Nitrogen Isotope Analysis Research Articles

Human land-use changes the diets of sympatric native and invasive mammal species.

The consequences of biological invasions and habitat degradation for native biodiversity depend on how species cope with the individual and synergetic challenges these processes present. To assess the impact of anthropogenic land-use on the food web architecture of an invaded community, we examine the diets of nine native and two highly invasive mammal species at different trophic levels, inhabiting different land-uses across six biogeographic regions in Tasmania, Australia. We use two complementary methods, environmental DNA metabarcoding analysis (eDNA) of faeces and stable isotope analysis (SIA) of nitrogen (N) and carbon (C) in whole blood, to account for the high interindividual and temporal variability in the diets of multiple species simultaneously. eDNA showed regionalisation in the diet of smaller species, with land-use further defining dietary taxa within each region. SIA revealed that bioregion and land-use influence the δ13C values of all carnivore species and omnivores, whereas the δ15N values of these species are influenced only by land-use and not bioregion. Including multiple species showed that native rats are changing their diet in response to the presence of invasive rats, an impact that would have otherwise been attributed to land-use. Our findings demonstrate that human activities and invasive species are moulding the diets of invaded communities, raising questions about the potential impacts that dietary modifications will have on the life-history traits and the evolutionary consequences these modifications might have on the survival of native species. This highlights the urgency of including human activities in ecological studies and the importance of targeting multispecies assemblages to gain a better understanding of synergetic impacts on native biodiversity.

Evolution of dissolved organic nitrogen chemistry during transportation to the marginal sea: Insights from nitrogen isotope and molecular composition analyses

Estuaries are hotspots where terrestrially originated dissolved organic matter (DOM) is modified in molecular composition before entering marine environments. However, very few research has considered nitrogen (N) modifications of DOM molecules in estuaries, limiting our understanding of dissolved organic nitrogen (DON) cycling and the associated carbon cycling in estuaries. This study integrated optical, stable isotopes (δ15N and δ13C) and molecular composition (FT-ICR MS) to characterize the transformation of DOM in the Yangtze River Estuary. Both concentration of dissolved organic carbon (DOC) and DON decreased with increasing salinity, while their δ13C and δ15N increased with the increasing salinity. A significant positive correlation was found between δ15N and δ13C during the transportation of DOM to marginal seas, indicating that the behavior of both DOC and DON are primarily controlled by the mixing of freshwater and the seawater in the YRE. During the mixing process, the DON addition was observed using the conservative mixing curves. In the view of molecular composition, DOM molecules became more aromatic as the number of N atoms increased. Spearman correlations reveal that DOM molecules with fewer N atoms exhibited a higher enrichment in protein-like components, while those with more N atoms were more enriched in humic-like components. In addition, the δ15N and δ13C tended to increase as the N content of DOM decreased. Therefore, DON molecules with fewer N atoms were likely to be transformed into those with more N atoms based on the isotopic fractionation theory. This study establishes a linkage between the molecular composition and the δ15N of DOM, and discovers the N transformation pattern within DOM molecules during the transportation to marginal seas.

Biomagnification and health risks of perflfluoroalkyl acids (PFAAs) in seafood from the Yangtze river estuary of China

Bioaccumulation and human health risk assessment of Perfluoroalkyl acids (PFAAs) is important for pollutant hazard assessment. In this study, 26 aquatic organisms were collected from the Yangtze River estuary, the PFAAs concentrations in organisms were detected by liquid chromatography-mass spectrometry, and the trophic levels of organisms were constructed using nitrogen isotope analysis. The results showed that Perfluorobutane sulfonate (PFBS) was predominant in organisms with the mean concentration of 6.43 ± 8.21 ng/g ww. The biomagnification of organisms along the food chain was widespread, and the biomagnification factor (BMF) of perfluorooctane sulfonic (PFOS) was the most prominent. Trophic magnifcation factors (TMFs) of PFAAs were estimated in the marine food web, and TMFs >1 were observed in Perfluorodecanoic acid (PFDA), Perfluoroundecanoic acid (PFUnDA), Perfluorododecanoic acid (PFDoDA), and PFOS, indicating the biomagnifcation effects of these 4 individual PFAAs in organisms at Yangtze River estuary. The estimated daily intake (EDI) of PFBS was highest in adolescents aged 6–18 years, with EDIs of 18.9 ng/kg·bw/day for males and 14.0 ng/kg·bw/day for females. The hazard ratio (HR) of PFAAs reported in different age and gender groups were lower than 1.

