MARINE ECOLOGY PROGRESS SERIES Vol. 189: 289-291. L999 1 Published November 26 Mar Ecol Prog Ser

15~enrichment as a method of separating the isotopic signatures of and its epiphytes for food web analysis

M. A. Winning1-*, R.M. Connolly2, N. R. Loneragan3, S. E. Bunn'

'centre for Catchment and In-Stream Research. Faculty of Environmental Sciences, Griffith University. Nathan. Queensland. 411 1. Australia 'School of Environmental and Applied Science, Gold Coast campus. Griffith University, PMB 50. Gold Coast Mail Centre, Queensland 9726. Australia 'CSIRO Division of Marine Research, Cleveland Marine Laboratories. PO Box 120. Cleveland. Queensland 4163. Australia

ABSTRACT: Stable isotope analysis of food webs is of limited primary sources, or where a consumer's signature use where there is little or no difference in the natural abun- could have resulted from assimilating either 1 source dance isotopic ratios of potential food sources. "N-enriched or a mixture of 2. potassium nitrate was used to differentially label 2 potential food sources for seagrass fauna: seagrass and its attached One method which overcomes the limitations of this epiphytes. Different combinations of exposure time to the technique, as well as the low level of resolution enriched substrate and different concentrations of enriched observed in natural abundance stable isotope food substrate were used to maximise the difference in 6lSN signa- web studies, is the manipulation of the isotopic ratio of ture between the 2 food sources. After adding the enriched substrate 615N values of epiphytes ranged from 87 to 713%., a particular primary food source, by adding a I5N- or and were consistently higher than the 6% values of sea- 13C-enriched substrate. This enriched signature can grass, which ranged from 25 to 90%. Enriched substrate addi- then be traced through the food web. Few studies of tions every 3 d resulted in the greatest sustained separation food webs have used enriched stable isotope tracers between seagrass and epiphytes over 18 d. The results de- (e.g. Kling 1994, Hall 1995), and this technique does monstrate that enriched 15N tracers are useful for separating the 6I5N signatures of previously difficult to distinguish pri- not appear to have been applied in estuarine or marine mary sources, and that thls technique has the potential to ecosystems. resolve ambiguous natural abundance isotope results. Seagrass and epiphytic algae attached to seagrass leaves are both potential food items for animals inhab- KEY WORDS: Algae - Stable isotopes . "N-enriched isotope iting seagrass meadows. Many animals have been tracers . Trophic webs - Zostera capricorni shown to ingest seagrass. McRoy & Helfferich (1980) list 154 such species including dugon, blue crabs Callinectes sapidus, numerous birds, and 59 species of fish. Seagrass, however, is gen- The past 2 decades have seen a rapid expansion in erally considered to be of little nutritional importance the use of natural abundance stable isotopes such as compared to other plant sources, such as epiphytic 6I3C, 8l5N and, to a lesser extent, 634S to analyse algae (Klumpp et al. 1989). Epiphytes can be of higher aquatic food webs (e.g. Peterson et al. 1985, Sullivan & nutritional quality than and are often Moncreiff 1990, Bunn & Boon 1993).The advantages of mechanically more accessible (Bell & Pollard 1989). In this approach over techniques such as gut content some cases, the greater importance of algae than sea- analysis are that it distinguishes assimilated rather grass to consumers has been supported by the 613C val- than ingested foods, and it can represent a history of ues of the consumers (e.g. Kitting et al. 1984), while in assimilation over longer periods (Gearing 1991). A other studies the isotope ratios of seagrass and epi- major limitation, however, is that potential food phytes could not be differentiated (Fry et al. 1982, Lon- sources must be isotopically distinct in 1 or more of the eragan et al. 1997). isotopes examined (Gearing 1991). Additional prob- In applying enriched-tracer techniques to a seagrass lems can arise when there are more than 2 distinctive system for the first time, we attempted to: (1) separate the isotopic signature of 2 potential food sources (sea- grass and its attached epiphytes) for seagrass fauna in the short term, using a 15N-enriched substrate, and

O Inter-Research 1999 Resale of fuU article notpermitted Mar Ecol Prog Ser 189: 289-294, 1999

