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Stable isotope analyses of archival specimens have revealed trophic declines over the past 150 yr in a growing number of predatory pelagic seabirds that breed in the Hawaiian Islands. However, they have not examined whether isotopic shifts occurred primarily during a specific phase of the annual cycle, which could allow us to better identify the causes of trophic declines and potential consequences for affected populations. We evaluated seasonal (breeding versus nonbreeding season) foraging habits of 2 ecologically distinct species, Newell’s shearwater Puffinus newelli and Laysan albatross Phoebastria immutabilis, and extended this analysis back 50 and 100 yr, respectively. Our assessment relied on amino acid δ15N proxies for nutrient regime use (δ15NPhe) and trophic position (Δδ15NGlu-Phe) from 2 tissues (feather and bone collagen) reflecting different time scales. Both study species exhibited season-specific isotopic shifts resulting in more pronounced seasonality in modern populations. We also identified inter-species differences in nutrient regime use, regardless of season. Laysan albatross experienced a trophic decline exclusive to the breeding season, while their nonbreeding season foraging ecology has remained constant over the past century. In contrast, the nutrient regime at the base of the food chain for Newell’s shearwaters during the nonbreeding season underwent a shift within the last 50 yr, and the trophic decline they experienced was heavily weighted toward the nonbreeding season. Efforts to mitigate potential fitness consequences of future trophic declines might benefit from focusing on fisheries management near the Hawaiian Islands, where susceptible seabirds forage during winter/spring, rather than the entire North Pacific.

To study the influence of different ocean currents on the trophic spectra found in a sympatric fish community, we analyzed the radiocarbon contents (Δ14C) and stable carbon (δ13C) and nitrogen (δ15N) isotopic compositions in their bulk muscle tissues and the δ15N of individual amino acids in 26 species of demersal fish collected from off Tohoku (Pacific coast), northeastern Japan. The Δ14C values of the fish varied from –42 to +41‰, consistent with the Δ14C of the dissolved inorganic carbon in the cold Oyashio Current (typically about –50‰) and the warm Kuroshio Current (typically about +50‰). The trophic positions (TPs) of the fish estimated from the δ15N values of amino acids increased from 3.1 to 4.5 with increasing snout length. A negative correlation was observed between Δ14C and TP in gadiform fish and flatfish, suggesting that the Oyashio Current delivers a high TP diet to these fish groups. These results suggest that the trophic ecology of marine fish in the coastal western North Pacific is primarily controlled by the two major surface water currents, but is also significantly influenced by a combination of species-specific feeding and migration strategies.

We investigated how foraging habits vary among three ecologically distinct wide-ranging seabirds. Using amino acid δ15N proxies for nutrient regime (δ15NPhe) and trophic position (Δδ15NGlu-Phe), we compared Newell’s shearwater (Puffinus newelli) and Laysan albatross (Phoebastria immutabilis) foraging habits over the past 50–100 years, respectively, to published records for the Hawaiian petrel (Pterodroma sandwichensis). Standard ellipses constructed from the isotope proxies show that inter-population and interspecific foraging segregation have persisted for several decades. We found no evidence of a shift in nutrient regime at the base of the food web for the three species. However, our data identify a trophic decline during the past century for Newell’s shearwater and Laysan albatross (probability ≥ 0.97), echoing a similar decline observed in the Hawaiian petrel. During this time, Newell’s shearwaters and Hawaiian petrels have experienced population declines and Laysan albatross has experienced range extension and apparent population stability. Counting other recent studies, a pattern of trophic decline over the past century has now been identified in eight species of pelagic seabirds that breed in the Hawaiian Islands. Because our study species forage broadly across the North Pacific Ocean and differ in morphological and behavioral traits and feeding methods, the identified trophic declines suggest a pervasive shift in food web architecture within the past century.

A novel species of the family Alepocephalidae (slickheads), Narcetes shonanmaruae, is described based on four specimens collected at depths greater than 2171 m in Suruga Bay, Japan. Compared to other alepocephalids, this species is colossal (reaching ca. 140 cm in total length and 25 kg in body weight) and possesses a unique combination of morphological characters comprising anal fin entirely behind the dorsal fin, multiserial teeth on jaws, more scale rows than congeners, precaudal vertebrae less than 30, seven branchiostegal rays, two epurals, and head smaller than those of relatives. Mitogenomic analyses also support the novelty of this large deep-sea slickhead. Although most slickheads are benthopelagic or mesopelagic feeders of gelatinous zooplankton, behavioural observations and dietary analyses indicate that the new species is piscivorous. In addition, a stable nitrogen isotope analysis of specific amino acids showed that N. shonanmaruae occupies one of the highest trophic positions reported from marine environments to date. Video footage recorded using a baited camera deployed at a depth of 2572 m in Suruga Bay revealed the active swimming behaviour of this slickhead. The scavenging ability and broad gape of N. shonanmaruae might be correlated with its colossal body size and relatively high trophic position.

Oceanic methane from global deep-sea sediment is largely consumed through microbially mediated sulfate-coupled oxidation, resulting in 13C-depleted cell biomass of anaerobic methanotrophic archaea (ANME). The general ecological importance of subseafloor ANME has been well recognized in the last two decades. However, the crucial biochemical pathways for the overall anaerobic oxidation of methane (AOM) still remain enigmatic. Here, methanotrophic pathways were analyzed to trace 13C-depleted amino acid biosynthesis in two clades of ANME (ANME-1 and ANME-2) from the Black Sea. Compound-specific analysis of ANME-dominated microbial mats showed a significant 13C-depletion trend in association with increasing carbon numbers in protein-derived amino acid families (e.g., the pyruvate family in the order of alanine, valine, isoleucine and leucine was down to −114‰). This result indicates a stepwise elongation of 13C-depleted carbon during amino acid biosynthesis. The overall results suggest that intracellular protein amino acids and the most 13C-depleted signature of leucine, which has a specific branched-chain structure, are potentially propagated as isoprenoid precursor molecules into archaeal biosynthesis, resulting in the extremely 13C- and 14C-depleted nature of ANME cells in the deep microbial oasis.