Compound-specific stable isotopes of amino acids reveal influences of trophic level and primary production sources on mercury concentrations in fishes from the Aleutian Islands, Alaska

Total mercury concentrations ([THg]) exceed thresholds of concern in some Steller sea lion (Eumetopias jubatus) tissues from certain portions of the Aleutian Islands, Alaska. We applied compound-specific stable isotope analyses of both carbon and nitrogen in amino acids from fish muscle tissue to quantify the proportional contributions of primary production sources and trophic positions of eight prey species (n = 474 total) that are part of Steller sea lion diets. Previous THg analyses of fish muscle, coupled with monomethylmercury analyses of a subset of samples, substantiated previous findings that fishes from the west of Amchitka Pass, a discrete oceanographic boundary of the Aleutian Archipelago, have higher muscle Hg concentrations relative to fishes from the east. The δ13C values of essential amino acids (EAAs) in fish muscle demonstrated that although most fishes obtained their EAAs primarily from algae, some species varied in the extent to which they relied on this EAA source. The δ15N values of phenylalanine (0.9 to 7.8 ‰), an indicator of the isotopic baseline of a food web, varied widely within and among fish species. Trophic position estimates, accounting for this baseline variation, were higher from the west relative to the east of the pass for some fish species. Trophic magnification slopes using baseline-corrected trophic position estimates indicated similar rates of Hg biomagnification to the east and west of Amchitka Pass. Multiple linear regression models revealed that trophic position was the most important driver of fish muscle [THg] with less variation explained by other parameters. Thus, higher trophic positions but not the rate of Hg biomagnification to the west of Amchitka Pass may play a role in the regional differences in both fish and Steller sea lion [THg]. Although, differences in Hg contamination and uptake at the base of the east and west food webs could not be excluded.

Compound-specific stable nitrogen isotope analysis of amino acids shows that bulk methods provide higher estimates of mercury biomagnification in the Gulf of St. Lawrence

Compound-specific stable isotope analysis of amino acids (CSIA-AA) provides a method to estimate baseline δ15N values of food chains, allowing less biased estimates of trophic positions for organisms. Greater accuracy in trophic positions can improve estimates of contaminant biomagnification. We calculated trophic positions with various CSIA-AA equations for four species of fish and northern gannets (Morus bassanus) from the Gulf of St. Lawrence. We examined the effect of CSIA-AA-derived trophic positions on mercury biomagnification metrics (trophic magnification factors (TMF) and biomagnification factors) and compared these with trophic position estimates and metrics obtained from traditional bulk stable isotope analysis. The TMFs for the CSIA-AA equations ranged from 10 to 19, and bulk stable isotope analysis produced TMFs of 43, one of the highest TMFs recorded yet in the literature. Biomagnification factors between prey and northern gannets ranged from 20 to 42 using dietary observations and stable isotope mixing models. Our study demonstrates that discrepancies in biomagnification assessed using different approaches may go undetected when using a single approach.

Trophic Transfer of Halogenated Organic Pollutants in a Wetland Food Web: Insights from Compound-Specific Nitrogen Isotope of Amino Acids and Food Source Analysis.

