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Evidence of microbial succession on decaying leaf litter in an arctic lake
Department of Biological Sciences, University of Cincinnati, Cincinnati, OH, U.S.A. 45221 Accepted March 8, 1982
FEDERLE, T. W., and J. R. VESTAL. 1982. Evidence of microbial succession on decaying leaf litter in an arctic lake. Can. J. Microbiol. 28: 686-695.
Microbial colonization and its relation to the decomposition of sedge-(Carex) leaf litter were studied in the littoral area of Toolik Lake, Alaska. Colonization was assessed using scanning electron microscopy and by measuring the concentration of ATP associated with the litter. Litter lost approximately 20% of its dry weight and 60% of its phosphorus by leaching during the first 4 days of exposure to lake water. Microbial activity was responsible for any additional decomposition. Microorganisms were first observed on the litter surface on the 3rd day of incubation in the lake, when isolated bacterialike forms and microbial filaments were present, but ATP was not yet detected. This initial appearance of microbes was followed by a dramatic rise in ATP concentration and a rapid proliferation of microbial forms, especially large filaments. Associated with this rapid colonization was a steep decline in nitrogen content, a twofold increase in protein content, and the appearance of microbially mediated weight loss of the litter. By day 13, microbial colonization declined by 50% and remained stabilized at this lower level for the duration of the study. This decline coincided with the demise of the large filamentous forms, a decrease in the protein content of the litter, and slower but continued decrease in the nitrogen content of the litter. As time progressed, the microbial community became increasingly characterized first by the presence of bacterialike forms enmeshed in slime and later by large numbers of pennate diatoms. These findings show that decomposition is dependent on the development and succession of the microbial community on the litter.
FEDERLE, T. W., et J. R. VESTAL. 1982. Evidence of microbial succession on decaying leaf litter in an arctic lake. Can. J. Microbiol . 28: 686-695.
La colonisation microbienne et ses relations avec la dCcomposition de la litiere' feuillCe de laiches (Carex) ont CtC CtudiCes dans le littoral du lac Toolik, en Alaska. La colonisation a Ctk CvaluCe a l'aide d'un microscope Clectronique a balayage et par des mesures de la concentration en ATP associCe a la littikre. La litikre perd environ 20% de son poids sec et 60% de son phosphore par dClavage au cours ders 4 premiers jours d'exposition a I'eau du lac. Toute dCcomposition ultkrieure relkve de I'activitC microbienne. PrCsents en surface de la litikre aprks 3 jours d'incubation dans l'eau, les microorganismes sont des structures ayant la forme de bactCries ou de filaments microbiens; mais I'ATP n'est pas encore dCcelCe. Cette apparition initiale des rnicroorganismes est suivie d'une importante augmentation de concentration en ATP et d'une rapide prolifkration des formes microbiennes, en particulier des gros filaments. A cette colonisation rapide est associk: un dCclin brusque de la teneur en azote, une augmentation au double de la teneur en protCine, et une diminution du poids de la litiere rksultant de I'activitC microbienne. Treize jours plus tard, la colonisation microbienne fut rkduite de 50% et elle est demeurCe a ce bas niveau pour le reste de I'ktude. Ce dCclin coincide avec une rCduction des grosses formes filamenteuses, une diminution de la teneur en protCine de la litiere et une disparition lente et graduelle de la teneur en azote de la litiere. La communautC microbienne Cvolue avec le temps; elle est d'abord caractCrisCe par une augmentation des structures de forme bactkrienne, entremelkes d'une substance visqueuse, suivie de la prCsence de grandes quantitks de diatomCes pennCes. Cette Ctude montre que la dCcomposition de la litiere est like au dkveloppement et a la succession de la communautC microbienne dans la litiere.
[Traduit par le journal]
Introduction In the shallow littoral habitats of a lake, the vast
majority of the carbon fixed by aquatic vascular plants during photosynthesis is not consumed by animals but enters the pool of dead organic materials in the form of large particulate detritus or litter (Berrie 1976; Fenchel and Jorgensen 1977). The degradation of this litter constitutes an important flux in the energy budget of a lake and can form the base of a detrital food chain. Both microorganisms and invertebrates inhabit the litter, and its presence physically and metabolically structures the littoral habitat (Wetzel 1975).