To adapt to ecological and environmental conditions, species can change their ecological niche (e.g., interactions among species) and function (e.g., prey‐predation, diet competition, and habitat segregation) at the species and guild levels. Stable isotope analysis of bulk carbon and nitrogen of organisms has conventionally been used to evaluate such adaptabilities in the scenopoetic and bionomic views as the isotopic niche width.Compound‐specific stable isotope analysis (CSIA) of nitrogen within amino acids provides trophic information without any disruption of scenopoetic views in the isotope ratios, unlike conventional bulk isotope analysis provides both information and therefore frequently hinders its usefulness for trophic information.We performed CSIA of amino acids to understand the trophic variability of the pike gudgeon Pseudogobio esocinus and largemouth bass Micropterus salmoides as representative specialist and generalist fish species, respectively, from 16 ecologically variable habitats in the four major rivers of Korea.There was little variation (1σ) in the trophic position (TP) among habitats for P. esocinus (± 0.2); however, there was considerably large variation for M. salmoides (± 0.6). The TP of M. salmoides was negatively correlated with the benthic invertebrate indices of the habitats, whereas the TP of P. esocinus showed no significant correlation with any indices. Thus, these two representative fish species have different trophic responses to ecological conditions, which is related to known differences in the trophic niche between specialists (i.e., small niche width) and generalists (i.e., large niche width).Over the past four decades, the conventional bulk isotope analysis has not been capable of deconvoluting “scenopoetic” and “bionomic” information. However, in the present study, we demonstrated that the CSIA of amino acids could isolate trophic niches from the traditional ecological niche composed of trophic and habitat information and evaluated how biological and ecological indices influence the trophic response of specialists and generalists., We first applied compound‐specific stable isotope analysis of amino acids to the ecological niche studies, in order to isolate trophic niche that is accompanied with habitat niche in the conventional bulk isotope method.

Quantifying resource partitioning is central to community and food web ecology and of increasing interest in an era of rapid global change disrupting biotic interactions. Multichannel feeding – consuming resources from both green and brown food webs – can be a stabilizing force in communities. While multichannel feeding has been well-documented in invertebrate and aquatic systems, it has been relatively under-studied in terrestrial vertebrate populations. Applied ecologists are seeking approaches to assess niche partitioning and cryptic trophic pathways, like multichannel feeding, which have been difficult to quantify, especially among vertebrates. Using both bulk (δ13C and δ15N) and compound specific stable isotope ratios (δ15N glutamic acid and phenylalanine), we tested how three common and competing tree squirrel partition resources. Our complementary analyses revealed that squirrels partitioned niche space and, because of differences in multichannel foraging, possessed different trophic identities. While all squirrels consumed food items from green and brown food webs, their dependence on each differed, revealing an important, yet cryptic, mechanism behind apparent stable co-occurrence of these competitors. Our work supports multichannel feeding as a potential mechanism promoting coexistence in this guild of terrestrial vertebrates, and provides a framework to quantify resource partitioning in other ecological communities.

The abundance and biomass of benthic foraminifera are high in intertidal rocky‐shore habitats. However, the availability of food to support their high biomass has been poorly studied in these habitats compared to those at seafloor covered by sediments. Previous field and laboratory observations have suggested that there is diversity in the food preferences and modes of life among rocky‐shore benthic foraminifera. In this study, we used the stable nitrogen isotopic composition of amino acids to estimate the trophic position, trophic niche, and feeding strategy of individual foraminifera species. We also characterized the configuration and structure of the endobiotic microalgae in foraminifera using transmission electron microscopy, and we identified the origin of endobionts based on nucleotide sequences. Our results demonstrated a large variation in the trophic positions of different foraminifera from the same habitat, a reflection of endobiotic features and the different modes of life and food preferences of the foraminifera. Foraminifera did not rely solely on exogenous food sources. Some species effectively used organic matter derived from endobionts in the cell cytoplasm. The high biomass and species density of benthic foraminifera found in intertidal rocky‐shore habitats are thus probably maintained by the use of multiple nitrogen resources and by microhabitat segregation among species as a consequence.

The nitrogen isotopic composition of amino acids (δ15NAA) is a promising tool for estimating the trophic position and nitrogen sources of organisms in grazing food webs. The utility of δ15NAA analysis of microbial processes in the biogeochemistry remains uncertain, because information about the δ15NAA values of chemotrophic microbes are still limited. In this study, we report the δ15NAA values in five cultured heterotrophic or chemolithoautotrophic microbes (one fungus, one bacterium, and three archaea) with controlled nitrogen sources in synthetic media. When the microbes were grown on ammonium, the apparent nitrogen isotopic fractionation of amino acids relative to glutamic acid in the microbes (e.g., −3.0 ± 1.0‰ for phenylalanine) was similar to those in algae. When the microbes were grown on a free amino acid mixture, the δ15N offsets of amino acids in the microbial cells relative to the amino acids in the culture media (e.g., +8.2 ± 0.8‰ for glutamic acid and +0.1 ± 0.2‰ for phenylalanine) were close to the standard isotopic fractionation between animals and their diets. These results suggest that the δ15NAA pattern can be used to distinguish two end-member metabolic modes in microbes (i.e., de novo synthesis of amino acids from inorganic nitrogen vs. assimilation of amino acids from environments). We also discussed the effects of metabolic pathways on the δ15NAA values and their fractionation with respect to the diversity of amino acid metabolism in microbes. These findings will form an important baseline for interpreting the δ15NAA values of organisms and detritus in natural environments.

Compound-specific nitrogen stable isotope analysis provides an approach for estimating animal trophic position that may overcome key issues associated with stable isotope analysis of bulk tissue. Yet compound-specific trophic discrimination factors have not been estimated for a broad range of habitats, taxa, and diets. We conducted a controlled-feeding experiment to characterize the variation in compound-specific (TDFAA) and bulk (TDFBulk) trophic discrimination factors of four freshwater fish species fed on three distinct diets. We also compared TDFAA of fish muscle and scale to evaluate the viability of scales for making food web inferences. Mean ± 1 SD TDFBulk was 2.2‰ ± 0.9‰, and there were significant effects of species and diet trophic position on TDFBulk. Mean ± 1 SD TDFAA was 6.9‰ ± 0.8‰. Although there was no effect of species on TDFAA, there were significant differences in TDFAA across the three diets. TDFAA from fish scales were not significantly different from those of muscle. Our study illustrates the advantages of estimating trophic position using compound-specific stable isotopes and the need for continued investigation of factors resulting in variation in TDF values.

