Individual variability in stable isotope turnover rates of epidermal mucus according to body size in an omnivorous fish

Epidermal mucus (‘mucus’) is increasingly applied to fish ecological studies based on stable isotope analysis (SIA) due to its non-invasive collection. However, knowledge on mucus SI turnover rates of individual fish remains limited, including uncertainty over how they are influenced by fish body sizes. Here, a diet switch experiment predicted mucus SI turnover rates (δ¹³C and δ¹⁵N) as a function of time using samples taken over 200 days from 10 individually tagged common carp Cyprinus carpio covering two size groups. Non-linear mixed effects models revealed rapid turnover of both δ¹³C and δ¹⁵N (T₅₀: 2–5 days; T₉₅: 9–22 days); δ¹⁵N turnover rates were slower for the larger cohort, while δ¹³C turnover rates were independent of body size. Within size groups, turnover rates were not expected to vary between individuals. These experimental results suggest that due to these fast turnover rates, epidermal mucus can provide insights into the diets of fish over very short timeframes, although for δ¹⁵N the body size of the fish needs consideration.

     

Stable isotopes reveal contrasting trophic dynamics between host–parasite relationships: A case study of Atlantic salmon (Salmo salar) and parasitic lice (Lepeophtheirus salmonis and Argulus foliaceus)

Across existing fish host–parasite literature, endoparasites were depleted in δ¹⁵N compared to their hosts, while ectoparasitic values demonstrated enrichment, depletion and equivalence relative to their hosts. δ¹³C enrichment varied extensively for both endo- and ectoparasites across taxa and host tissues. In our case study, sea lice (Lepeophtheirus salmonis) were enriched in δ¹⁵N relative to their farmed Atlantic salmon (Salmo salar) hosts, although the value contradicted the average that is currently assumed across the animal kingdom. Common fish lice (Argulus foliaceus) did not show a consistent trend in δ¹⁵N compared to their wild S. salar hosts. Both parasitic species had a range of δ¹³C enrichment patterns relative to their hosts. Farmed and wild S. salar had contrasting δ¹³C and δ¹⁵N, and signals varied across muscle, fin and skin within both groups. L. salmonis and A. foliaceus subsequently had unique δ¹³C and δ¹⁵N, and L. salmonis from opposite US coasts differed in δ¹⁵N. Given the range of enrichment patterns that were exhibited across the literature and in our study system, trophic dynamics from host to parasite do not conform to traditional prey to predator standards. Furthermore, there does not appear to be a universal enrichment pathway for δ¹³C nor δ¹⁵N in parasitic relationships, which emphasizes the need to investigate host–parasite linkages across species.     

Isotope analysis reveals proportional change and site-selection variation of river- and lake-produced eggs of a landlocked migratory fish

Changes in the proportions of river- and lake-produced eggs of a landlocked amphidromous fish, ayu (Plecoglossus altivelis altivelis) in the Lake Biwa water system, Japan, were monitored by stable isotope analysis, based on different δ¹⁵N and δ¹³C values of prey organisms between the lake and its tributaries. During the 3 month reproduction season, the δ¹⁵N values of spawned eggs decreased with time. This result implies that there was a shift from lake-produced eggs to river-produced eggs within a reproductive season, based on the observation that adult fish in the lake had previously been shown to have eggs with distinctly higher δ¹⁵N values in their ovaries than those in the tributaries. This explanation was also supported by the change in δ¹³C values of the spawned eggs. Furthermore, eggs with lower δ¹⁵N and higher δ¹³C values tended to be spawned at less variable depths, suggesting that females spawning river-produced eggs selected the spawning sites from a narrower range. We conclude that stable isotope ratios of spawned eggs can be indicators of the relative contributions of different food chains and can enable comparisons of reproductive characteristics between types of egg.     