A trophic position (TP) model (TPmix model) that simultaneously considered trophic discrimination factor and βGlu/Phe variations was developed in this study and was first applied to investigate the trophic transfer of halogenated organic pollutants (HOPs) in wetland food webs. The TPmix model characterized the structure of the wetland food web more accurately and significantly improved the reliability of TMF compared to the TPbulk, TPAAs, and TPsimmr models, which were calculated based on the methods of stable nitrogen isotope analysis of bulk, traditional AAs-N-CSIA, and weighted βGlu/Phe, respectively. Food source analysis revealed three interlocking food webs (kingfisher, crab, and frogs) in this wetland. The highest HOP biomagnification capacities (TMFmix) were found in the kingfisher food web (0.24-82.0), followed by the frog (0.08-34.0) and crab (0.56-11.7) food webs. The parabolic trends of TMFmix across combinations of log KOW in the frog food web were distinct from those of aquatic food webs (kingfisher and crab), which may be related to differences in food web composition and HOP bioaccumulation behaviors between aquatic and terrestrial organisms. This study provides a new tool to accurately study the trophic transfer of contaminants in wetlands and terrestrial food webs with diverse species and complex feeding relationships.

Tropical and temperate differences in the trophic structure and aquatic prey use of riparian predators.

The influence of aquatic resource-inputs on terrestrial communities is poorly understood, particularly in the tropics. We used stable isotope analysis of carbon and nitrogen to trace aquatic prey use and quantify the impact on trophic structure in 240 riparian arthropod communities in tropical and temperate forests. Riparian predators consumed more aquatic prey and were more trophically diverse in the tropics than temperate regions, indicating tropical riparian communities are both more reliant on and impacted by aquatic resources than temperate communities. This suggests they are more vulnerable to disruption of aquatic-terrestrial linkages. Although aquatic resource use declined strongly with distance from water, we observed no correlated change in trophic structure, suggesting trophic flexibility to changing resource availability within riparian predator communities in both tropical and temperate regions. Our findings highlight the importance of aquatic resources for riparian communities, especially in the tropics, but suggest distance from water is less important than resource diversity in maintaining terrestrial trophic structure.

Isotopic and proteomic evidence for communal stability at Pre-Pottery Neolithic Jericho in the Southern Levant

As one of the key, long-term occupied sites in the Southern Levant, Jericho was one of the most important early Neolithic centres to witness social and economic changes associated with the domestication of plants and animals. This study applies strontium (87Sr/86Sr), oxygen (δ18O) and carbon (δ13C) isotope analyses to the enamel of 52 human teeth from Pre-Pottery Neolithic (PPN) layers of Jericho to directly study human diet and mobility and investigate the degree of consolidation and the flexibility of social organization of Jericho society in the PPN period. The results indicate only two non-local individuals out of the 44 sampled inhabitants identified by strontium isotope analysis and are consistent with the presence of a largely sedentary community at PPN Jericho with no evidence for large-scale migration. We also construct strontium spatial baselines (87Sr/86Sr map) with local 87Sr/86Sr signatures for the sites across the Southern Levant based on systematic compilation and analysis of available data. In addition, we apply proteomic analysis of sex-specific amelogenin peptides in tooth enamel for sex estimation of the sampled individuals (n = 44), the results of which showed a sex-biased ratio (more male than female detected in this sample pool) in Jericho society during the PPN period, which may be due to the limited sample size or selective ritual practices like particular burial zones used for specific groups. We also pretreated a batch of human bone samples recovered from PPNB Jericho for stable carbon and nitrogen isotope analyses for dietary investigations. However, the extracted collagen showed poor preservation and no valid δ13C or δ15N data were obtained.

Farming and feasting during the Bronze Age–Iron Age transition in Britain (ca. 900–500 bce): multi-isotope evidence for societal change