It is widely held that sterols are key cyclic triterpenoid lipids in eukaryotic cell membranes and are synthesized through oxygen-dependent multi-enzyme pathways. However, there are known exceptions―ciliated protozoans, such as Tetrahymena, along with diverse low-oxygen adapted eukaryotes produce, instead of sterols, the cyclic triterpenoid lipid tetrahymanol that does not require molecular oxygen for its biosynthesis. Here, we report that a number of anaerobic microbial eukaryotes (protists) utilize neither sterols nor tetrahymanol in their membranes. The lack of detectable sterol-like compounds in their membranes may provide an opportunity to reconsider the physiological function of sterols and sterol-like lipids in eukaryotes. This article is protected by copyright. All rights reserved.

Sterols are key cyclic triterpenoid lipid components of eukaryotic cellular membranes, which are synthesized through complex multi-enzyme pathways. Similar to most animals, Bathymodiolus mussels, which inhabit deep-sea chemosynthetic ecosystems and harbor methanotrophic and/or thiotrophic bacterial endosymbionts, possess cholesterol as their main sterol. Based on the stable carbon isotope analyses, it has been suggested that host Bathymodiolus mussels synthesize cholesterol using a sterol intermediate derived from the methanotrophic endosymbionts. To test this hypothesis, we sequenced the genome of the methanotrophic endosymbiont in Bathymodiolus platifrons. The genome sequence data demonstrated that the endosymbiont potentially generates up to 4,4-dimethyl-cholesta-8,14,24-trienol, a sterol intermediate in cholesterol biosynthesis, from methane. In addition, transcripts for a subset of the enzymes of the biosynthetic pathway to cholesterol downstream from a sterol intermediate derived from methanotroph endosymbionts were detected in our transcriptome data for B. platifrons. These findings suggest that this mussel can de novo synthesize cholesterol from methane in cooperation with the symbionts. By in situ hybridization analyses, we showed that genes associated with cholesterol biosynthesis from both host and endosymbionts were expressed exclusively in the gill epithelial bacteriocytes containing endosymbionts. Thus, cholesterol production is probably localized within these specialized cells of the gill. Considering that the host mussel cannot de novo synthesize cholesterol and depends largely on endosymbionts for nutrition, the capacity of endosymbionts to synthesize sterols may be important in establishing symbiont-host relationships in these chemosynthetic mussels.

Numerous studies have used stable isotope analysis (SIA) of carbon and nitrogen within the bulk tissues of organisms to determine the trophic structure among organisms in a food web. Recently, SIA has evolved to compound-specific stable isotope analysis (CSIA) of nitrogen within amino acids to significantly reduce the uncertainty in the estimated trophic position (TP) of organisms based on the isotopic difference between glutamic acid and phenylalanine within a single organism. However, because the initial offset (β) between glutamic acid and phenylalanine differs between aquatic algae (ca. +3.4‰) and vascular plants (ca. −8.4‰) in food webs that rely on both resources, β should be replaced by a value adapted to the admixture of primary producers for each specimen. In this study, we established a new method involving the β value (βmix) of each consumer specimen determined based on its bulk tissue δ13C value and successfully obtained realistic TPs (TPmix) for organisms in a complex seagrass meadow food web. Remarkable differences between the TPmix and traditional TPalgal values were found in deposit feeders due to the large contribution of seagrass to their basal resources. The estimated TPs of organisms increased by up to 1.5 units (from TPalgal to TPmix), in terms of trophic transfer, when their diets included substantial seagrass-derived contributions. Thus, combinatorial analysis of the amino acid δ15N and specimen-specific βmix values provides better understanding of the trophic interactions in food webs, even in complex seagrass meadow ecosystems.

The trophic discrimination factor (TDF) of nitrogen isotopes (15N/14N) within amino acids, between a stream-dwelling dobsonfly larva (Protohermes grandis: Megaloptera; Corydalidae) and its diet (chironomid larvae), was determined in controlled feeding experiments. Last-instar larvae of P. grandis were collected from the Yozawa-gawa River, central Japan, and reared in the laboratory. After fed to satiation for 1 month, one group of larvae was each fed one living chironomid larva per day for 4 weeks, while a second group was starved for 8 weeks. The larvae were harvested at intervals and the nitrogen isotopic composition of glutamic acid (δ15NGlu) and phenylalanine (δ15NPhe) were determined to calculate TDF. The mean TDF of satiated and starved larvae were 7.1‰ ± 0.5‰ (n = 3) and 7.3‰ ± 0.5‰ (n = 5), respectively. Thus, the TDF for P. grandis larvae in this study was similar to that reported for other arthropods (approximately 7‰) and was independent of satiation or starvation. A previous study of wild P. grandis larvae, based on the δ15NGlu and δ15NPhe values, estimated its trophic position (TP) as approximately 2.0 ± 0.1 (n = 5), a low value close to that of algivores, although they are generally characterized as carnivores (usually accepted as TP ≥ 3). The TDF for P. grandis larvae suggests that their low TPs in nature were caused by incorporation of vascular plant-derived amino acids (with a different δ15N profile from that of algae) and not by an unusually low TDF or by the effects of the satiation/starvation on amino acid metabolism.

In most ecosystems, microbes are the dominant consumers, commandeering much of the heterotrophic biomass circulating through food webs. Characterizing functional diversity within the microbiome, therefore, is critical to understanding ecosystem functioning, particularly in an era of global biodiversity loss. Using isotopic fingerprinting, we investigated the trophic positions of a broad diversity of heterotrophic organisms. Specifically, we examined the naturally occurring stable isotopes of nitrogen ((15)N:(14)N) within amino acids extracted from proteobacteria, actinomycetes, ascomycetes, and basidiomycetes, as well as from vertebrate and invertebrate macrofauna (crustaceans, fish, insects, and mammals). Here, we report that patterns of intertrophic (15)N-discrimination were remarkably similar among bacteria, fungi, and animals, which permitted unambiguous measurement of consumer trophic position, independent of phylogeny or ecosystem type. The observed similarities among bacterial, fungal, and animal consumers suggest that within a trophic hierarchy, microbiota are equivalent to, and can be interdigitated with, macrobiota. To further test the universality of this finding, we examined Neotropical fungus gardens, communities in which bacteria, fungi, and animals are entwined in an ancient, quadripartite symbiosis. We reveal that this symbiosis is a discrete four-level food chain, wherein bacteria function as the apex carnivores, animals and fungi are meso-consumers, and the sole herbivores are fungi. Together, our findings demonstrate that bacteria, fungi, and animals can be integrated within a food chain, effectively uniting the macro- and microbiome in food web ecology and facilitating greater inclusion of the microbiome in studies of functional diversity.

