using pyrolysis mass spectrometry and Parker 1976), therefore, p-hydroxy phenols are not reported that peak intensities for the lignin-derived treated further in this manuscript. pyrolysis products were 'rather unusual'. A more Many of the individual phenols produced from lignin comprehensive survey of other seagrass species, which during CuO oxidation are also present in soluble form includes quantification of soluble and ester-bound or may be ester-bound to cell wall constituents (this phenols, is needed to determine whether other species study, Table 2; Higuchi et al. 1967, Hartley 1973). As of seagrass contain lignin. demonstrated above, monomeric soluble and ester- Green and senescent tissue had higher yields of bound phenols are readily extracted in mild base, lignin-derived phenols (C8; Table 2) than 24 d tissue whereas lignin-derived phenols are not. In this study, indicating losses of lignin during the leaching stage of we have taken a new approach to more accurately decomposition. Between 24 and 419 d, 28 was variable quantify the CuO oxidation products of lignin by and did not change significantly during decomposition separating the total phenol yields produced upon (p> 0.05). Vanillin and syringaldehyde were the pre- Opsahl & Benner. Decomposition of senescent Halodule wrightii 199

dominant components produced from Table 3. Halodule wrightii.Cutin acid yields (mg g-' AFDW) for green, ~~l~d~l~wfightjj lignin throughout the senescent, decomposing and photobleached seagrass blades. Deviation: average percent mean deviation for decomposing samples. o-C,,: 16- study, Vanillyl yields were slightly higher hydroxyhexadecanolc acid; C,,,DA: hexadecane-1,16-dioic acid; x, WC,, than syringyl which in turn were (8, 9 or 10),16-d~hydroxyhexadecanoicac~d,x-CI6DA: (7 or 8)-hydroxy- higher than cinnamyl phenol yields, and this hexadecane-l,l6-dioic acid; LCA: sum of cutin-derived acids pattern varied little during decomposition.

Total yields for green and senescent material Sample w-Clb ClbDA X, w-Clb X-CIBDA ZCA were 3 to 8 times lower than most other herbaceous angiosperm tissues (Hedges & Green 0.09 0.05 0.58 1.23 1.95 Mann 1979) due to a much lower lignin con- Senescent 0.09 0.03 0.56 l .03 1.71 tent in seagrasses. Decomposing (d) Particulate material which had been hy- 24 0.13 0.04 0 73 1.17 2 07 49 0.09 0.04 0.68 1.12 1 93 drolyzed in 1 N NaOH was subjected to 136 0.12 0.04 0.89 1.44 2.48 CuO oxidation and produced consistently 166 0.11 0.04 0.76 1.21 2.11 lower individual phenol yields (data not 195 0.11 0.04 0.91 1.35 2.41 shown) and a substantially lower total phe- 227 0.12 0.04 1-01 1.49 2.66 0.05 1.15 1.85 3.15 nol content for green, senescent and 24 d 269 0.11 305 0.07 0.04 0.94 1.39 2.44 detritus [2.49, 3.21 and 3.08 mg (10 g 339 0.09 0.03 0.88 1.13 2.12 AFDW)-' respectively] when compared to 367 0.08 0.03 0.82 0.61 1.54 lignin-derived phenols [7.63, 6.00 and 395 0.05 0.08 0.60 0.91 1.64 4.89 mg (10 g AFDW)-'; Table 21 from un- 419 0.03 0.14 0.39 0.84 1.40 %) (10 %) (12 X) (12 %) treated starting material. This indicates that Deviation: (16 mild base hydrolysis removes some poly- Photobleached 0.03 0.04 0.33 1.28 1.68 meric lignin in addition to soluble and ester- bound phenols. These results agree with those from dioic acid (C16DA);(8, 9 or 10),16-dihydroxyhexade- Benner et al. (1984). Using a variety of estuarine canoic acid (X, 0-CI6); and (7 or 8)-hydroxyhexa- macrophytes, these authors found that less than 30 % decane-1,16-dioic acid (X-C16DA)]were obtained from of base soluble lignin was determined to be ether sol- senescent tissues in yields of 0.09, 0.03, 0.56 and uble (and potentially identifiable as phenolic acids), 1.03 mg g-l AFDW, respectively (Table 3). The lack of leaving the majority of this base-soluble lignin un- CIBacids and predominant X, U-C,~and X-CIGDAacids characterized at the molecular level. Mild base make H. wrightii cutin rather unusual with respect to hydrolysis can be used to measure the soluble and other included in a survey of 67 plant ester-bound phenol content, which, when subtracted species subjected to CuO oxidation (Goni & Hedges from total phenols produced upon CuO oxidation of 1990b). H. wrightii also differs from the seagrass whole tissue, will provide a measure of lignin-derived Zostera marina which produced only trace amounts of phenols. However, due to the substantial quantities of CI,DA (Goni & Hedges 1990b). base-soluble lignin lost from the particulate material Both the C16DA and U-C,, acids occur near the upon hydrolysis, sequential treatment of the same detection limit, making trends in abundance difficult sample of dried plant material to measure both base- to detect. Yields of the more abundant X, wCI6and soluble phenols and lignin-derived phenols is not X-C16DAacids (Table 3) exhibited 2 apparent trends. possible unless the additional base-soluble lignin is During the first 269 d of the study, which corresponds accounted for. to ca 72 O/oorganic mass loss, yields of both of these hydroxy-acids increased to an even greater extent than did lignin. Beyond 269 d, these yields declined to Cutin-derived fatty acids a point In which the highly degraded detritus was slightly depleted in these compounds relative to the CI6and Cle hydroxylated fatty acids are the 2 major starting material. These 2 phases which characterize families of cutin acids produced upon CuO oxidation of the decomposition of cutin appear to be unique cutin polymer (Goni & Hedges 1990a). The complete among measured classes of major biochemical con- absence of any cutin-derived CI8 fatty acids from stituents in this as well as other studies. It appears Halodule wrightii tissue suggests that this angiosperm that the physical integrity of the cuticle remains at species may no longer utilize the biochemical path- least partially intact during leaching and the earlier ways for synthesis of the CI8cutin acids (Kolattukudy & stages of decomposition, but eventually breaks down Espelie 1985). Four members of the C,, family 116- at which time the component hydroxy fatty acids hydroxyhexadecanoic acid (0-CI6);hexadecane-1,16- degrade more rapidly. 200 Mar. Ecol. Prog. Ser. 94. 191-205, 1993