Following a brief period of leaching, the degradation of plant litter in aquatic environments is totally depend- ent on its colonization by microorganisms (Boling et al. 1975; Federle and Vestal 1980; Fenchel and Jorgensen 1977; Kaushik and Hynes 197 1 ; Petersen and Cummins 1974). Associated with this colonization process is a succession in the microbial community as decomposi- tion progresses. Microbial succession has been studied on decaying terrestrial plant litters in temperate streams (Barlocher and Kendrick 1974; Suberkropp and Klug 1974, 1976) and in a semitropical estuary (Morrison et al. 1977), but this process on aquatic plant litter in a large lake has received virtually no attention. Furthermore,
l~u tho r to whom reprint requests should be addressed. information on the earliest stages of the colonization
OOO8-4166/82/060686-10$01 .OO/O 0 1982 National Research Council of Canada /Conseil national de recherches du Canada
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process is also limited, and these previous studies have been limited to temperate climates.
In this study, the sequence of events occurring during the microbial colonization and decomposition of aquatic macrophyte litter in the littoral zone of a large arctic lake was examined. Colonization was assessed by measuring the concentration of ATP associated with the litter and examining the litter surface by scanning electron microscopy. Decomposition was determined by mea- suring weight loss and changes in the carbon, nitrogen, phosphorus, and protein content of the litter.
Materials and methods The study site
Toolik Lake is located approximately 230 km north of the arctic circle and 190 km south of Prudhoe Bay in the Trans-Alaska Pipeline corridor (68'38' N; 149'38' W). It is covered with ice from October to June each year and is extremely oligotrophic because of the very low concentrations of dissolved nitrogen ( ~ 3 . 0 ~g-atom nitratell = 1.0 ~g-atom arnmonia/L) and phosphorus (= 1.0 ~g P/L). Scattered around the margins of the lake are numerous beds of emergent aquatic plants growing in the shallow littoral zone. These plants are primarily sedges, with the dominant genus being Carex. More than 70g/m2 of sedge litter is generated each year in these areas.
During the growing season, in excess of 80% of the leaf area of Carex is above the water. Upon death and loss of turgor, these leaves fall into the water. Senescence of Carex leaves in the arctic has been reported to be completed in as little as 12 h and as long as 32 days (Tieszen 1978). As a consequence, it was impossible to obtain leaves that had completed senes- cence, but had not yet entered the water. Green leaves, there- fore, were used in this study.
Preparation of the litter Fresh green Carex leaves were collected from the margins
of Toolik Lake, cut into 4-cm segments, and oven-dried to constant weight at 65'C. Leaf segments for weight loss and chemical analyses were randomly selected, and 300-400 mg was sewn into litter bags (8 x 8 cm) consisting of fiber glass window screening (1.5-mm mesh). Leaf segments for micro- biological analysis were hand-selected to insure uniformity in width, thickness, and weight. Segments chosen in this manner usually varied less than 3 mg in weight. Twenty-five to 30 of these segments were enclosed together in a litter bag.
In situ incubation and sampling The sequence of colonization and decomposition of Carex
litter was studied in the littoral zone of Toolik Lake during the summer of 1980. Bags were placed in a shallow emergent Carex bed on June 25, and these bags were periodically sampled, initially every day and later every 3 days. At each sampling, two bags for weight loss determination and one bag for microbiological analysis were recovered. Abiotic controls consisted of litter bags incubated in aquaria containing lake water at ambient lake water temperature supplemented with 0.01% mercuric chloride to inhibit microbial growth. Upon recovery, bags for weight determination were gently washed in
lake water and brushed to remove any surface materials, which were generally inconsequential. Weight loss was measured gravimetrically after the litter was oven-dried to a constant weight. This dried litter was later pooled and used for determinations of carbon, nitrogen, phosphorus, and protein content. Immediately following sampling, litter for microbio- logical analysis was cut into 1-cm segments and stored in filter-sterilized (0 .45 -~m Millipore) lake water until analysis was completed, which was never more than 4 h.