Utility of fish scales from stock assessment surveys in stable isotope analysis for initial assessments of trophic relationships in riverine fish communities

The utility of using fish scales collected during stock assessment exercises to assess the trophic relationships of riverine fishes using their stable isotopes of d¹³C and d¹⁵N was tested using three riverine fish communities in England (Rivers Great Ouse, Ivel and Goyt). In each river, European barbel Barbus barbus was an important species, with other cyprinid species, including chub Squalius cephalus, present. Stable isotope analyses was completed using relatively small sample sizes per species (<11) from fish samples collected in 2001, 2005 and 2006 when up to 5 scales were collected from each fish. The calculation of standard ellipse areas (as a measure of trophic niche size) revealed that relative to other fishes, B. barbus occupied high trophic positions with minimal overlap in their trophic niche with other species, especially S. cephalus. As the analysed fish samples comprised species of various length ranges and as length has strong ontogenetic consequences for fish diet composition, generalized linear models were developed in which length was the covariate; model outputs included length‐adjusted mean δ¹³C and δ¹⁵N for each species. In each fish community, significant differences in δ¹³C and δ¹⁵N were apparent between B. barbus and S. cephalus, but were less apparent between B. barbus and other fishes. Thus, whilst the utility of using fish scales from stock assessments in stable isotope analyses are limited due to the differing length ranges of the sampled fishes, they can be useful in identifying trophic differences between species when methods such as stomach content analyses are unavailable.     

Nitrogen isotopes in otoliths reconstruct ancient trophic position

The ratio of ¹⁵N/¹⁴N (δ¹⁵N) from consumer and prey tissue is commonly used in ecological studies to determine trophic level, food web structure, and mean trophic level in aquatic ecosystems. There is a predictable positive relationship between the δ¹⁵N values in tissue and trophic level, caused by the bioaccumulation of ¹⁵N in tissues of consumers with each step up the food chain. Reconstructing trophic structure or food chain length over time may provide resource managers with insights about ecosystem biodiversity and resilience. Yet, in many marine systems the absence of baseline information before anthropogenic disturbances makes comparative studies addressing ecosystem responses extremely difficult. Here we attempt to retrospectively reconstruct trophic position in four species of fish from the upper Gulf of California, Mexico before perturbations such as overfishing or the damming of the Colorado River. We first validated if otolith δ¹⁵N approximates the δ¹⁵N observed in fish tissue. We then used the δ¹⁵N encapsulated in ancient fish otoliths that are between 1,000 and 5,500 years old to define the food web structure. Our results suggested that δ¹⁵N in otoliths has slightly more positive δ¹⁵N than soft tissue. The δ¹⁵N values from ancient otoliths appropriately defined the fishes’ relative trophic position. We found significant differences in δ¹⁵N between functional groups, apex predator versus intermediate predators. Juveniles and adult fishes displayed trophic separation between functional groups. Our findings advocate the application of δ¹⁵N analysis of prehistoric otoliths for establishing pre-disturbance ecological benchmarks.     

Use of a δ13C–δ15N relationship to determine animal trophic positions in a tropical Australian estuarine wetland

Stable isotope composition of organisms from different trophic groups collected from a semi‐isolated wetland pool in the Ross River estuary, northern Australia, was analysed to determine if there was a consistent relationship between δ¹³C, δ¹⁵N and trophic level that could be used to assign trophic positions. A strong linear negative relationship between δ¹³C and δ¹⁵N was detected for the three trophic levels considered (primary producers, primary consumers and secondary consumers). This relationship was consistent among trophic levels, differing only in height, that is, on δ¹⁵N values, which indicate trophic positions. A difference of 3.6–3.8‰ between trophic levels was present, suggesting a δ¹⁵N fractionation of approximately 3.7‰, a value slightly higher than the commonly assumed δ¹⁵N fractionation of approximately 3.4‰. The relationship between δ¹³C and δ¹⁵N was similar for invertebrate and fish primary consumers, indicating similar δ¹⁵N trophic fractionation for both groups, meaning trophic positions and trophic length could be reliably calculated based on either invertebrates or fish.     