(2) determine an enriched substrate addition regime concentrations (low = 0.35 mg 1-l K15N03 or 3.25 pM N; which creates the greatest separation in 615N values of high = 0.7 mg l-' K15N03 or 6.5 pM N) were tested seagrass and its epiphytes over an extended period. (Table 1).Three tanks were randomly assigned to each Materials and methods. Zostera capn-corni is the treatment, with the proviso that each large holding dominant shallow-water seagrass species along the container had only 1 tank of each treatment. east coast of Australia and within . Pieces Following the addition of I5N-KNO, to each tank, the of seagrass turf, including the substrate, the root sys- flow was turned off for the duration of the spiking tem, and the epiphytes attached to the seagrass leaves, period, and a plastic stick was used to thoroughly mix were removed from Toondah Harbour, in Moreton Bay, the body of water for approximately 5 S, to ensure an Australia (27" 38'S, 153" 25' E), using a spade. The sea- even mix of the enriched material throughout the grass was placed immediately into plastic tanks water. Except for those tanks collected immediately (415 mm long, 270 mm wide, and 255 mm high), where after 15 min (Combination l), the flow-through seawa- it remained for the duration of the isotopic enrichment ter system was turned on at a faster rate (the flushg experiment. The seagrass was predominantly the long- rate of approximately 80 1 h-') after the spiking period leaved Z. capricorni morph (200 to 300 mm long, (Table 1). All other combinations were collected l h approximately 5 mm wide) as identified by O'Dono- after the initial spike, and were then placed into plastic hue et al. (1991). Seagrass shoot density averaged 425 bags. shoots m-' (n = 20). Longer-term I5N enrichment using multiple addi- At the same time as seagrass turf was collected for tions: The second experiment was designed to exam- the enrichment experiments, 4 samples of the follow- ine 2 aspects of lSN enrichment of seagrass and epi- ing material were collected for the measurement of phytes: (1) to determine which 15N-addition interval natural abundance stable isotope values: (1) seagrass, (every 3 or 6 d) creates the greatest and most consis- cleaned of epiphytes; (2) seagrass, with epiphytes tent separation between the 615N values of seagrass attached; (3) epiphytes removed from the seagrass. and epiphytes over an 18 d period, and (2) to examine Laboratory procedures: All tanks of seagrass turf the loss of enrichment (or decay) of seagrass and epi- were retained indoors for the duration of the isotopic phyte 6I5N values after 1 initial addition. enrichment experiments. Four tanks of seagrass were Three treatments were examined: (1) 1 initial contained within 1 large holding container. The vol- 15N addition only (i.e. decay tanks); (2) 15N additions ume of water in individual tanks ranged from 11 to 19 1, every 3 d; and (3) 15N additions every 6 d. Samples of depending on the amount of seagrass substrate con- seagrass, epiphytes and seagrass with epiphytes were tained in each tank. Each tank was connected to a sep- collected from the 3 treatments at various intervals arate flow-through seawater system. Seawater was over 18 d (Table 2).Three tanks were assigned to each pumped from 2 km offshore in Moreton Bay, and was collection time for each treatment. Two-way analysis of sand filtered before it reached the tanks. The flow rate variance (ANOVA), followed by Tukey pairwise com- into each tank was adjusted to a rate of about 11 1 h-'. parisons where appropriate, were used on log-trans- Fluorescent lights attached directly above the tanks formed isotope ratios to test for significant differences provided a light intensity of approximately 400 ~Einst between seagrass and epiphyte values (seagrass with m-2 s-l at the water surface from 06:OO to 18:OO h. epiphytes attached was excluded) and amongst times. Water temperatures were maintained between 25 and The initial concentration of 15N-enriched potassium 27°C. nitrate added was 0.7 mg 1-l. All subsequent 15N addi- The 15N was added as "N-enriched potassium tions were 0.5 mg 1-l. Following each addition, flowing nitrate (>98% '$N). The enriched substrate was dis- water was turned off for 15 min, followed by a fast solved in 5 m1 of distilled water, and then distributed flushing rate for another 45 min to remove the enriched over the surface of each individual tank. water. After this time, the flow-through seawater sys- Short-term I5N enrichment: The aim of this experi- ment was to gain the greatest separation between sea- Table 1. Comb~nations of spiking period, flushing time ana grass and epiphyte 615N values within a l h period by sampling time examining the effects of 2 factors: (1) the time that each tank is exposed to the "N-enriched addition with no Combination Spilung Flushing Sampling time (after flowing seawater (the spiking period), before flushing period time initial lSN-addition) with a faster than normal flow rate, and (2) the concen- tration of "N-enriched potassium nitrate (KI5NO3). 1 15 min - 15 rnin 2 15min 45m lh Four combinations of spiking period and flushing 3 30 min 30 min lh time (amount of time the water was turned on at a 4 lh - lh faster rate to remove enriched water) and 2 substrate Winning et al.: I5N enrichment separates isotopic signatures 291

Table 2. Samphg days for 3 15N-addition treatments ermass, Crewe, UK). Ratios of "N/14N were expressed as the relative per m11 (%o) difference between the sam- Treatment Day of tank san~pling ple and recognised international standards (N2 in air). These differences are expressed in &-notation: (l) l initial 15N-addition 1 3"