Chemical composition of the litter Carbon and nitrogen content Small (1-2 mg) samples of the dried Carex litter were
ground to a fine powder and then combusted and analyzed in a Carlo Erba element analyzer, standardized with cysteine. Triplicate deteminations were made for each sample.
Phosphorus content All glassware was rinsed with 2 N hydrochloric acid, and
only deionized distilled water was used. Approximately 100 mg of the dried pooled litter was combusted in a ceramic crucible at 550°C for 2 h. The methods of Likens and Bormann (1970) were used to dissolve the ash, and the phosphorous concentration was measured by the molybdate blue method (Anonymous 1 979).
Protein content Approximately l00mg of dried litter was crushed, ex-
tracted, and analyzed for protein using the procedure described by Kaushik and Hynes (1968). Protein was measured by the procedure of Lowry et al. (195 1) using bovine serum albumin as the standard.
Microbiological analysis ATP ATP was extracted using a modifed version of Bulleid's
(1978) method. Ten or twelve 1-cm leaf segments were extracted in 8 mL of boiling McIlvaine buffer (0.04 M dibasic sodium phosphate adjusted to pH 7.7 with 0.02 M citric acid). The extract was cooled and transferred to a 10-mL graduated test tube and brought to volume with distilled water. One- millilitre samples of the extract were transferred to 8-mL plastic minivials and frozen until further analysis. ATP was quantified photometrically using the firefly lantern extract assay of Karl and LaRock (1975). Firefly lantern extract (FLE-50; Sigma Chemical Company, St. Louis, MO) was reconstituted with 12.5 mL of distilled water, 7.5 mL of arsenate buffer (AS- 100; Sigma), and 5 mL of 0.05 M magnes- ium sulfate. The solution was placed in the dark for 12 h prior to use to allow hydrolysis of any endogenous ATP. One half millilitre of the l%~-50 solution was combined with 1 mL of the sample extract in a minivial. The minivial was vortexed and placed in the counting chamber of a LSC-2 miniscintilla- tion counter equipped with an ST3 timer-scaler (Nuclear Enterprises, San Carlos, CA) operating in the noncoincidence mode. Exactly 1 min following addition of the FLE-50 reagent, the sample was counted for 10 s. These counts were used to determine ATP concentration based on a standard curve. ATP was expressed as nanograms per 4-cm leaf segment. ATP was expressed on a per-segment basis rather than a per-weight basis to eliminate the bias caused by differential weight loss of the samples. Blanks were run
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simultaneously and subtracted for the correction of the samples. Efficiency was determined by means of internal standards and was found to approximate 80%. Triplicate determinations were made on each sample of colonized litter.
Scanning electron microscopy (SEM) Leaf segments were fixed in 3% glutaraldehyde (Eastman
Kodak Co., Rochester, NY) in 0.1 M phosphate buffer (pH 7.4) for 1 h. The fixed samples were washed three times with buffer and then three times with distilled water (10 minlwash). They were dehydrated in a stepwise succession of 30, 50, 70, 90,95, and 100% ethanol (10 min each) and washed three times with 100% ethanol. The dehydrated samples were critical point dried using liquid carbon dioxide in a pressure bomb (Parr Instruments, Moline, IL), mounted with silver paint, and coated with 500A (50 nm) of gold in an IS1 PE-5000 sputter coater (International Scientific Instruments, Mountain View, CA). Samples were examined with an IS1 Mini-SEM. Leaf segments were examined in their entirety at low power ( 100-200 x ) and representative areas were examined at higher powers. Conclusions about the microflora were based on the examination of at least five leaf segments. Photographed areas were chosen as being representative of the overall flora.