Bats perform important ecosystem services, but it remains difficult to quantify their dietary strategies and trophic position (TP) in situ. We conducted measurements of nitrogen isotopes of individual amino acids (δ15NAA) and bulk-tissue carbon (δ13Cbulk) and nitrogen (δ15Nbulk) isotopes for nine bat species from different feeding guilds (nectarivory, frugivory, sanguivory, piscivory, carnivory, and insectivory). Our objective was to assess the precision of δ15NAA-based estimates of TP relative to other approaches. TPs calculated from δ15N values of glutamic acid and phenylalanine, which range from 8.3–33.1‰ and 0.7–15.4‰ respectively, varied between 1.8 and 3.8 for individuals of each species and were generally within the ranges of those anticipated based on qualitative dietary information. The δ15NAA approach reveals variation in TP within and among species that is not apparent from δ15Nbulk data, and δ15NAA data suggest that two insectivorous species (Lasiurus noctivagans and Lasiurus cinereus) are more omnivorous than previously thought. These results indicate that bats exhibit a trophic discrimination factor (TDF) similar to other terrestrial organisms and that δ15NAA provides a reliable approach for addressing questions about variation in the TP of bats that have heretofore proven elusive.

Compound-specific stable isotope analysis of amino acids (AA method) has recently been used to estimate the trophic level (TL) of animals. The individual TL (TLGlu/Phe) of Japanese anchovy Engraulis japonicus was precisely determined using the AA method without sampling or analysis of primary producers. Previously, TLs of organisms were determined using bulk nitrogen stable isotopic composition (δ15Nbulk) of tissues (bulk method). The δ15Nbulk for Japanese anchovies varied widely because of their migratory behavior, and the TL of anchovies in Sagami Bay remained ambiguous. We showed that the TLGlu/Phe of anchovy was almost 3 and that food-web structures differed between inshore and offshore waters. Furthermore, there was little difference in TLGlu/Phe for representative Japanese anchovies in Sagami Bay, despite the large intra-sample variation in δ15Nbulk for anchovies ranging over a trophic shift of 3–4 ‰ in Sagami Bay. We speculated that this variation in δ15Nbulk resulted from differences in food sources among the different food webs. Estimation of individual TLs in some simple food chains using the AA method may enable the determination of the food-web structure of migratory fishes, which is difficult to determine using the traditional bulk method.

We evaluated nitrate utilization by benthic foraminifera at the redox boundary around the sediment-water interface by examining the stable nitrogen isotopic composition (δ15N, ‰ vs. AIR) of amino acids. Five foraminiferal species collected from bathyal Sagami Bay (water depth, 1430 m) in three different sampling seasons were analyzed by determining the δ15N values of amino acids from whole cells and those in test (shell) proteins remaining after H2O2 treatment. The δ15N values of phenylalanine fell within a narrow range (typically 1–3‰) in the test proteins of all five species, but the whole-cell δ15N values of phenylalanine were substantially different from those of the test proteins in two species, Globobulimina affinis (by 6.3‰) and Uvigerina akitaensis (by 4.5‰). These differences in the δ15N values of amino acids suggest that these species utilize nitrate in their cells probably for nitrate respiration (i.e., denitrification) to adapt to oxygen-depleted environments, as was previously observed in the shallow water benthic foraminifera Ammonia sp. Apparent trophic positions determined using the δ15N values of amino acids also differed between whole cells and tests in G. affinis, U. akitaensis, and Ammonia sp., perhaps because they have different microbial associations, as observed by cellular ultrastructural analysis. These differences in nitrate utilization and microbial associations among benthic foraminifera suggest that foraminifera adapt in diverse ways to conditions in dysoxic to anoxic sediments.

Among eukaryotes, some benthic foraminiferal species have been reported to be capable of nitrate respiration, although little is known about their denitrification processes. In this study, we incubated the shallow-water benthic foraminifer Ammonia beccarii with isotopically labeled sodium nitrate (273% ,0 % ,o r+73%) under oxic or anoxic conditions to investigate how nitrate is used in foraminiferal cells and whether those signatures remain in amino acids. The d15N values of amino acids from bulk cells incubated under anoxic conditions were correlated with those of nitrate in seawater and were enriched in 15 Nb y up to, 50% compared to the isotopic compositions in seawater. There was no such relationship or enrichment in the cases of organic matter in the calcite tests under oxic or anoxic conditions or bulk cells under oxic conditions, suggesting that benthic foraminifera take up ambient nitrate under anoxic conditions and use it for denitrification; the remaining 15N-enriched intracellular nitrate pool is used for amino acid synthesis, probably by endosymbiotic microbes, as suggested by observation of the cellular ultrastructure. The degree of 15N enrichment may depend on denitrification rates of the intracellular nitrate pool. Because the amino acids in the calcite test are synthesized by foraminifera, they were not enriched in 15N, even under anoxic conditions. Thus, differences between amino acid d15N of bulk foraminiferal cells and