18 (2)15N additions every 3 d 3d Results. Short-term 15~enrichment: Mean &15Nval- 6 ues for seagrass and epiphytes from the first experi- 12 ment (Fig. 1) were enriched (seagrass &15N range = 18 25 i 4 to 90 + epiphyte range = 87 * 29 to (3) 15N additions every 6 d 6 713 i 118%0)compared to the natural abundance 615N 12 values from the field (3.7 + 0.2 and 3.8 i 0.4%0respec- 18 tively). The uptake of 15N by seagrass and epiphytes "Treatments 1 and 2 share the same tank data for Day 3 was evident within 15 min of adding the enriched sub- bTreatments 1 and 3 share the same tank data for Day 6 strate (Combination 1). The 615N values for seagrass and epiphytes continued to increase after the 15 min spiking period despite a flushing rate of 80 1 h-' (Com- tem was returned to the normal slow rate of approxi- bination 2). Seagrass with epiphytes attached, and epi- mately 11 l h-'. phytes alone were generally more enriched after the Sample collection and preparation: At each sam- addition of the high concentration than the low con- pling time, the following were collected: (1) seagrass, centration. The proportional differences between the cleaned of epiphytes; (2) seagrass with epiphytes 615N values for epiphytes at the high and low concen- attached, and (3) epiphytes only. All seagrass leaves trations were greater than those observed for seagrass, were removed at the shoot base and placed in a plastic except for Combination 3. In tanks with a hgh concen- bag. Leaves were then gently washed in distilled tration added, epiphyte 615N values were generally water. Some diatoms may have been lost during the double those spiked with the low concentration. The rinsing process, but rinsing was necessary to remove &15Nvalues for seagrass with epiphytes attached were salt and excess detritus (Ott 1990). Epiphytes were intermediate between those of seagrass and epiphytes. removed by gently scraping the seagrass leaves with a Large differences between the 615N values of sea- dull razor blade. This scraping method was used grass and epiphytes (>200%0) were recorded in all instead of the acid wash technique (5% hydrochloric acid) (e.g. Kitting et al. 1984), which can alter the nitrogen composition and Seagrass Seagrass wlth ep~phytes 615N isotope ratios of seagrass (Bunn et al. Ep~phytes 1995). The epiphyte sample was collected in a YUU small amount of distilled water in an alu- 800 rninunl foil container. Epiphytes consisted of 3 700 main types: encrusting calcareous red algae, 600 short filamentous algae and diatoms. 500 Stable isotope analysis: All samples were g 400 dried in an oven at 60°C for 24 to 48 h. Enriched 5 300 and natural abundance samples were dried in separate ovens to reduce the risk of contami- 200 nation. All natural abundance samples were loo ground in a ring grinder. Small amounts of the o dried sam~lesfrom the enrichment ex~eri- low h~gh low h~gh low hlgh low high ments were ground using a porcelain mortar Combination 1 Combination 2 Combination 3 Combination 4 (15 0 (15 45 (60 mm, 0 rnin) and pestle. After each sample was crushed, the min, min) min, rnin) (30 min, 30 min) mortar and pestle were washed in 10 % hydro- Treatment chloric acid, and then rinsed twice in dishlled water to prevent contamination. Fig. 1. Mean 615N values (+l SE) for seagrass, seagrass with epiphytes, and epiphytes using 2 substrate concentrations (low = 0.35 mg 1-l, and Dried were weighed and high = 0.7 mg 1-' of 15N-enriched potassium nitrate), and 4 spiking pe- for 15N/14N and %Nvusing a continuous flow- ,od and flushina- combinations. Duration of S~iklIla.-and flushins are isotope ratio mass spectrometer (Europa Trac- shown in parentheses (see Table 1 for details); n = 3 for each mean Mar Ecol Prog Ser 189: 289-294, 1999

a) one '%-addition only than natural abundance samples in the Seagrass field (4.2 0.02 and 4.1 + 0.81%0 respec- =0° l Seagrass wlth epiphytes tively). The mean 6I5N values for epiphytes Epiphytes were higher than those for seagrass in all treatments at all times (Fig. 2). Following a single addition of 15N- enriched substrate on Day 0, the epiphyte 615N signature ranged from 279 to 452% until Day 6, before decreasing to 64%0 by Day 18 (Fig. 2a). The seagrass 615N signa- ture only declined from 96% at Day 6 to b) '%-additions every 3 d 49%0 by Day 18. The 6I5N values of sea- grass with epiphytes attached generally 600 1 more closely resembled those of seagrass than those of epiphytes. ANOVA results demonstrated a significant interaction between plant type and day (p = 0.033): epiphytes had significantly higher FI5N values than seagrass at all times, except Day 18. In the treatment with multiple I5N addi- tions every 3 d, the mean 615N values for