δ15N of estuarine fishes as a quantitative indicator of urbanization

Nitrogen stable isotope values (δ¹⁵N) have commonly been used as a qualitative indicator of catchment urbanization in estuaries, but no quantitative relationship has to date been established between δ¹⁵N and degree of urbanization. We sampled five species of common estuarine fish (Mugil cephalus, Acanthopagrus australis, Sillago ciliata, Gerres subfasciatus and Herklotsichthys castelnaui) of different trophic levels from six estuaries in Southeast Queensland, Australia, to test if a quantitative relationship exists between fish δ¹⁵N and urbanization in the catchment. Degree of urbanization (urban %) in the catchment adjacent to the tidal estuary was measured using polygons constructed in Google Earth images, and verified using data from the detailed Queensland Land Use Mapping Project (QLUMP). Significant linear relationships exist between fish δ¹⁵N and urban % in most cases irrespective of the section of the estuary (upper, middle and lower) where the fish were caught, with no difference in slope and elevation between lines established for different sections. Inclusion of additional fish δ¹⁵N data from other Australian estuaries in a combined multiple regression model adding trophic level as a predictor generated a significant relationship predicting increase in fish δ¹⁵N values with increasing urban % and trophic level. There is therefore a generic quantitative relationship between estuarine fish δ¹⁵N values and degree of urbanization applicable to different fish species, geographic location or feeding habit. While the generality of this relationship needs to be tested further in other geographic locations, our findings potentially improve the applicability of fish δ¹⁵N values as a quantitative indicator of urban influence in estuaries.     

Application of non‐lethal stable isotope analysis to assess feeding patterns of juvenile pallid sturgeon Scaphirhynchus albus: a comparison of tissue types and sample preservation methods

Traditional techniques for stable isotope analysis (SIA) generally require sacrificing animals to collect tissue samples; this can be problematic when studying diets of endangered species such as the pallid sturgeon Scaphirhynchus albus. Our objectives were to (i) determine if pectoral fin tissue (non‐lethal) could be a substitute for muscle tissue (lethal) in SIA of juvenile pallid sturgeon, and (ii) evaluate the influence of preservation techniques on stable isotope values. In the laboratory, individual juvenile pallid sturgeon were held for up to 186 day and fed chironomids, fish, or a commercially available pellet diet. Significant, positive relationships (r² ≥ 0.8) were observed between fin and muscle tissues for both δ¹⁵N and δ¹³C; in all samples isotopes were enriched in fins compared to muscle tissue. Chironomid and fish based diets of juvenile pallid sturgeon were distinguishable for fast growing fish (0.3 mm day^?1) using stable δ¹⁵N and δ¹³C isotopes. Frozen and preserved fin tissue δ¹⁵N isotopes were strongly related (r² = 0.89) but δ¹³C isotopes were weakly related (r² = 0.16). Therefore, freezing is recommended for preservation of fin clips to avoid the confounding effect of enrichment by ethanol. This study demonstrates the utility of a non‐lethal technique to assess time integrated food habits of juvenile pallid sturgeon and should be applicable to other threatened or endangered species.     

Spatial,temporal, age,and sex related variation in the diet of South American sea lions in southern Chile

We analyzed spatial, temporal, and age/sex variation in the diet of the South American sea lions (SASL, Otaria byronia) in southern Chile via δ¹³C and δ¹⁵N isotope analysis, with emphasis on exotic farmed and feral salmonids as a source of prey for this generalist predator. Isotope mixing models show that the primary prey consumed by SASL were demersal and benthopelagic fishes. Individuals sampled in summer and in the outer coast had higher δ¹⁵N values than those from winter or from the inner coast due to consumption of high trophic level demersal fish. We also found evidence of an ontogenetic shift in isotopic niche width as measured by δ¹³C and δ¹⁵N standard ellipse areas, with juveniles showing a restricted isotopic niche width (1.0‰²) in comparison to subadult (2.2‰²) and adult males (1.9‰²). The proportion of salmonids in the diet of the SASL was negligible and lower than reported in previous studies, which may be related to a recent drop in the production of salmon aquaculture in the region. Our study suggests that the SASL are able to adapt to shifts in spatiotemporal variations in the abundance of potential prey, including feral and farmed salmonids.     