Intra‐species variability in foraging strategies may be common, which has significant implications for efforts to understand and manage enigmatic species like the whale shark Rhincodon typus. The ecological relevance of differences in tissue isotopes within and between individuals in the context of foraging however depends on understanding tissue turnover times and carbon (Δ¹³C) and nitrogen (Δ¹⁵N) discrimination, which can vary with physiology, metabolism, and diet quality. Here, we examine isotope dynamics in captive R. typus as a basis for enhanced ecological insights into wild populations of the world's largest fish and other enigmatic species. A variable diet, principally consisting of two krill (Euphausia pacifica and Euphausia superba) provided an average of 48 ± 20 MJ/d (mean ± SD), or 2.7 ± 1.3 times basal metabolic requirements. On this diet, in agreement with allometric relationships, large body sizes (3,000–4,000 kg) were matched by slow plasma and cartilage turnover rates (empirically derived as 9 months and 3 yr, respectively), which provide tissue‐specific limits on the timescales over which we can isotopically detect diet changes in this species. Average diet‐to‐tissue discrimination showed significant variation between tissues (plasma and cartilage), and among growing and fasting individuals (Δ¹³C range, 1.5 to 5.5‰; Δ¹⁵N range, −0.1 to 2.9‰). Assimilation rates increased with temperature and were higher for the smaller E. pacifica (15 ± 2 mm) than E. superba (48 ± 2 mm). Growth significantly lowered both Δ¹⁵Nₚₗₐₛₘₐ and Δ¹⁵Ncₐᵣₜᵢₗₐgₑ, with inappetence markedly reducing Δ¹⁵Nₚₗₐₛₘₐ and Δ¹³Cₚₗₐₛₘₐ, as well as significantly altering blood biochemistry. Captive findings facilitated the first robust multi‐tissue growth‐ and nutrition‐corrected isotope analysis of a wild R. typus population, suggesting individual foraging specialization on low trophic level mid‐ocean or coastal prey. Long‐term fasting during ocean‐basin‐scale migrations may be common and such metabolic effects should be carefully quantified when isotopically assessing intra‐species foraging differences. The metabolically constrained multi‐tissue, multi‐isotope approach described can facilitate ecological insights that are indispensable for effective conservation and management of globally threatened, but poorly understood, species by identifying differences in key foraging areas and target prey within and between individuals.

The ram’s horn squid, Spirula spirula (Spirulida, Coleoidea), inhabits subsurface waters of the tropical and subtropical oceans. Because of the presence of an internal chambered shell in this species, it has frequently been used as a model species to investigate the paleobiology of fossil coleoids. However, the feeding and dietary habits of S. spirula in the nature are poorly known. In this study, we applied a new method (amino acid nitrogen isotopic analysis) to estimate the trophic position of S. spirula specimens captured off Suriname, as well as three cuttlefish Sepia species (Sepia officinalis, S. latimanus, and S. esculenta), with a calcified chambered shell from the shallower water. The trophic position of S. spirula was estimated to be 2.5–2.8, which was significantly lower than that for the three Sepia species (3.4–3.6). The results and available data on the gastric contents of S. spirula suggest that it feeds mainly on detritus and zooplankton, including crustaceans, from the overlying water column. The method used in this study can potentially be applied to the estimation of the trophic position of the fossil cephalopods having calcified chambered shells.

To evaluate the influence of formalin-fixation on the nitrogen isotopic composition of individual amino acids, we conducted a formalin-fixation experiment over 62 weeks using muscle tissues of largemouth bass (Micropterus salmoides) and freshwater minnow (Zacco platypus) collected in Lake Biwa. The results indicated that the formalin-fixation does not affect the nitrogen isotopic composition of amino acids including glutamic acid and phenylalanine from muscle protein of the fish. We applied this technique to formalin-fixed gobiid fish “Isaza” (Gymnogobius isaza, Tanaka) specimens that had been collected from Lake Biwa through the twentieth century. The pattern of the δ15N values for these amino acids remains relatively constant, even though each amino acid exhibits a gradual increase by ~3 ‰ from 1916 to 1992. With a formula proposed by Chikaraishi et al. (Limnol Oceanogr Method 7:740–750, 2009), we estimated the temporal variations in the trophic position of Isaza in Lake Biwa. The trophic position of Isaza remained quite constant (3.2–3.3) across a major eutrophication period in 1960–1980. The estimated trophic position of Isaza fish is consistent with the stomach content analysis in the previous reports. The constant trophic position suggests that the eutrophication did not apparently affect the trophic position of Isaza fish, although Isaza’s dietary preference changed from zooplankton to gammarids around 1970.

Archaea are prevalent in the deep sea, and comprise a major fraction of the biomass in marine sediments. 13C-labelling experiments on the sea floor suggest that benthic archaea use sedimentary organic compounds to construct their membranes.

The relative contribution of marine-derived carbon in the ancient diet is essential for correcting the marine reservoir effect on the radiocarbon age of archaeological human remains. In this study, we evaluated the marine protein consumption of 3 human populations from the Okhotsk culture (about AD 550–1200) in Hokkaido, Japan, based on stable carbon and nitrogen isotopic compositions in bulk bone collagen as well as the nitrogen isotopic composition of glutamic acid and phenylalanine. Despite the similarity of carbon and nitrogen isotopic composition of bulk collagens, nitrogen isotopic composition of their constituent amino acids suggests differences in fur seal contributions among northern Hokkaido (0–24% for Kafukai 1, 0–10% for Hamanaka 2) and eastern Hokkaido (78–80% for Moyoro) populations. It suggests that nitrogen composition of glutamic acid and phenylalanine could provide a detailed picture of ancient human subsistence.

We investigated the nitrogen isotopic compositions of individual amino acids from brown and red macroalgae and gastropods in a natural marine coastal environment, to further eval- uate them as a tool for ecological studies and to understand the factor(s) controlling the isotopic com- positions in terms of biosynthetic and metabolic processes. The isotopic compositions of 12 amino acids range from -2.1 to + 8.4 ‰ for brown algae, from -3.3 to +12.9 ‰ for red algae, and from -0.6 to +16.6 ‰ for gastropods. The isotopic distributions between algae and gastropods and within algal classes are consistent with those in previous reports, suggesting them to be quite useful for studying food web structure. The nitrogen isotopic variation between amino acids strongly reflects their biosynthetic and metabolic processes. In particular, we suggest here that metabolic fate is an impor- tant factor in producing distinct trophic relationships in nitrogen isotopic compositions between amino acids.