The Late Bronze Age saw the rise of a new site type in southern Britain, commonly termed a midden. These vast monumental mounds, some comprising tens of thousands of artefacts/ecofacts dominated by animal bone and ceramics, signal a societal refocus towards agricultural intensification and communal feasting on a grand scale. These sites point to agricultural produce being the mainstay of the economy, with bronze having a reduced social and economic importance. This likely created new regimes of managing landscapes and livestock and new networks and agricultural catchments anchored on the sites. Major questions remain surrounding the strategies employed to enhance agricultural productivity, how landscapes and livestock were managed, and how different sites and regions met these challenges at a time of climatic deterioration. This article employs multi-isotope analyses on domestic and wild fauna to address these questions. It presents carbon, nitrogen, and sulfur (δ13C, δ15N, and δ34S, respectively) isotope analyses on 235 animals from 6 midden sites in 2 major regions of midden accumulation: the Vale of Pewsey in Wiltshire and the Thames Valley. The results demonstrate distinct differences in approaches to maximising agricultural productivity, with varied strategies apparent at a site, species, and regional level. Some sites, such as Potterne and Runnymede, clearly drew domestic fauna from a wide catchment where wide-ranging management and foddering regimes were employed. Other sites, such as East Chisenbury, had a more restricted catchment but a tightly controlled, intensive management regime. These data provide new insights into regional responses to the reorganisation of the economy, landscape use, and developing agricultural networks during the Bronze Age–Iron Age transition in Britain, revealing dynamic and evolving societal change.

Effects of lipid extraction on human bone collagen: Comparing stable carbon and nitrogen isotope values with and without lipid extraction

This study analyzes 24 human bone fragments to assess how lipid extraction affects stable carbon and nitrogen isotope ratios. Eighteen samples were archaeological bone and six were modern bone. Each bone was split into two pieces, producing 48 bone samples, and half underwent lipid extraction and half did not. This was done to assess whether the two techniques yielded statistically significant differences in δ13C and δ15N. The results are an important contribution for establishing standard stable isotope preparation protocols, which affect time and cost of laboratory procedures. Further, given that many dietary stable isotope studies compare original results to published material, this study addresses whether those comparisons are suitable if some samples undergo lipid extraction while others do not.We also examine 144 articles that report stable carbon and nitrogen isotope results to evaluate the proportion of studies that conduct lipid extraction versusthose that do not. We explore those outcomes by archaeological geographic region and by stable isotope laboratories. Findings show that 15% of the published studies do lipid extraction, and they tend to be grouped by researchers and laboratories who conduct research in specific global regions. The meta-analysis of the published articles and the findings that some laboratories conduct lipid extraction, while others do not, prompted the experimental portion of this study.Results of the experimental study show no significant differences in δ13C between samples prepared with and without lipid extraction and no significant differences in δ15N between samples prepared with and without lipid extraction. These results suggest that stable carbon and nitrogen isotope analysis of human bone collagen may not require lipid extraction for certain dietary reconstruction studies, specifically for non-coastal contexts (i.e., non-marine based diets). The isotope data was analyzed overall and among subgroups (e.g., archaeological bone, modern bone, mid-valley Peruvian ecological zones, and highland Peruvian ecological zones). In all cases, stable isotope ratios from samples prepared with and without lipid extraction were not significantly different.

Distribution of siphonophores in the Northwest Pacific Ocean and links to environmental conditions

Despite their abundance in marine ecosystems, studies on siphonophores are limited. In this study, 26 species of siphonophores in the Northwest Pacific Ocean were identified during multiple cruises of the R/V ISABU from 2018–2020, and various factors that may affect the occurrence of siphonophores, including water temperature, salinity, zooplankton biomass, and trophic niche were investigated. Statistical analysis revealed that the distribution of siphonophores and their biomass could be divided into two water mass groups, affected by the Kuroshio and Oyashio Currents. The species with high contributions to distinguishing the water mass groups (including Chelophyes contorta, Dimophyes arctica, Bassia bassensis, and Eudoxoides spiralis—mainly belonging to the Diphyidae) showed species-specific correlations with water temperature and salinity. This suggests that diphyids can be used as indicator species for currents and hydrological factors that influence water mass. The biomass of siphonophores exhibited a trend opposite to that of non-gelatinous zooplankton and showed no association with other gelatinous zooplankton. These results can be interpreted from an ecological niche perspective. Through nitrogen and carbon stable isotope analyses, the dietary sources of siphonophores could potentially overlap with those of chaetognaths or non-gelatinous zooplankton. Because the trophic position of siphonophores (2.4–3.2) also falls in the range of those of chaetognaths (2.8–3.4) and non-gelatinous zooplankton including copepods, euphausiids, and amphipods (2.4–3.5), diet competition with carnivorous mesozooplankton could be predicted. Considering that the diversity and biomass of most siphonophores are strongly positively correlated with water temperature and salinity, expansion of the Kuroshio Current is expected to lead to an increase in siphonophores in the Northwest Pacific in the future. The findings of this study are anticipated to provide novel insights into climate change prediction and response and enhance our understanding of siphonophore communities.