The trophic niche of sculpins Cottus spp. in forage fish assemblages of boreal lakes

We compared the trophic niches of freshwater sculpins Cottus spp. with those of other co-habiting forage fishes in two groups of boreal lakes with distinct habitats and fish communities. Near North Lakes (45° 00′ to 47° 30′ N) were deeper, stratified and contained lake trout Salvelinus namaycush as the apex piscivore, whereas Far North Lakes (51° 10′ to 52° 20′ N) were shallower, did not stratify and contained pike Esox lucius and walleye Sander vitreus as the apex piscivores. Trophic niches of sculpins and other forage fishes were compared based on niche metrics calculated from muscle stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope ratios. In Near North Lakes, sculpins were found almost exclusively in deep, offshore waters and their niche positions reflected a greater reliance on pelagic production (lower δ¹³C) and a higher trophic elevation (higher δ¹⁵N) compared with most other forage fishes. Furthermore, sculpins in Near North Lakes tended to have larger trophic niches (occupied greater area in δ¹³C– δ¹⁵N space), particularly in the food chain (δ¹⁵N) dimension, than other cohabiting forage fishes. In contrast, sculpins in Far North Lakes were commonly found in the nearshore and had trophic niche positions and sizes that were similar to those of the other cohabiting forage fishes. This study illustrates the flexibility in the realised trophic niches of sculpins in relation to habitat availability and fish community composition in boreal lakes.     

Relationships between growth traits and scale stable isotopes (δ13C, δ15N) of adult chum salmon Oncorhynchus keta in Hokkaido,Japan

Stable isotope analysis (SIA) in combination with growth analysis using scales collected from adult chum salmon Oncorhynchus keta migrating back to Hokkaido, Japan, was performed to describe the variation of isotopic composition of carbon (δ¹³C) and nitrogen (δ¹⁵N) in scales and to examine relationships with growth traits [age, fork length (FL), and relative growth ratio in the last growth period [(RGR^last)]. Scale stable isotope (SI) values in 3‐ to 6‐year‐old fish were highly variable, ranging from ?17.6‰ to ?14.3‰ for δ¹³C and 9.5‰ to 13.4‰ for δ¹⁵N. The δ¹⁵N was positively correlated with FL, and this tendency may indicate changes in trophic level with growth. Significant effect was not detected between δ¹⁵N and RGR^last, it can be inferred that factors potentially yielding high δ¹⁵N may not necessarily result in higher growth rates. No trend found between FL and δ¹³C may imply that there is no clear segregation in feeding locations between the 3‐ to 6‐year groups. This study provided basic information for scale SI values of chum salmon adults and indicated that SIA using scales could be a new approach to elucidating the trophic ecology of chum salmon.     

Patterns of soil and plant nitrogen isotope composition and their relationships with climate factors in Xilingol League,Inner Mongolia,China

Background: Plant and soil nitrogen stable isotope (δ¹⁵N) can integrate several fundamental biogeochemical processes in ecosystem nitrogen dynamics, and reflect characteristics of ecosystem nitrogen cycling.

Aims: We investigated how climate change influenced plant-soil nitrogen cycling by relating soil δ¹⁵N, plant δ¹⁵N and Δδ¹⁵N (difference between soil and plant δ¹⁵N) with climatic factors.

Methods: Field investigation was conducted in temperate grasslands in Inner Mongolia during August 2015. Plant δ¹⁵N, soil δ¹⁵N and Δδ¹⁵N were determined, and their relationships with climatic factors were examined by simple regression analyses and general linear models.

Results: Soil δ¹⁵N was significantly higher than plant δ¹⁵N, and there was a positive linear correlation between them. Soil and plant δ¹⁵N were negatively related with mean annual precipitation (MAP) and positively with mean annual temperature (MAT); conversely, Δδ¹⁵N was positively related with MAP and negatively with MAT.

Conclusion: Soil δ¹⁵N was dominantly controlled by MAT, while it was MAP for plant δ¹⁵N. Climate factors influenced plant δ¹⁵N not only through their effects on soil nitrogen dynamics but also strategies of plant nitrogen acquisition. Thus, compared with plant δ¹⁵N, soil δ¹⁵N can more accurately reflect soil nitrogen dynamics, while plant δ¹⁵N may integrate soil nitrogen dynamics and plant nitrogen acquisition.     