Grasses (Poaceae) are distributed across the world in broad latitudinal belts and are an important source of C4 biomass in the geological record of soils as well as lake and marine sediments. We examined long-chain leaf wax components of thirty-five C4 grasses of the subfamilies Aristidoideae, Chloridoideae and Panicoideae from the southern African grasslands and savannas and three C3 grasses of the subfamily Pooideae from Peru and Australia and review the relevant botanical, phytogeographic and leaf wax compositional background information. Contents, distribution patterns and molecular stable carbon isotopic compositions of long-chain n-alkanes (n-C27 to n-C35) and n-alkan-1-ols (n-C22 to n-C32) were used to estimate the chemotaxonomic relevance of wax signatures of whole plants, separately for different subfamilies and for members of the three C4 subtypes (NADP–ME, NAD–ME and PCK). Two grass species were separated into flower heads, leaves and stems and the parts analysed separately. Grass flowers contain remarkable amounts of short-chain n-alkanes, which may have a significant influence on the chemical signature of the whole plant, whereas n-alkanol distribution patterns exhibit no systematics. The stable carbon isotopic composition of both biomarker types in different plant parts is remarkably uniform. Chemotaxonomic differentiation was not possible on a species level based on whole plant samples, but was more successful for averages of subfamily and photosynthetic subtype data. Wax signatures of C4 grasses are generally distinguishable from those of C3 species by heavier isotopic values, higher contents of n-C31 and n-C33 alkanes and the abundance of the n-C32 n-alkanol, which is largely absent in C3 grass waxes. Especially the waxes of the NAD–ME and PCK C4-subtype grasses, which thrive in extremely arid tropical and subtropical areas, contain high relative amounts of longer-chain n-alkane homologues. The chemical classification on a subfamily level, which is in agreement with previously reported subfamilial phylogeny of grasses, implies an evolutionary wax adaptation of C4 grasses to warm and arid habitats. Our results confirm the validity of the contents, distribution patterns and molecular stable carbon isotopic compositions of long-chain n-alkanes and alkan-1-ols as indirect proxies of continental climate conditions in environmental studies of the tropics.

Stable carbon (δ13C) and hydrogen (δD) isotopic compositions of n-alkanes, anteiso-alkanes, n-alkanoic acids, n-alkanols, phytol and sterols in raw leaves of Acer argutum and Acer carpinifolium, their fallen leaves, mold and soils from a natural Acer forest were measured in order to: (1) understand isotopic variation of the plant biomarkers in a plant–soil system and (2) evaluate which biomarker is the most effective recorder of soil vegetation. Long-chain (> C24) n-alkanes, n-alkanoic acids and n-alkanols are gradually enriched in 13C up to 12.9‰ (average of 4.3‰) and depleted in D up to 94‰ (average of 55‰) from raw leaves to soils. However, anteiso-alkanes, phytol and sterols show little variation in both δ13C (

Carbon (δ13C) and hydrogen isotopic compositions (δD) of sterols in natural marine brown (Sargassum filicinum and Undaria pinnatifida), red macroalgae (Binghamia californica and Gelidium japonica), and in shellfish (Binghamia californica, Haliotis discus and Omphalius pfeifferi) feeding on the brown algae have been investigated as a first attempt to understand the isotopic compositions of sterols in natural algae and heterotrophs. Brown algae have 24-methylcholesta-5,24(28)-dien-3β-ol with δ13C value of −19.1 ± 0.1‰ and δD value of −298 ± 4‰, and 24-ethylcholesta-5,24(28)E-dien-3β-ol with δ13C value of −20.3 ± 0.2‰ and δD value of −301 ± 1‰. Red algae have cholest-5-en-3β-ol with δ13C value of −20.9 ± 3.0‰ and δD value of −289 ± 0‰. The δ13C and δD values of these sterols in algae are consistent with those of algal sterols found in marine sediments, suggesting that the isotopic compositions of algal sterols are well preserved in sediments with little change due to heterotrophic effects. The three shellfish have a wide diversity of sterol types but most sterols, except cholest-5-en-3β-ol, have quite similar isotope compositions (δ13C value of −19.5 ± 0.4‰ and δD value of −296 ± 5‰) to the dietary algal sterols. On the other hand, cholest-5-en-3β-ol is slightly depleted in 13C by up to 1.7‰ and significantly enriched in D by up to 49‰ relative to the other shellfish sterols. This may suggest that shellfish sterols are derived from dietary algal sterols with no substantial isotopic fractionation during either assimilation or modification, but cholest-5-en-3β-ol is derived from an admixture of processes including de novo biosynthesis in shellfish.

We determined hydrogen, carbon and nitrogen isotopic compositions of chlorophylls a and b isolated from leaves of five C3 higher plant species (Benthamidia japonica, Prunus japonica, Acer carpinifolium, Acer argutum and Querus mongloica), and hydrogen and carbon isotopic compositions of phytol and chlorophyllides in the chlorophylls to understand isotopic fractionations associated with chlorophyll biosynthesis in these species. Chlorophylls are depleted in D relative to ambient water by approximately 189 per thousand and enriched in (13)C relative to bulk tissue by approximately 1.6 per thousand. These data can be explained by the contribution of isotopic fractionations during phytol and chlorophyllide biosyntheses. Phytol is more depleted in both D (by approximately 308 per thousand) and (13)C (by approximately 4.3 per thousand), while chlorophyllides are less depleted in D (by approximately 44 per thousand) and enriched in (13)C (by approximately 4.8 per thousand). Such inhomogeneous distribution of isotopes in chlorophylls suggests that (1) the phytol in chlorophylls reflects strong D- and (13)C-depletions due to the isotopic fractionations during the methylerythritol phosphate pathway followed by hydrogenation, and (2) the chlorophyllides reflect D- and (13)C-enrichments in tricarboxylic acid cycle. On the other hand, chlorophylls are slightly ( approximately 1.2 per thousand) depleted in (15)N relative to the bulk tissue, indicating that net isotopic fractionation of nitrogen during chlorophyll biosynthesis is small compared with those of hydrogen and carbon.

Compound-specific hydrogen isotopic compositions ( δ D) of saturated, monounsaturated and polyunsaturated fatty acids have been determined for natural marine macroalgae including two brown algae (Heterokontophyta) and two red algae (Rhodophyta). δ D values of individual fatty acids from four macroalgae exhibit a wide variation ranging from −189‰ to +48‰. Generally, stearic (18:0), arachidic (20:0) and behenic acids (22:0) are much more enriched in D by up to ∼180‰ relative to myristic (14:0), palmitic (16:0), octatetraenoic [18:4( n − 3)] and eicosapentaenoic acids [20:5( n − 3)]. Other fatty acids such as oleic [18:1( n − 9)], lenoleic [18:2( n − 6)] and linolenic acids [18:3( n − 3)] fall isotopically between these fatty acids. This wide δ D variation of fatty acids is probably explained by the hydrogen isotopic fractionation during desaturation being much larger than that during elongation in the network of polyunsaturated fatty acid biosynthesis. A large hydrogen isotopic fractionation during desaturation may cause D-enrichment in the remaining hydrogen of the residual fatty acids, which could be controlled by the relative flux into their desaturates.