Relative stabilities

In order to compare the short- and long- AFDW term relative stabilities of individual con- A ARA - RHA 0 stituents, early losses which result from O FUC leaching must first be considered. D MAN + GAL Whereas the largest loss of total organic C 40- GLU -4 X XYL mass occurred during the first 24 d of a B MY0 decomposition, many of the specific 20- compounds measured did not show this pattern. For example, vanillin yields ,, o--* :: I LL7#+3 -m 7.- F - m-q z changed little during the first 24 d of 0 100 200 300 4 00 500 decomposition. This apparent stability Days in Field was also evidenced by the fact that vanillin did not appear to be released in Fig. 2. Halodule wrightii.Percent remaining of individual neutral sugars ester or soluble form after treatment with from decomposing seagrass blades. Each symbol represents average values 1 N NaOH. Therefore, early losses due to for replicate litter bags. See Table 1 for list of abbreviations leaching are not an important factor in determining the decomposition dynamics of the phe- each component must also be considered in order to nolic precursors of vanillin. Alternatively, galactose estimate relative inputs of individual sugars to the yields were considerably reduced during the first 49 d dissolved pool. Senescent H. wrightii lost 35.5 % (i.e. of decomposition, but beyond Day 49, yields progres- 355 mg g-' AFDW) of its total organic mass after 24 d sively increased through the duratlon of the study. In in the field (Fig. 1). Of this, 63.1 mg g-l, or 18 % of the this instance, it is likely that there are 2 distinct sources total organic mass was accounted for as neutral sugars, within Halodule wrightil tissue which yield galactose leaving 82 % of the organic matter lost during leachlng upon hydrolysis: one which is relatively labile and con- as yet chemically undefined. Glucose was the major tributes to the observed early losses and a second sugar removed during leaching, accounting for 61 % which is stable relative to bulk tissue and is selectively (38.6mg g-' AFDW) of the total neutral sugar fraction preserved throughout decomposition.These variations lost. Losses for other sugars range from 1.2 mg g.' in the decomposition kinetics of individual compounds AFDW for fucose to 6.4 mg g-l AFDW for galactose may be important depending on which phase of de- after the first 24 d of decomposition. It is interesting to composition is being studied. Large early losses due to note that glucose, which is least susceptible to leach- leaching processes are important in terms of vascular ing, still accounts for the majority of the neutral sugar plants as sources of dissolved organic matter and also lost during the first 24 d of decomposition. Sources in for potential changes which may influence biomarker addition to structural polymers, such as sucrose, are applications. Such losses, however, are not necessarily present in relatively fresh seagrass detritus and may representative of the latter stages of decomposition. account for some of the early rapid neutral sugar In order to properly evaluate the temporal behavior losses. of the various constituents, the percentages of initial A considerably different pattern of distribution of mass remaining are calculated as: individual sugars becomes evident when highly de- graded samples are compared to the initial senescent % remaining = (yieldhnitial yield) material. Using the Day 419 data from Fig. 2, a long- X 11 - (% AFDW loss / loo)] X 100. term stability series for the individual sugars in highly Compounds which have a higher '% remaining' than degraded Halodule wrlghtli detritus from most to '% AFDW remaining' have become enriched relative least stable is: mannose > fucose > arabinose = rham- to initial organic mass while those with a lower '% nose = AFDW = galactose = xylose > glucose S myo- remaining' are relatively depleted. inositol. Clearly, patterns of loss for individual neutral Neutral sugars comprise the largest measured pool sugars during the early stage of decomposition (0 to of organic matter lost during leaching (Table l), and 24 d) did not reflect these overall trends, indicating a the percent initial yields of individual neutral sugars shift in the neutral sugar content of detritus between from decomposing Halodule wrightii blades are the early and latter (post-leaching) stages of decom- depicted in Fig. 2. Changes during the first 24 d of position. This most likely reflects different processes decomposition provide an indication as to which con- (solubilization versus microbial decomposition) that stituents are most susceptible to leaching, however, are selective for certain forms of individual sugar total neutral sugar ylelds as well as initial yields of sources. Opsahl & Benner: Decomposition of senescent Halodule wr~ght~i 20 1