Using hydrogen isotopes of freshwater fish tissue as a tracer of provenance

Hydrogen isotope (δ²H) measurements of consumer tissues in aquatic food webs are useful tracers of diet and provenance and may be combined with δ¹³C and δ¹⁵N analyses to evaluate complex trophic relationships in aquatic systems. However, δ²H measurements of organic tissues are complicated by analytical issues (e.g., H exchangeability, lack of matrix‐equivalent calibration standards, and lipid effects) and physiological mechanisms, such as H isotopic exchange with ambient water during protein synthesis and the influence of metabolic water. In this study, δ²H (and δ¹⁵N) values were obtained from fish muscle samples from Lake Winnipeg, Canada, 2007–2010, and were assessed for the effects of species, feeding habits, and ambient water δ²H values. After lipid removal, we used comparative equilibration to calibrate muscle δ²H values to nonexchangeable δ²H equivalents and controlled for H isotopic exchange between sample and laboratory ambient water vapor. We then examined the data for evidence of trophic δ²H enrichment by comparing δ²H values with δ¹⁵N values. Our results showed a significant logarithmic correlation between fork length and δ²H values, and no strong relationships between δ¹⁵N and δ²H. This suggests the so‐called apparent trophic compounding effect and the influence of metabolic water into tissue H were the potential mechanisms for δ²H enrichment. We evaluated the importance of water in controlling δ²H values of fish tissues and, consequently, the potential of H isotopes as a tracer of provenance by taking account of confounding variables such as body size and trophic effects. The δ²H values of fish appear to be a good tracer for tracking provenance, and we present a protocol for the use of H isotopes in aquatic ecosystems, which should be applicable to a broad range of marine and freshwater fish species. We advise assessing size effects or working with fish of relatively similar mass when inferring fish movements using δ²H measurements.     

Variations in stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) in different body parts and eggs of adult stoneflies (Plecoptera)

Variations in δ¹³C and δ¹⁵N might arise from differences in nutrient allocation. Residence times of δ¹³C and δ¹⁵N vary among tissues depending on metabolic turnover rates. However, because of their small size, entire individual insects are generally used as single samples in isotope analyses. The present study aimed to determine the degree of isotope similarity among regions of the adult body and eggs in four species of Plecoptera (Amphinemura sp., Sweltsa sp., Kamimuria tibialis Pictet and Ostrovus sp.). Levels of δ¹³C and δ¹⁵N differ between the four species, being lowest in Amphinemura sp., and with δ¹⁵N being highest in Sweltsa sp. Egg masses contain consistently the lowest values of δ¹³C in the four species, with the δ¹⁵N value of eggs being highest in K. tibialis and Ostrovus sp., and lower in Amphinemura and Sweltsa spp. In Sweltsa sp., the δ¹⁵N levels of the dermal layers and cuticle are lowest, whereas the δ¹³C values of the dermal layers and cuticle are almost equal to those in other regions of the body, except egg masses. Oviposited individuals of Amphinemura and Sweltsa spp. have lower δ¹⁵N levels than individuals that have not oviposited. The rates of metabolism and incorporation of dietary metabolites will differ depending on the body regions and species. Differences in egg ecology such as egg developmental period and egg buoyancy among species are considered to impact on the values of δ¹³C and δ¹⁵N. These results will be useful for understanding the nutritional status of aquatic insects and their energy allocation.     

Isotopic niche differs between seal and fish‐eating killer whales (Orcinus orca) in northern Norway