Results Weight loss
During the incubation period, average daily water temperature increased from an initial temperature of 1 1 "C on June 25 to a maximum (1 7°C) on day 10, and then declined and plateaued at approximately 12- 15°C following cooler weather. Carex leaf litter rapidly
After day 17, no additional weight loss was observed until day 28, when total weight loss equaled 59%.
Carbon and nitrogen content The carbon content of the decomposing Carex litter
remained relatively constant throughout the study, accounting for 45-46% of the total dry weight. Nitrogen content of fresh litter equaled 3.4% of the dry weight and fluctuated between 3.3 and 3.9% during the first 4 days of exposure to lake water (Fig. 2). The abiotic control, similarly, initially fluctuated, but then stabilized at 3.65% nitrogen. In contrast, the nitrogen content of the litter incubated in the lake declined dramatically be- tween days 4 and 7, reaching 2.75%. This decline continued throughout the study, but at an incresingly slower rate. By day 28, the nitrogen content of the litter was only 2.0%. The C:N ratio increased predictably with the decreases in nitrogen concentration. Initially equaling 12.5, it increased to 17 by day 7 and to 22 by the end of the study.
Phosphorus content Phosphorus initially accounted for 0.79% of the dry
weight of the litter but rapidly decreased in importance following immersion in lake water (Fig. 3). The abiotic control lost phosphorus more quickly than litter in the lake and stabilized at 0.25% on day 7. The phosphorus content of the litter in the lake was 0.38% on day 7 and stabilized at 0.28% after day 17.
decreased in dry weight following immersion in ~ b o l i k Lake water (Fig. 1). After only 12 h, 9% of the total Protein content
weight was lost, and after 4 days a total of 19-20% of Carex leaf litter initially contained 5.5% protein.
the litter had disappeared. This same pattern and amount During the first 3 days of incubation, protein content
of initial weight loss was observed for the abiotic increased to 8% (Fig. 4). The abiotic control similarly
control, but no additional weight loss occurred after day increased in protein content, but remained at this level
4. Litter incubated in the lake, however, continued to permanently. Litter incubated in the lake dramatically
lose weight at a rapid rate. Approximately 3 1 % of the increased in protein between days 3 and 7, reaching
litter was lost after 7 days, and 52% was gone by day 17. 15%. It remained at this level until day 13, when it began
FIG. 1 . Decomposition (percent weight loss) of Carex leaf litter incubated in the littoral zone of Toolik Lake as a function of time (x + SD; n = 2).
TI ME (DAYS)
FIG. 2. Nitrogen content and C:N ratio of Carex leaf litter incubated in the littoral zone of Toolik Lake as a function of time (x + SD; n = 3).
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I 0 ABIOTIC CONTROL
1 I I I I I 5 10 I5 20 25
TIME (DAYS)
FIG. 3. Phosphorous content of Carex leaf litter incubated in FIG. 5 . ATp and biologically mediated weight the littoral zone of Toolik Lake as a function of time. loss of Carex leaf litter incubated in the littoral zone of Toolik
Lake as a function of time.
FIG. 4. Protein content of Carex leaf litter incubated in the littoral zone of Toolik Lake as a function of time.
a sustained period of decline. By day 24, protein content in the litter had returned to 8.7%.
ATP No ATP was detected on litter placed in Toolik Lake
until the 4th day of incubation when it equaled 52 ng/ segment (Fig. 5). Between days 4 and 7, ATP increased in concentration by more than an order of magnitude reaching 670 nglsegment. This dramatic increase was followed by a subsequent 50% decline between days 10 and 13. Throughout the remainder of the study, ATP concentration remained at this lower level fluctuating approximately 350 ng /segment. The dramatic increase in ATP at day 4 corresponds with the beginning of the biotic weight loss.
(Fig. 7). At this time, a variety of scattered and isolated forms were observed on the litter surface. Bacterialike forms were observed as isolated cells and in localized microcolonies. Similar forms could be observed invad- ing stomates. Filamentous forms were also observed, but were extremely rare.