stability of the major polymers from which they arise. -+ AFDW In this study, the entire glucose pool is taken to repre- v S sent cellulose (see above),all remaining neutral sugars except myo-inositol are grouped and designated as hemicellulose, lignin is represented by the sum of all lignin-derived V, S and C phenols and cutin is taken as the sum of all 4 cutin-derived hydroxy acids. In a similar manner as above, the % remaining have been calculated and plotted in order to provide the clearest representation of the long-term behavior of these

0, O\O 0, .- I major biochemical pools (Fig. 4). We have included 0 100 200 300--A 400 500 soluble and ester-bound phenols for comparison to the Days in Field other measured constituents. Note, however, that their relative stabilities are determined during the first 0 to Fig. 3. Halodule wrightii. Percent remaining of individual 200 d of decomposition (Table 2). With the exception of lignin-derived phenol families from decomposing seagrass cutin, the major biochemical constituents follow AFDW blades. Each symbol represents average values for replicate losses beyond about 100 d of decomposition. A stability litter bags. V: total yield of vanillyl phenols; S: total yield of series for these constituents, based on the most highly syringyl phenols; C: total yield of cinnamyl phenols degraded san~ples,from most to least stable is: hemi- cellulose = lignin = AFDW > cutin > cellulose 9 ester- Likewise, the % remaining has been calculated for bound phenols > soluble phenols. Overall, the relative lignin-derived phenol families and plotted over the abundance of the major polymers appears only slightly time series (Fig. 3). While considerable variation in % different in the most highly degraded detritus when remaining for V, S and C is evident throughout most of compared to freshly senescent material. the study, the yields for the last several time points in- dicate that lignin phenols are present at near initial yields in highly degraded Halodule wrightii detritus Biomarker applications resulting in a stability series: C = S = V = AFDW for lignin-derived phenols. Unlike neutral sugars, distrib- The abundance and composition of phenols derived utional patterns of lignin-derived phenols in highly from CuO oxidation are diagnostic of the presence and degraded detritus were similar to that of the initial type of vascular plant tissues in environmental samples senescent material. and have received considerable attention as biomarker Individual biochemical constituents may also be compounds in marine environments (Hedges & Mann grouped in order to evaluate the long-term relative 1979). Previous studies have used CuO oxidation data to determine vascular plant inputs to

pp p depositional environments (Gardner & 100 T- Menzel 1974, Hedges & Parker 1976, - 4.- % AFDW Remaining A ',, L Cellulose Hedges et al. 1984, Wilson et al. 1985, O Hemicellulose 8O4\o Lignin Requejo et al. 1986, Haddad & Martens h l# 0 0 0 Cutin 1987, Hamilton & Hedges 1988) and to f=t X Soluble Phenols - 0 + Ester-Bound Phenols pools of dissolved organic carbon in both m 60 ' freshwater and marine environments c (Ertel et al. 1984, Meyers-Schulte & .r( d Hedges 1986, Moran & Hodson 1990). -4 40- K! In addition to providing a biomarker which is unique to vascular plants, the