Ecological diversity has been reported for killer whales (Orcinus orca) throughout the North Atlantic but patterns of prey specialization have remained poorly understood. We quantify interindividual dietary variations in killer whales (n = 38) sampled throughout the year in 2017–2018 in northern Norway using stable isotopic nitrogen (δ¹⁵N: ¹⁵N/¹⁴N) and carbon (δ¹³C: ¹³C/¹²C) ratios. A Gaussian mixture model assigned sampled individuals to three differentiated clusters, characterized by disparate nonoverlapping isotopic niches, that were consistent with predatory field observations: seal‐eaters, herring‐eaters, and lumpfish‐eaters. Seal‐eaters showed higher δ¹⁵N values (mean ± SD: 12.6 ± 0.3‰, range = 12.3–13.2‰, n = 10) compared to herring‐eaters (mean ± SD: 11.7 ± 0.2‰, range = 11.4–11.9‰, n = 19) and lumpfish‐eaters (mean ± SD: 11.6 ± 0.2‰, range = 11.3–11.9, n = 9). Elevated δ¹⁵N values for seal‐eaters, regardless of sampling season, confirmed feeding at high trophic levels throughout the year. However, a wide isotopic niche and low measured δ¹⁵N values in the seal‐eaters, compared to that of whales that would eat solely seals (δ_N‐measured = 12.6 vs. δ_N‐expected = 15.5), indicated a diverse diet that includes both fish and mammal prey. A narrow niche for killer whales sampled at herring and lumpfish seasonal grounds supported seasonal prey specialization reflective of local peaks in prey abundance for the two fish‐eating groups. Our results, thus, show differences in prey specialization within this killer whale population in Norway and that the episodic observations of killer whales feeding on prey other than fish are a consistent behavior, as reflected in different isotopic niches between seal and fish‐eating individuals.     

Use of stable isotopes to understand food webs in Macao wetlands

In this study, components of the food-web in Macao wetlands were quantified using stable isotope ratio techniques based on carbon and nitrogen values. The δ¹³C and δ¹⁵N values of particulate organic matter (δ¹³C_POM and δ¹⁵N_POM, respectively) ranged from ?30.64 ± 1.0 to ?28.1 ± 0.7 ‰, and from ?1.11 ± 0.8 to 3.98 ± 0.7 ‰, respectively. The δ¹³C values of consumer species ranged from ?33.94 to ?16.92 ‰, showing a wide range from lower values in a freshwater lake and inner bay to higher values in a mangrove forest. The distinct dietary habits of consumer species and the location-specific food source composition were the main factors affecting the δ¹³C values. The consumer ¹⁵N-isotope enrichment values suggested that there were three trophic levels; primary, secondary, and tertiary. The primary consumer trophic level was represented by freshwater herbivorous gastropods, filter-feeding bivalves, and plankton-feeding fish, with a mean δ¹⁵N value of 5.052 ‰. The secondary consumer level included four deposit-feeding fish species distributed in Fai Chi Kei Bay and deposit-feeding gastropods in the Lotus Flower Bridge flat, with a mean δ¹⁵N value of 6.794 ‰. The tertiary consumers group consisted of four crab species, one shrimp species, and four fish species in the Lotus Flower Bridge Flat, with a mean δ¹⁵N value of 13.473 ‰. Their diet mainly comprised organic debris, bottom fauna, and rotten animal tissues. This study confirms the applicability of the isotopic approach in food web studies.     

δ15N values of macroalgae as an indicator of the potential presence of waste disposal from land-based marine fish farms

The nitrogen isotope ratio (δ¹⁵N) in tissues of native macroalgae was evaluated as a means of indicating the intensity and spatial extent of organic contamination due to disposal of waste from land-based marine fish farms (LBMFFs). Three species of macroalgae from the genus Fucus and the green macroalgae Codium tomentosum were selected for study. The study was carried out at seven flat marine fish farms located in Galicia (NW Spain). Tests were carried out to determine the intra-annual variation in δ¹⁵N values and any differences between selected macroalgae. The δ¹⁵N values enrichment was observed close to the disposal point, and δ¹⁵N values varied more widely throughout the year (±5.57 ‰) at sites affected by the marine fish farm effluent compared to natural conditions (±2 ‰). No significant differences in the isotopic signals were observed in the different species studied (standard major axis). The δ¹⁵N values of macroalgae may be an ideal means of detecting the presence of LBMFFs effluents.     