By day 4 (Fi'g. 8), the surface of the litter was colonized by an increasing variety and density of microbial forms, with rod-shaped bacterialike forms remaining the most common. At this time, stomates were increasingly invaded by bacterialike forms, and the guard cells showed signs of degradation.
On day 7, large filaments (visible at 30x) were observed covering the surface of the litter (Fig. 9). Stomates became difficult to locate because of the overgrowth of microorganisms. Besides the large fila- ments, actinomycetelike forms and a bacteriaiike form, enmeshed in slime, were first.observed at this time. The microbiota did not significantly change again until day 1 2 1 2 .
On day 13, the large filaments, previously dominating the litter surface, were observed to be in a fragmented and deteriorated condition (Fig. 9). Increasingly con- spicuous were bacterialike forms enmeshed in slime. By day 17, the entire surface of the litter was covered by this slime, and the only observable forms were diatoms and bacterialike forms (Fig. 10).
As time progressed, the microbiota became increas- ingly characterized by the presence of pennate diatoms of different sizes and species. On day 24, large numbers of diatoms were observed on the litter surface (Fig. 10).
SEM observations By day 28, the slime was largely gone, and the diatoms Prior to immersion in Toolik Lake, no microbial were even more obvious. Litter from a previous experi-
forms were observed on the surface of Carex leaf litter ment, incubated in the littoral site for 1 year, exhibited a (Fig. 6). After 12 h, debris began to accumulate on the surface characterized by diatoms and bacterialike forms litter surface, but still no microbial forms were apparent inhabiting the hollowed out remains of the Carex cell (Fig. 6 ) . Microbial forms were first observed on day 3 walls (Fig. 10).
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FIG. 6. Scanning electron micrographs of Carex leaf litter (A) prior to incubation (bar = 100 pm), (B) after 0.5 day of incubation (bar = 50 pm), and (C) after I day of incubation (bar = 50 pm) in the littoral zone of Toolik Lake.
FIG. 7. Scanning electron micrographs of Carex leaf litter incubated for 3 days in the littoral zone of Toolik Lake showing (A) scattered bacterialike forms (bar = 50 pm) and (B) microbial colonization of a stomate (bar = 50 pm).
Discussion The findings of this study indicate that the decomposi-
tion of Carex leaf litter in Toolik Lake occurred as the result of leaching and microbial activity. Futhermore, changes in the weight loss and chemical composition of the litter were found to coincide with changes in the ATP biomass associated with the litter and the microbiota observed on the litter surface by SEM. Colonization of Carex litter in the littoral zone of Toolik Lake appeared to occur in four distinct phases as a function of time. These stages included ( I ) a period of initial colonization, (2) a period of explosive growth, (3) a period of decline, and (4) a period of stabilization and continued succession.
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FIG. 8 . Scanning electron micrographs of Carex leaf litter incubated for 4 days in the littoral zone of Toolik Lake showing (A) large numbers of rod-shaped bacterialike forms (bar = 50 pm) and (B) colonization and deterioration of a stomate by bacterialike forms (bar = 10 pm).
The first stage of colonization occurred during the first 3 days of exposure to the environment. During this period, weight loss could be accounted for by leaching as observed in the abiotic control. A large amount of phosphorus was leached, but nitrogen concentration remained relatively unchanged. In agreement with the present study, Hobbie et al . (1980) found that Carex aquatilis litter lost 20.8% of its dry weight and almost all of its phosphorus during the 1st week of incubation in a tundra pond. Other workers have also observed similar periods of abiotic weight loss (Hobbie et al . 1980; Howard-Williams and Davies 1979).
Colonization of the litter by microorganisms did not being immediately. Debris but no microbial forms accumulated on the litter during the first 2 days. It was not until day 3 that the first scattered microbial forms were present on the litter, although at a density too low to be detected as ATP. Nevertheless, signs of cell division were already present. The time lag that oc- curred in the development of ATP biomass probably was related to the time involved for microorganisms to accumulate on the litter surface by either active or passive means.