Pr; 20-x A a CuO oxidation products of lignin contain

o\o 1 + more detailed information characteristic of the plant tissue source Vanillyl phe- nol~are predominant reaction products of woody and herbaceous tissue. Vanillyl Days in Field phenols are characteristic of both gymnosperm and angiosperm CuO oxi- Fig. 4. Halodule wrighfii. Percent remaining of major polymers and soluble and ester-bound phenols associated wlth decomposiny seaqrass blades. dation products, but syringyl phenols are Each symbol represents average values for replicate l~tterbags virtually absent from gymnosperm tis- Mar Ecol. Prog. Ser. 94: 191-205, 1993

Table 4. Halodule wrightii.Lignin-derived parameters for Other lignin parameters can be used as indicators of green, senescent and decomposing seagrass blades. Green. the diagenetic state of vascular plant organic matter. senescent and 24 to 136 d tissue calculated using total For example, Hedges et al. (1988) demonstrated ele- phenols produced upon CuO oxidation minus soluble and ester-bound phenols. Beyond Day 136, whole material is vated vanihc acid to vanillin ratios, (Ad/Al),, in woods used because soluble and ester-bound are virtually absent. which had been degraded by terrestrial white-rot S/V- weight ratio of syringyl phenols (SAL + SON + SAD) to fungi. In the case of Halodule wrightii, a non-woody vanillyl phenols (VAL + VON + VAD); C/V: weight ratio of tissue which had been decomposed in the water cinnamyl (CAD + FAD) phenols to vanillyl (VAL + VON + VAD) phenols; (Ad/Al),. weight ratio of vanillic acid to column, no significant trend (p > 0.05) was apparent in vanillin; (Ad/AI),: weight ratio of syringic acid to syringalde- (Ad/Al), or (Ad/Al),, syringic acid to syringaldehyde hyde. Other abbreviations as in Table 2 ratio, throughout the duration of the study (Table 4). Results from the decomposition of other non-woody Sample S/V C/V (Ad/Al), (Ad/Al), plant tissues including conifer needles (Hedges & Weliky 1989), mangrove leaves (Benner et al. 1990a) Green 0.46 0.29 2.93 0.50 and cordgrass (Benner et al. 1991), have indicated non- Senescent 0.71 0.23 0.62 0.49 selective degradation of the lignin subunits which pro- Decomposing (d) duce these acids and aldehydes upon CuO oxidation. 24 0.82 0.35 0.16 0.62 Bacteria are the primary organisms responsible for the 4 9 0.78 0.32 0.22 0.80 136 0.88 0.30 0.39 0.35 degradation of lignin in marine environments (Benner 166 0.79 0.37 0.4 1 0.41 et al. 1986a), and the associated mechanisms appear to 195 0.87 0.37 0.38 0.57 be nonselective for the macromolecular precursors 227 0.72 0.37 0.36 0.55 which yield vanillyl and syringyl phenols upon CuO 269 0.97 0.34 0.29 0.43 305 0.76 0.39 0.40 0.45 oxidation. 339 1.10 0 24 0.29 0.36 Earlier studies which have investigated the mole- 367 0.72 0 22 0.31 0.43 cular-level composition of lignin using CuO oxidation 395 0.40 0.23 0.27 0.32 have not distinguished soluble and ester-bound phe- 419 0.66 0.24 0.40 0.79 nol~from the corresponding total individual phenol yields. We found soluble and ester-bound phenols to sues (Hedges h Mann 1979). Lignin-derived cinnamyl be quantitatively important in fresh and relatively phenols are produced almost exclusively from the recent Halodule wrightii detritus. Total lignin-derived oxidation of herbaceous tissues and therefore the phenols (L8), C/V and (Ad/Al), ratios would be ele- presence of cinnamyl phenols indicates the presence vated and the S/V ratio reduced relative to the of vascular plant herbaceous tissue (Hedges & Mann reported values if these corrections had not been 1979). made. Therefore, measurement of soluble and ester- Ratios of syringyl to vanillyl (S/V = sum of synngyl bound phenols was necessary to accurately calculate phenols divided by sum of vanillyl phenols) and cin- total lignin-derived phenols and associated lignin- namyl to vanillyl (CN = sum of cinnamyl phenols derived parameters. If a similar trend of elevated divided by sum of vanillyl phenols) indicate plant type phenolic acids holds for other plant species, then it (angiosperm or gymnosperm) and tissue type (herba- may be necessary to reevaluate the phenol content of ceous or woody), and are key parameters which can fresh tissue endmembers in order to explain rapid help to decipher source inputs to heterogeneous mix- early changes in the total phenol content. Using this tures (Hedges & Mann 1979). Previous investigations new approach to characterize individual pools of using a variety of plants have shown that associated phenols, we are now able to distinguish between lignin parameters are subject to selective decomposi- changes primarily due to solubilization versus tion processes (Hedges & Weliky 1989, Benner et al. those related to the biodegradation of specific lignin 1990a, 1991). For example, C/V in particular has been subunits. shown to decrease with increasing decomposition owing to the preferential loss of C phenols (Hedges & Weliky 1989, Benner et al. 1991. Haddad et al. 1992). In Photobleached detritus this study, no significant linear trends (p> 0.05) were evident in either the S/V or C/V ratios (Table 4; green Although the absolute age of the photobleached tissue is excluded from the decomposition series). detritus is not known, Newel1 et al. (1986) point out Although variable, when these parameters are calcu- that photobleached detritus at the supratidaI level of lated exclusively from lignin-derived phenols, S/V the shoreline may degrade more slowly than that and C/V ratios are similar in senescent and highly found at mid-tidal level. In the Laguna Madre, this degraded tissue. material is abundant and represents detritus which has Opsahl & Benner. Decompos~t~on of senescent Halodule wnghtil 203