C) 'S~-additionsevery 6 d epiphytes always exceeded 200%0, and 600 1 were significantly higher than seagrass val- ues (p < 0.001). The 6'" values of seagrass ranged from 60x0 over the first 6 d to 150%0 at Day 18, but differences among days were not significant (p = 0.122) and there was no significant interaction (p = 0.373). The 6% values for seagrass in the 6 d 15N addition treatment ranged from 64 to

101 %O over the 18 d (Fig. Zc), whereas those f t .) of epiphytes were always significantly higher (p < 0.001), ranging from 160 to Samplingtime (days) 450%0. Differences among days were not Fig. 2. Mean 615N values (+l SE) for seagrass, seagrass with epiphytes, and significant (p = 0.094)1 was there a sig- epiphytes for the 3 I5N-addition treatments (see Table 2 for details). f : 15N nificant interaction (p = 0.053). The differ- addition. Samples collected at each time had not been respiked at that ences between the 6I5N values of seagrass time. No samples were taken at Day 9 or Day 15 and epiphytes at Days 12 and 18 were greater with a 15N addition every 3 d (245 + 145%0and 197 +. 88%0difference at Days 12 spiking periods at the high substrate concentration and 18 respectively) than with an addition every 6 d (Fig. 1). However, a spiking period of 15 min at a high (83 rt 34 and 89 + 13%0). concentration was chosen for the second experiment Discussion. Nitrogen has been considered a limiting involving repeated 15N additions because this combi- nutrient in some seagrass meadows (Hillman et al. nation resulted in good separation of seagrass and epi- 1989), and in the present study it was taken up within phyte 15N signatures. It also offered a lower risk that 15 min of the I5N addition by both seagrass and epi- seagrass, which we considered would retain an phytes. Within 1 h, epiphytes were considerably more enriched signature for longer than epiphytes, would "N enriched than seagrass. Algae have been found to increase In their 6I5N values over time. take up nitrogen from the water column more effi- Long-term ''N enrichment using multiple additions: ciently than seagrass, possibly because algae rely As with the first experiment, the 615N values of sea- totally upon nutrients from the water column, whereas grass and epiphyte samples in the second experiment seagrasses also take up nutrients from the sediment (seagrass 6I5N range = 46 + 13 to 149 ? 31 %0; epiphyte (Paling & McComb 1994). Epiphytes are also highly range = 64 * 19 to 452 -r- 31 %o) were more enriched productive, with faster turnover times compared to Winning et al.: I5N enrichme mnt separates isotopic signatures 293

seagrass leaves (Klun~ppet al. 1992, Pollard & Kogure to use this technique to determine the relative impor- 1993). They would therefore replace a greater propor- tance of seagrass and epiphytes as food sources for tion of their biomass with I5N more quickly than sea- juvenile P. esculentus, a species known to feed on sea- grass. The higher surface area to volume ratio of the grass seeds (Wassenberg 1990, O'Brien 1994). When epiphytic algae probably also contributes to the rapid developed and refined, enriched stable isotope tracers divergence in 6I5N values for seagrass and their epi- have the potential to resolve food web questions that phytes. The substantial increase in the 6I5N value of cannot be answered by natural abundance levels of epiphytes after 45 min of flushing (Combination 2) stable isotopes alone. indicates that I5N was still available for uptake by plant sources despite the flow of new water through the tanks. This is probably due to 15N availability Acknowledgements. We thank MichelIe Jenkins. Mick Sutton decreasing in tanks under an exponential dilution and Fraser Smith for advise on the use of the mass spectrom- eter, Bob Pendrey for assistance in the laboratory, and function. However, the seawater inlets of the tanks Michele Burford and James Udy for comments on the manu- were near the surface of the water, and it is possible script. The study was funded by the Australian Research that only the top few centimetres of water were con- Council, using the mass spectrometer at Griffith University stantly replaced and some of the enriched water was and laboratories at CSIRO Marine Research at Cleveland, Queensland. still available for uptake. The high turnover of the epiphytic comn~unities(e.g. Morgan & Kitting 1984, Pollard & Kogure 1993) proba- LITERATURE CITED bly explains the faster decrease in the epiphyte 615N compared with seagrass. 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Editorial responsibility: Otto Kinne (Editor), Submitted: March 1, 1999; Accepted: September 15, 1999 OldendorfLuhe, Germany Proofs received from author(s): November 1, 1999