Once the first microorganisms were observed on the litter surface, colonization proceeded at a rapid rate, beginning the next phase of the colonization process. Within 24 h of the observation of the first microbial forms, ATP was detected and the litter surface became covered by increased numbers of primarily bacterialike forms. Associated with the appearance of ATP was a decline in the rate at which phosphorus was lost from the litter in the lake compared with the abiotic controls. Microoganisms were probably immobilizing this critical nutrient as cellular material, preventing its loss from the litter. It has been demonstrated that radioactive phos- phorus is taken up from the environment quite rapidly by colonized compared with sterilized litter, and the rate is related to the respiration associated with the litter (Gregory 198 1).
Following the appearance of ATP associated with the litter, weight loss, which could not be accounted for by the leaching observed in the abiotic controls, was detected for the first time. This finding is in agreement with previous observations that weight loss of Carex leaf litter in Toolik Lake correlated with various measurements of microbial activity associated with the litter (Federle and Vestal 1980). ATP concentration continued to increase by more than an order of magni- tude, and the surface of the litter became covered by large microbial filaments. During this time, protein content of the litter increased twofold, probably due to the presence of large amounts of microbially produced protein. Surprisingly, however, nitrogen content of the litter decreased dramatically.
Other workers have similarly noted an explosive increase in the amount of microbial colonization on plant litter in aquatic environments during the early stages of decomposition. Suberkropp and Klug ( 1976) reported a dramatic rise in the amount of ATP on oak and hickory leaves after 2 weeks in a stream. Morrison et al . (1977) observed a similar dramatic incrase in the ATP associated with oak and pine litter in an estuary. Kaushik and Hynes (1971) reported a threefold increase in the protein content of elm leaves after 3 weeks in stream water.
These rapid increases in colonization have usually been associated with the appearance and growth of
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fungal forms. Kaushik and Hynes (197 1) reported that the dramatic increase in the protein content of elm leaves was inhibited by antifungal but not antibacterial antibio- tics. Suberkropp and Klug (1976) noted that the rise in ATP corresponded to increasing density and frequency of hyphomycete isolates. Morrison et a / . (1977) also reported that the rapid rise in ATP coincided with the appearance of mats of funguslike filaments, observed by SEM. In this study, it was impossible to positively determine that the large filaments observed were of fungal nature because of the absence of reproductive structures. However, the increased rate of weight loss coinciding with their appearance would support the contention that they are fungal, rather than algal, filaments.
The next stage of colonization involved a 50% decline in the amount of ATP associated with the litter and occurred after day 10. Associated with this decline was a decline in the protein content of the litter and the demise of the large filamentous forms. Nitrogen continued to be lost, however, at an increasingly slower rate. In contrast with previous studies, the peak biomass persisted for only 1 week. Other workers have observed declines in the microbiota, which were often associated with declines in the fungal populations, but occurring much later. Morrison et a1. ( 1977) observed that ATP concentration on oak and pine leaves declined by approximately 50% after 3-5 weeks in an estuary. In a similar manner, ATP concentration on hickory leaves decreased more than 50% after 12-14 weeks in a woodland stream (Suber- kropp an Klug 1976). This decline coincided with a decrease in the frequency of isolation of aquatic hypho- mycetes from the litter. In another study, Lee et a1. (1980) noted an 80% decline in the fungal biomass (measured as ergosterol) on Spartina litter after 26 weeks in a salt marsh.
The last stage of colonization observed to occur on decomposing Carex litter in Toolik Lake was one in which the size of the microbiota remained relatively constant but its composition continued to change. This continued succession involved a shift from a community dominated by slime enmeshed bacterialike forms to one increasingly dominated by diatoms. Following the demise of the large filamentous forms, the litter surface became covered by slime. Associated with the appear- ance of this material was a temporary cessation of
FIG. 9. Scanning electron micrographs of Carex leaf litter incubated for 7 days (A) and 13 days (B and C) in the littoral zone of Toolik Lake showing (A) large microbial filaments, (B) remains of large funguslike filaments, and (C) deteriorated - - fragment of a large funguslike filament and bacteralike forms enmeshed in slime.