Neolithic herding practices in the Southern Caucasus: Animal management in the early 6th millennium BCE at Masis Blur in Armenia’s Ararat Valley

The Southern Caucasus is traditionally an important area of livestock production and a key region in the spread of herding from southwestern Asia to Eurasia’s western steppe. Neolithic settlements appeared across the Southern Caucasus in the early 6th millennium BCE. How these communities used the landscape and managed livestock in the context of mixed farming is not well understood. Stable carbon (δ13C) and nitrogen (δ15N) isotope analysis of bone collagen and stable carbon and oxygen (δ18O) isotope analysis of tooth enamel from faunal remains from Masis Blur, one of the earliest Neolithic sites in the Southern Caucasus, clarify how communities maintained herds in this topographically varied region. δ13C and δ15N values indicate hunting wild fauna in diverse environments. δ13C and δ18O values from sequentially sampled livestock teeth demonstrate markedly different strategies for managing cattle and caprines. Negative covariation in δ18O and δ13C values in most caprines suggest seasonal mobility to C3-dominated high elevation pastures in the summers, and/or winter foddering with water-stressed C3 or wild C4 plants. These seasonal strategies were in place in the earliest known phase of occupation at Masis Blur, indicating that Southern Caucasus Neolithic communities likely exploited a range of environments linking the lowland plains and mountainous zones.

Unprecedented shift in Canadian High Arctic polar bear food web unsettles four millennia of stability

Stable carbon (δ13C) and nitrogen (δ15N) isotope analysis was conducted on modern and archaeological polar bear bone collagen from the Canadian Arctic Archipelago to investigate potential changes in polar bear foraging ecology over four-millennia. Polar bear δ13C values showed a significant decline in the modern samples relative to all archaeological time-bins, indicating a disruption in the sources of production that support the food web, occurring after the Industrial Revolution. The trophic structure, indicated through δ15N, remained unaltered throughout all time periods. The lower δ13C observed in the modern samples indicates a change in the relative importance of pelagic (supported by open-water phytoplankton) over sympagic (supported by sea ice-associated algae) primary production. The consistency in polar bear δ13C through the late Holocene includes climatic shifts such as the Medieval Warm Period (MWP, A.D. 950-1250) and the early stages of the Little Ice Age (LIA, A.D. 1300-1850). These findings suggest that polar bears inhabit a food web that is more pelagic and less sympagic today than it was through the Late Holocene. We suggest that modern, anthropogenic warming has already affected food web structure in the Canadian Arctic Archipelago when modern data are contextualized with a deep time perspective.

Seasonal variation in trophic structure and community niche dynamics of an Arctic coastal community of marine vertebrates

Temporal variation in food web structure is widespread among highly seasonal environments, such as the Arctic, and is driven by changes in resource availability. Variation in resource availability can lead to species differences in diet composition, isotopic niche width, and trophic position (TP) across seasons. Here, we used tissue samples that represent two distinct turnover rates for diet (liver = shorter term, muscle = longer term) from 18 fish and three marine mammal species to investigate seasonal (i) variation in TPs within the Southampton Island marine ecosystem of Hudson Bay, (ii) variation in consumer isotopic niche width within this part of the food web, and (iii) variation in community niche dynamics among a fish and marine mammal community using stable carbon (δ13C) and nitrogen (δ15N) isotope analyses. Many zooplanktivores and piscivores increased in TP in summer (i.e., shorter-term turnover period), whereas benthic feeders dependent on ice-obligate prey decreased in TP. Most isotopic niche widths and community metrics (δ15N range, total ellipse area, mean centroid distance) were higher in liver than muscle. Our findings demonstrate seasonal changes in TPs, which suggests that Arctic communities may be differentially affected by longer ice-free periods and earlier onset of primary production due to accelerated climate change.