Consistency of elemental and isotope‐ratio patterns across multiple scales from individual fish

Assays of elemental and stable‐isotope ratios across growth increments of scales have the potential to provide a non‐lethal alternative to otolith chemistry for identifying migration and ontogenetic trophic shifts. A central assumption when employing scales as otolith analogues is that any scale from an individual will provide equivalent information about the chemical history of that fish. This assumption was investigated with multiple scales from wild and captive euryhaline Atlantic tarpon Megalops atlanticus from the north‐west Gulf of Mexico. Elemental (Sr:Ca) and isotope‐ratio (δ¹³C and δ¹⁵N) life‐history profiles were compared among multiple scales from each fish. All three chemical proxies showed highly consistent patterns among non‐regenerated scales, while patterns in regenerated scales diverged, indicating rapid regrowth of interior scale material at the onset of regeneration. Patterns of Sr:Ca and δ¹³C covaried, supporting their use as salinity proxies, while δ¹⁵N patterns were consistent with ontogenetic diet shifts. Water samples taken from aquaria holding captive fish were used to calculate partition coefficients for a suite of elements in M. atlanticus scales for future quantification of migratory movements in the region. Together, these results support the assumption that non‐regenerated scales from individual M. atlanticus provide equivalent chemical histories, further validating their use as a viable non‐lethal alternative to otoliths.     

VARIATION IN INTERNAL δ15N AND δ13C DISTRIBUTIONS AND THEIR BULK VALUES IN THE BROWN MACROALGA PADINA AUSTRALIS GROWING IN SUBTROPICAL OLIGOTROPHIC WATERS1

The utility of δ¹⁵N measurements in Padina australis Hauck as a probe for its external nitrogen (N) sources was tested by monitoring the bulk values of chemical components [δ¹⁵N, δ¹³C, and N and carbon (C) contents] and their internal distributions during a 12 d incubation in a controlled environment. Under the saturated conditions of isotopically heavier nitrate than that of original algal tissue, the bulk δ¹⁵N in P. australis was enriched, but less than what was predicted from a simple mixing model, signaling possible isotopic discrimination during N assimilation and subsequent N efflux from the cells. The enhanced N content (%), which occurred simultaneously with this δ¹⁵N shift, was a useful signal indicating this phenomenon. Bulk δ¹⁵N was enriched, especially around the meristem, in tissues growing under conditions of higher irradiance and temperature, probably due in part to dissolved organic nitrogen (DON) excretion. The δ¹³C enhancement in bulk algal tissues, also associated with high photosynthetic activity, may be an additional signal indicating this unbalanced internal δ¹⁵N distribution. However, in summer and winter environmental conditions with periodic nitrate supplies simulating typical fringing reef waters, the difference in measured algal bulk δ¹⁵N from theoretical predictions was within ±1.0‰. This difference is very small compared with the variation in δ¹⁵N in possible N sources in coastal areas. In the field, therefore, δ¹⁵N in Padina can be used effectively to trace N sources in both space and time after determining algal N content and δ¹³C to determine whether large alterations occur in algal δ¹⁵N.     

Investigating patterns of symbiotic nitrogen fixation during vegetation change from grassland to woodland using fine scale δ15N measurements

Biological nitrogen fixation (BNF) in woody plants is often investigated using foliar measurements of δ¹⁵N and is of particular interest in ecosystems experiencing increases in BNF due to woody plant encroachment. We sampled δ¹⁵N along the entire N uptake pathway including soil solution, xylem sap and foliage to (1) test assumptions inherent to the use of foliar δ¹⁵N as a proxy for BNF; (2) determine whether seasonal divergences occur between δ¹⁵N_{xylem sap} and δ¹⁵N_{soil inorganic N} that could be used to infer variation in BNF; and (3) assess patterns of δ¹⁵N with tree age as indicators of shifting BNF or N cycling. Measurements of woody N‐fixing Prosopis glandulosa and paired reference non‐fixing Zanthoxylum fagara at three seasonal time points showed that δ¹⁵N_{soil inorganic N} varied temporally and spatially between species. Fractionation between xylem and foliar δ¹⁵N was consistently opposite in direction between species and varied on average by 2.4‰. Accounting for these sources of variation caused percent nitrogen derived from fixation values for Prosopis to vary by up to ～70%. Soil–xylem δ¹⁵N separation varied temporally and increased with Prosopis age, suggesting seasonal variation in N cycling and BNF and potential long‐term increases in BNF not apparent through foliar sampling alone.