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weight loss. Nevertheless, protein content and nitrogen content of the litter continued to decline. It was therefore likely that decomposition was continuing, but weight loss was masked by the slime. Weight loss was again observed when the slime showed signs of deterioration. Prior to its degradation, the surface of the slime became colonized by large numbers of diatoms and bacteria. It is possible that the slime may have prompted the coloniza- tion of the litter by diatoms by providing a sticky surface for adherence.
The most commonly reported succession on plant litter introduced into an aquatic environment has been a primary fungal colonization followed by a broader microbial community (Kaushik and Hynes 197 1; Lee et al. 1980; Suberkropp et al . 1976). Morrison et al . (1977) reported a different pattern of succession for oak litter in an estuary, where a primarily bacterial micro- biota was succeeded by one dominated by fungi and other more complex forms. In the present study, bacteria dominated the microbiota initially but only for a short time. Various other workers have observed diatoms colonizing plant litter, but in no previous instance did they become such a major component of the microbiota (Iversen 1973; Morrison et al . 1977; Suberkropp et al . 1976). This high density of diatoms in the later stages of colonization suggests that the litter was increasingly becoming a substrate for attachment rather than nutri- tion.
In contrast with many previous studies (Hodkinson 1975; Howard-Williams and Davies 1979; Hunter 1976; Mathews and Kowalczewski 1969; Suberkropp et al . 1976), Carex litter decreased rather than increased in nitrogen content as decomposition progressed. In- creasing nitrogen content has been generally attributed to immobilization of nitrogen from the water by the litter microbiota. Because the concentration of dissolved nitrogen in Toolik Lake is so low and the nitrogen con- tent of Carex litter is so high, the microbiota probably obtained sufficient nitrogen from the litter and had no need to immobilize further nutrient. Recently, Odum et al. (1979) suggested that much of the nitrogen that becomes associated with decaying litter is not protein, but probably is in the cell walls of fungi as chitin. If this is true, the lack of evidence for extensive fungal growth during much of the study could also partially explain the increased C:N ratio as decomposition proceeds.
FIG. 10. Scanning electron micrographs of Carex leaf litter incubated for 17 days (A), 24 days (B), or I year (C) in the littoral zone of Toolik Lake showing (A) numerous bacteria- like forms enmeshed in slime, (B) bacterialike forms and mnnate diatoms on the surface of the slime, and (C) hollowed remains of the plant cell walls and numerous p k a t e diatoms (bar = 50 pm).
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Although ideally the litter used for a study such as this one should be leaves that have just completed senes- cence, the life cycle of emergent Carex made it impos- sible to obtain leaves that had completed senescence but had not become submerged. Green Carex leaves were used because (1) they were readily available, (2) their age and history could be easily controlled, (3) they were more homogenous in chemical composition, and (4) they were virtually uncolonized by microorganisms. Other available litters would have been of terrestrial origin or, having become submerged, would have begun the process of colonization and decomposition. Green Carex leaves, therefore, were the best available model to study the colonization and decomposition of emergent macrophytes in Toolik Lake. Although the handling and preparation of the litter may have influenced the rates of decomposition and colonization, it allowed measure- ments of these processes to be replicated with small variances using small sample sizes (300 mg). Without prior standardization of the litter, large amounts of litter would have been needed to obtain reasonable replica- tion. The inclusion of such large amounts of litter together would itself be unnatural and could possibly lead to localized anaerobiosis and nutrient depletion, which could have even more dramatically influenced the rates of colonization and decomposition.
Acknowledgements We wish to thank Dr. Robert Pfister for SEM
assistance, Robert Findlay for help with the CHN analy- sis, Dr. D. C. White for the use of the CHN analyzer, and Vicky McKinley for numerous discussions and assistance in the preparation of the manuscript. This work was supported by grant DPP78-27574 from the National Science Foundation and subcontract G C 80043 from the Marine Biological Laboratory which was part of a grant to Dr. J . E. Hobbie from the United States Department of Energy.
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