Stable hydrogen and carbon isotopic compositions of individual n-alkanes were determined for various terrestrial plants (33 samples including 27 species) and aquatic plants (six species) in natural environments from Japan and Thailand. In C3 plants, n-alkanes extracted from angiosperms have a deltaD value of -152+/-26 per thousand (relative to Standard Mean Ocean Water [SMOW]) and delta13C value of -36.1+/-2.7 per thousand (relative to Peedde Belemnite [PDB]), and those from gymnosperms have a deltaD value of -149+/-16 per thousand and delta13C value of -31.6+/-1.7 per thousand. Angiosperms have n-alkanes depleted in 13C relative to gymnosperms. n-Alkanes from C4 plants have a deltaD value of -171+/-12 per thousand and delta13C value of -20.5+/-2.1 per thousand, being a little depleted in D and much enriched in 13C compared to C3 plants. n-Alkanes of CAM plants are a little depleted in D and vary widely in delta13C relative to those of C3 and C4 plants. In aquatic plants, n-alkanes from freshwater plants have a deltaD value of -187+/-16 per thousand and delta13C value of -25.3+/-1.9 per thousand, and those from seaweeds have a deltaD value of -155+/-34 per thousand and delta13C value of -22.8+/-1.0 per thousand. All n-alkanes from various plant classes are more depleted in D and 13C relative to environmental water and bulk tissue, respectively. In addition, the hydrogen and carbon isotopic fractionations during n-alkane synthesis are distinctive for these various plant classes. While C3 plants have smaller isotopic fractionations in both D and 13C, seaweed has larger isotopic fractionations.

Human-induced ecological change in the open oceans appears to be accelerating. Fisheries, climate change and elevated nutrient inputs are variously blamed, at least in part, for altering oceanic ecosystems. Yet it is challenging to assess the extent of anthropogenic change in the open oceans, where historical records of ecological conditions are sparse, and the geographical scale is immense. We developed millennial-scale amino acid nitrogen isotope records preserved in ancient animal remains to understand changes in food web structure and nutrient regimes in the oceanic realm of the North Pacific Ocean (NPO). Our millennial-scale isotope records of amino acids in bone collagen in a wide-ranging oceanic seabird, the Hawaiian petrel ( Pterodroma sandwichensis ), showed that trophic level declined over time. The amino acid records do not support a broad-scale increase in nitrogen fixation in the North Pacific subtropical gyre, rejecting an earlier interpretation based on bulk and amino acid specific δ 15 N chronologies for Hawaiian deep-sea corals and bulk δ 15 N chronologies for the Hawaiian petrel. Rather, our work suggests that the food web structure in the NPO has shifted at a broad geographical scale, a phenomenon potentially related to industrial fishing.

What eel larvae feed on in the surface layer of the ocean has remained mysterious. Gut contents and bulk nitrogen stable isotope studies suggested that these unusual larvae, called leptocephali, feed at a low level in the oceanic food web, whereas other types of evidence have suggested that small zooplankton are eaten. In this study, we determined the nitrogen isotopic composition of amino acids of both natural larvae and laboratory-reared larvae of the Japanese eel to estimate the trophic position (TP) of leptocephali. We observed a mean TP of 2.4 for natural leptocephali, which is consistent with feeding on particulate organic matter (POM) such as marine snow and discarded appendicularian houses containing bacteria, protozoans and other biological materials. The nitrogen isotope enrichment values of the reared larvae confirm that the primary food source of natural larvae is consistent only with POM. This shows that leptocephali feed on readily available particulate material originating from various sources closely linked to ocean primary production and that leptocephali are a previously unrecognized part of oceanic POM cycling.

The sea slug Plakobranchus ocellatus (Sacoglossa, Gastropoda) retains photosynthetically active chloroplasts from ingested algae (functional kleptoplasts) in the epithelial cells of its digestive gland for up to 10 months. While its feeding behavior has not been observed in natural habitats, two hypotheses have been proposed: 1) adult P. ocellatus uses kleptoplasts to obtain photosynthates and nutritionally behaves as a photoautotroph without replenishing the kleptoplasts; or 2) it behaves as a mixotroph (photoautotroph and herbivorous consumer) and replenishes kleptoplasts continually or periodically. To address the question of which hypothesis is more likely, we examined the source algae for kleptoplasts and temporal changes in kleptoplast composition and nutritional contribution. By characterizing the temporal diversity of P. ocellatus kleptoplasts using rbcL sequences, we found that P. ocellatus harvests kleptoplasts from at least 8 different siphonous green algal species, that kleptoplasts from more than one species are present in each individual sea slug, and that the kleptoplast composition differs temporally. These results suggest that wild P. ocellatus often feed on multiple species of siphonous algae from which they continually obtain fresh chloroplasts. By estimating the trophic position of wild and starved P. ocellatus using the stable nitrogen isotopic composition of amino acids, we showed that despite the abundance of kleptoplasts, their photosynthates do not contribute greatly to the nutrition of wild P. ocellatus, but that kleptoplast photosynthates form a significant source of nutrition for starved sea slugs. The herbivorous nature of wild P. ocellatus is consistent with insights from molecular analyses indicating that kleptoplasts are frequently replenished from ingested algae, leading to the conclusion that natural populations of P. ocellatus do not rely on photosynthesis but mainly on the digestion of ingested algae.