Análisis isotópico seriado de una muestra de cabello del individuo inhumado en el volcán Quehuar (Salta, Argentina)

In 1999 the frozen remains of a young female individual of Inca affiliation were found on the summit of volcano Quehuar (Salta, Argentina) at an elevation of 6130 masl. The individual, part of a capacocha ritual, was partially destroyed due to a dynamite explosion executed by looters. In this work, the stable isotope analysis of carbon and nitrogen on a serial sample of hair is presented and discussed to investigate the eating patterns of the last year and a half of the individual's life. The results indicate a diet that included resources under the C4 photosynthetic pathway and slight dietary variation during the period considered. These results constitute a contribution to the reconstruction of the life histories of the individuals associated with high-altitude Inca burials, accounting for the diversity of experiences they went through in the months before their death.

Exploitation shifted trophic ecology and habitat preferences of Mediterranean and Black Sea bluefin tuna over centuries

AbstractDuring recent decades, the health of ocean ecosystems and fish populations has been threatened by overexploitation, pollution and anthropogenic‐driven climate change. Due to a lack of long‐term ecological data, we have a poor grasp of the true impact on the diet and habitat use of fishes. This information is vital if we are to recover depleted fish populations and predict their future dynamics. Here, we trace the long‐term diet and habitat use of Atlantic bluefin tuna (BFT), Thunnus thynnus, a species that has had one of the longest and most intense exploitation histories, owing to its tremendous cultural and economic importance. Using carbon, nitrogen and sulphur stable isotope analyses of modern and ancient BFT including 98 archaeological and archival bones from 11 Mediterranean locations ca. 1st century to 1941 CE, we infer a shift to increased pelagic foraging around the 16th century in Mediterranean BFT. This likely reflects the early anthropogenic exploitation of inshore coastal ecosystems, as attested by historical literature sources. Further, we reveal that BFT which migrated to the Black Sea–and that disappeared during a period of intense exploitation and ecosystem changes in the 1980s–represented a unique component, isotopically distinct from BFT of NE Atlantic and Mediterranean locations. These data suggest that anthropogenic activities had the ability to alter the diet and habitat use of fishes in conditions prior to those of recent decades. Consequently, long‐term data provide novel perspectives on when marine ecosystem modification began and the responses of marine populations, with which to guide conservation policy.

Energy channelling, food chain length and body condition in a northern lake predator

Abstract Identifying potential links between food web structure and animal body condition is fundamental for predicting the long‐term persistence of populations under rapidly changing environments. Northern lakes, and the cold‐water adapted species that inhabit them, are particularly vulnerable to a warming climate. We explored relationships among body condition and energy channel use in lake trout (Salvelinus namaycush) and seasonal variation in food‐chain length in the Lake of Two Rivers, Ontario, Canada. Bayesian mixing models using carbon isotope values (δ13C) of essential amino acids showed that individuals utilising multiple energy channels had higher gonad mass, suggesting that dietary diversity has reproductive fitness benefits. Amino acid nitrogen isotope (δ15N) analysis revealed seasonal variation in food chain length, with extension of the food web during winter and spring and truncation of the food web during summer. Although these findings illustrate increased omnivory of lake trout during summer, there was no clear evidence that seasonal changes in food chain length had an impact on trout condition. These findings provide new insight into potential relationships between food web structure, energy flow and consumer condition in freshwater ecosystems, thereby revealing potential responses of species to shifting ecosystem states under intensifying climate change at high latitudes.