been subjected to periodic submergence, desiccation exceptionally high weight loss due to leaching during and exposure to direct sunlight. Lignin phenol yields the first 24 d of submergence. Exponential and linear from photobleached Halodule rvrightii blades were decay models both yield a highly significant fit dramatically different than those from the litter bag (p< 0.01) to the data set . The exponential decay model samples and unlike any reported for other vascular predicts that 5% of the original AFDW will remain plant tissues since only ferulic acid was detected in the after 930 d while the linear model predicts 5'h) of the photobleached detritus (Table 2). This virtual lack of original AFDW remaining after only 536 d in the field, lignin phenols may be attributed to photodegradation a difference of about 400 d to account for 20 %, of the of aromatic structures in the seagrass blades because remaining detritus. This difference would be even phenolic constituents are susceptible to photooxidation greater if the calculation were taken to l of the (Sarkanen & Ludwig 1971). This is further supported inltial starting material remaining. While this study is by the fact that seagrass blades lying directly below significant in that 76 % of the decomposition has been the photobleached layer contained lignin-derived documented, an important but as yet unpredictable phenol yields comparable in magnitude to litter bag phase of decomposition remains unstudied. samples (data not shown). Soluble and ester-bound Processes which are involved in the deconlposition phenolic acids released from detritus during leaching of vascular plants, including leaching, fragmentation, are also likely to be susceptible to photooxidation and photooxidation and biodegradation, all potentially photobleaching may facilitate solution of partially result in selective decomposition thereby altering the degraded phenols. Photooxidation may represent a ultrastructure of the residual particulate material. major mechanism for degradation of phenolic com- Interestingly, in the case of Halodule wrjghtii, the pounds in the dissolved phase. neutral sugar, lignin and cutin fractions were all found In contrast to the absence of phenols, increased at similar levels in highly degraded tissue compared to neutral sugar yields, due primarily to elevated glucose freshly senescent material. At best, only minor trends and xylose yields, indicate that neutral sugars are not for selective decomposition or enrichment were evi- susceptible to photooxidation. The higher yields for dent for any of these components, with the possible this class of conlpounds are well within the range exception of cutin which may be predicted to selec- expected for passive concentration and thus likely tively decompose beyond 419 d. Apparently, with result from selective loss of other biochemical con- respect to these measured components, the ultrastruc- stituents within the detntus. The higher concentration ture of the highly degraded H. wrightij detritus does of neutral sugars may also be an indication that little not reflect changes due to selective deconlposition of microbial degradation is taking place under the these polymers. extremely dry and high ultraviolet light conditions. Cellulose, hemicellulose and lignin typically account Cutin acid yields in photobleached detritus were simi- for >75% of the biomass of woody plant tissues lar to those of partially degraded litter-bag samples (Sjostrom 1981). In the case of Halodule wrightij, cellu- and, like neutral sugars, do not have the same suscep- lose and hemicellulose together compnsed ca 20 % of tibility to photodegradation as the lignin component. the total organic mass and lignin and cutin collectively Therefore, pathways of deconlposition whlch alter the accounted for only a few percent. Similar concentra- chemical composition of photobleached detritus have a tions of these biopolymers have been reported for different net result than those acting on completely other herbaceous plant tissues (Hedges & Weliky 1989, submerged material demonstrating that photodegra- Benner et al. 1990a). Given the biochemical com- dation is an environmentally important degradative plexity of herbaceous tissues and the lack of suitable mechanism which may considerably alter the chemical n~olecular-level analyses for components such as composition of plant detritus. tannins, it is unlikely that a comprehensive view of the chemical composition can be obtained using this approach. Solid-state I3C nuclear magnetic resonance Ecological implications (NMR) has recently been used to provide a compre- hensive view of the chemical constituents comprising Estimations of decay rates from litter bag studies, leaf tissues (Hatcher 1987, Wilson et al. 1987, Benner et while useful for making comparisons among plant al. 1990b). These studies have revealed that aliphatic species treated in a similar manner, are limited in their polymers, tannins and polysaccharides not accounted predictive capacity. Questions still remain regarding for by the neutral sugar analysis, such as the poly- decay rates for highly degraded detritus and the types uronic acids of pectin and other polymers, can be major of models which are most representative of long-term constituents of herbaceous plant tissues. This kind of decomposition (Moran et al. 1989).We omitted the first approach is needed to gain a better understanding of data point for modeling decay rates because of the the qualitative changes that take place during the 204 Mar. Ecol. Prog. Ser.

decomposition of seagrasses and the influence of Buchsbaum, R,Valiela, I.,Swain, T., Dzierzeski, M.. Allen, S. detritus quality on bioavailability and organic matter (1991). Available and refractory nitrogen in detritus of coastal vascular plants and macroalgae. Mar. Ecol. Prog. preservation. Ser. 72: 131-143 Chin-Leo, G , Benner, R. (1991). Dynamics of bacterioplank- ton abundance and production In seagrass communities of Acknowledgements. The authors thank John Hedges and a hypersaline lagoon. Mar. Ecol. Prog. Ser. 73: 219-230 Miguel Goni for offering their expertise and advice and for Cowie. G. L.. Hedges, J. I. (1984a). Determination of neutral use of their GC/MS fac~lltyat the University of Washington. sugars in plankton, sediments, and wood by capillary gas We are especially grateful to John Hedges and 2 anonymous chromatography of equilibrated isomeric mixtures Anal. reviewers for thoughtful comments. We also appreciate help- Chem. 56: 497-504 ful reviews from Miguel Goni, Dean Pakulski, Bopi Biddanda Cowie, G. L.. Hedges, J. I. (1984b). Carbohydrate sources and Rainer Amon. We would also like to thank Brenda in a coastal marine environment. Limnol. Oceanogr. 48: Hamrnan for the cellulose determ~nations.This research was 2075-2087 supported by grant BSR 8910766 from the NSF, grant Drew, E. A. (1980). Soluble carbohydrate composition of sea- NA89AAD-SG139 from the NOAA Office of Sea Grant, and grasses. In: Phillips, R. C., McRoy, C. P. (eds.) Handbook grants 4541 and 3658-264 from the Texas Higher Education of seagrass biology. Garland STPM Press, New York, Coordinating Board Advanced Technology Program. This is p. 247-260 contribution number 871 from The University of Texas at Eglington, G., Hunneman, D. H. (1968). Gas chromato- Austin Marine Science Institute. graphic-mass spectrometric studies of long-chain hydroxy aclds-I. 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This article was presented by S. Y. Newell, Sapelo Island, h4anuscript first received: November 24, 1992 Georgia, USA Revised version accepted: February 1, 1993