Reproductive roles as likely drivers of sexual dimorphism in New Zealand’s endangered mountain parrot, the Kea

Abstract New Zealand’s endangered mountain parrot, the Kea (Nestor notabilis), exhibits moderate male-biased sexual size dimorphism in linear body measurements (~5%) and a pronounced dimorphism in bill size (12–14%). Using stable isotope analyses of carbon and nitrogen in Kea feathers and blood sampled from a significant portion (~10%) of the extant population, we determined that Kea bill dimorphism may be an ecologically selected trait that enhances male Kea’s ability to forage at a higher trophic level in order to provision females and offspring during nesting. Sexual dimorphism can arise through sexual selection, ecological drivers, or a combination of both. Ecological selection is associated with foraging niche divergence between the sexes to reduce inter-sexual competition or due to differing dietary needs associated with reproductive role. Despite the widespread occurrence of sexual dimorphism throughout the animal kingdom, empirical evidence for ecological causation is rare. We conducted the first molecular confirmation of sexual size dimorphism in Kea. We then employed Bayesian mixing models to explore potential correlations between diet and bill size to determine whether the dimorphism is linked to diet partitioning throughout all age classes (fledgling, juvenile, subadult, and adult). Female Kea foraged at a consistent, relatively low, trophic level throughout their lifetime, whereas male trophic level increased with age to a maximum at subadult stage, prior to breeding for the first time—a time in which males may have been actively learning extractive foraging techniques associated with a high protein diet. Adult males foraged at a high trophic level relative to all groups except subadult males. As males provision females on the nest, which in turn provision young, these results highlight that the evolution of morphology and reproductive output may be linked in circuitous ways.

Application of a porous graphitic carbon column to carbon and nitrogen isotope analysis of underivatized individual amino acids using high-performance liquid chromatography coupled with elemental analyzer/isotope ratio mass spectrometry.

Isolation of underivatized amino acids (AAs) using high-performance liquid chromatography (HPLC) is becoming a popular method for carbon (δ13 C) and nitrogen isotope (δ15 N) analyses of AAs because of the high analytical precision and for performing dual-isotope analysis. However, some AAs in natural samples, especially small, hydrophilic AAs, are not suitably separated using reversed-phase columns (e.g., C18) and ion-exchange columns (e.g., Primesep A). We developed a new method for HPLC using a porous graphitic carbon column for the separation of nine hydrophilic AAs. After purification, δ13 C and δ15 N values of AAs were determined using elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). We demonstrated the application of this method by determining δ13 C and δ15 N values of individual hydrophilic AAs in a biological sample, the muscle of blue mackerel (Scomber australasicus). Chromatographically, the baseline separation of hydrophilic AAs was achieved in both the standard mixture and the biological sample. We confirmed that δ13 C and δ15 N values of AA standards remained unchanged during the whole experimental procedure. The δ13 C values of AAs in mackerel muscle are also in good agreement with the values obtained using another verified method for δ13 C analysis. The good separation performance of hydrophilic AAs and the reliability of δ13 C and δ15 N analyses of individual AAs using the porous graphite column offer a significant advantage over conventional settings. We suggest that, in the future, the HPLC × EA/IRMS method can be used for reliable δ13 C and δ15 N analyses of AAs in natural samples.

Killer whales at northern Patagonia, Argentina: Evidence of different foraging groups from stable isotopes

AbstractKiller whales (Orcinus orca) are highly mobile, large marine apex predators that inhabit all oceans. Despite being the most conspicuous top predator, little is known about their ecology along Patagonia, Argentina. Here, we used carbon (δ13C) and nitrogen (δ15N) isotope analysis of bone and dentine collagen from killer whales (n = 13) stranded in northern Patagonia during the period 1970–2014 to assess their trophic ecology. A model‐based clustering analysis identified three distinct groups related with three large marine ecosystems: the Patagonia (P), Subtropical (ST), and Antarctica (A) groups. Group P (n = 8) is characterized by individuals with high δ13C and δ15N values fitting within the isotopic ratios observed over the Patagonian shelf. Group ST (n = 3) is composed of individuals with high δ13C and low δ15N values, similar to those reported for conspecifics in southern Brazil. Group A (n = 2) is composed of individuals with low δ13C and δ15N values, typical from high latitude areas like sub‐Antarctic/Antarctic waters. The finding of different killer whales' isotopic groups over the Patagonian shelf suggests the existence of individuals or groups exploiting different habitats. Our results expand the limited ecological knowledge for the species while presenting the basis to infer more complex ecological hypotheses.