We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (R)-(-)-2-butanol or (S)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of D- and L-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; Enterococcus faecalis, Staphylococcus aureus, Staphylococcus staphylolyticus, Lactobacillus acidophilus, Bacillus subtilis, Micrococcus luteus, and Streptomyces sp.), natural whole bacterial cells (Bacillus subtilis var. natto), (pseudo)-peptidoglycan from archaea (Methanobacterium sp.), and cell wall from eukaryota (Saccharomyces cerevisiae). We observed statistically significant differences in nitrogen isotopic compositions; e.g., delta15N ( per thousand vs air) in Staphylococcus staphylolyticus for d-alanine (19.2 +/- 0.5 per thousand, n = 4) and L-alanine (21.3 +/- 0.8 per thousand, n = 4) and in Bacillus subtilis for D-alanine (6.2 +/- 0.2 per thousand, n = 3) and L-alanine (8.2 +/- 0.4 per thousand, n = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in 15N-depleted D-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers.

Although analyses of chlorophyll d (Chl d)‐dominated oxygenic photosystems have been conducted since their discovery 12 years ago, Chl d distribution in the environment and quantitative importance for aquatic photosynthesis remain to be investigated. We analyzed the pigment compositions of surface sediments and detected Chl d and its derivatives from diverse aquatic environments. Our data show that the viable habitat for Chl d‐producing phototrophs extends across salinities of 0 to 50 practical salinity units and temperatures of 1� to 40�C, suggesting that Chl d production can be ubiquitously observed in aquatic environments that receive near-infrared light. The relative abundances of Chl d derivatives over that of Chl a derivatives in the studied samples are up to 4%, further suggesting that Chl d‐based photosynthesis plays a quantitatively important role in the aquatic photosynthesis.

Nitrogen (N) isotopic compositions of individual hydrolysable amino acids (δ 15 N AAs ) in N pools have been increasingly used for trophic position assessment and evaluation of sources and transformation processes of organic matter in marine environments. However, there are limited data about variability in δ 15 N AAs patterns and how this variability influences marine bacteria, an important mediator of trophic transfer and organic matter transformation. We explored whether marine bacterial δ 15 N AAs profiles change depending on the type and C:N ratio of the substrate. The δ 15 N AAs profile of a marine bacterium, Vibrio harveyi , was examined using medium containing either glutamate, alanine or ammonium as the N source [substrate C:N ratios (range, 3 to 20) were adjusted with glucose]. The data were interpreted as a reflection of isotope fractionations associated with de novo synthesis of amino acids by bacteria. Principal component analysis (PCA) using the δ 15 N offset values normalized to glutamate + glutamine δ 15 N revealed that δ 15 N AAs profiles differed depending on the N source and C:N ratio of the substrate. High variability in the δ 15 N offset of alanine and valine largely explained this bacterial δ 15 N AAs profile variability. PCA was also conducted using bacterial and phytoplankton (cyanobacteria and eukaryotic algae) δ 15 N AAs profile data reported previously. The results revealed that bacterial δ 15 N AAs patterns were distinct from those of phytoplankton. Therefore, the δ 15 N AAs profile is a useful indicator of biochemical responses of bacteria to changes in substrate conditions, serving as a potentially useful method for identifying organic matter sources in marine environments.

We conducted an in situ feeding experiment using 13C-labeled unicellular algae in Sagami Bay, Japan (water depth, 1450 m), in order to understand the fate of lipid compounds in phytodetritus at the deep-sea floor. We examined the incorporation of excess 13C into lipid compounds extracted from bulk sediments and benthic foraminiferal cells. 13C-enriched fatty acids derived from 13C-labeled algae were exponentially degraded during 6 days of incubation in the sediment. Subsequent enrichments in 13C in sedimentary n-C15, anteiso-C17, and C17 fatty acids indicated the microbial degradation of algal material and production of bacterial biomass in the sediment. We observed the incorporation of 13C-labeled algal phytol and fatty acids into foraminiferal cells. The compositions of 13C-labeled algal lipids in foraminiferal cells were different from those in the bulk sediments, indicating that foraminiferal feeding and digestion influenced the lipid distribution in the sediments. Furthermore, some sterols in Globobulimina affinis (e.g., 24-ethylcholesta-5,22-dien-3β-ol, 24-ethylcholest-5-en-3β-ol, and 23,24-dimethylcholesta-5,22E-dien-3β-ol) were newly produced via the modification of dietary algal sterols within 4–6 days. In addition to the effects of bacteria, feeding by benthic foraminifera can result in a significant reorganization of the composition of organic matter and influence benthic food webs and carbon cycling at the deep-sea floor.

The sources (marine algae, terrestrial C3 and C4 plants) of sterols deposited to sediments along a riverine‐marine transect from Ohtsuchi River in Iwate Prefecture, Japan, to the northwestern Pacific Ocean were estimated using carbon (d 13 C) and hydrogen isotopic compositions (dD) in surface sediments. In marine sediments, algal sterols such as 24-methylcholesta-5,22-dien-3b-ol had d 13 C values of 222.7 6 0.4‰ and dD values of 2292 6 3‰. In contrast, sterols derived from multiple potential sources, such as 24-methylcholest-5-en-3 b-ol and 24-ethylcholest5-en-3b-ol, were gradually enriched in 13 C from riverine (230.7‰ and 230.5‰, respectively) to marine sediments (222.3‰ and 224.4‰, respectively), but showed little variation in dD values (2262 6 1‰). These isotopic signatures suggest that 24-methylcholest-5-en-3b-ol and 24-ethylcholest-5-en-3b-ol are derived from C3 plants (;90%) and C4 plants (;10%) in the riverine sediments, and marine algae (;30%), C3 plants (;30%), and C4 plants (;40%) in the open marine sediments. Thus, dual isotopic compositions (d 13 C-dD) of sterols allow determinations of the proportions of three biological sources, in which the relative contribution of C3 plants decreases while that of marine algae and C4 plants increases from riverine to marine sediments. These results indicate that d 13 C-dD signatures of sterols provide a useful tool for interpreting the sources of sedimentary organic matter and for understanding the transport and mixing processes of distinct biological sources in riverine‐marine environments. Quantitative assessment of organic matter inputs from marine algae and terrestrial plants to riverine and marine sediments is important for understanding the burial and mixing processes affecting organic matter at continental margins and for estimating marine primary productivity and carbon flux associated with biogeochemical cycles in surface environments. Marine algae and terrestrial C3 and C4 plants are major primary producers and potential sources for sedimentary organic matter in river-dominated coastal environments. Molecular and isotopic compositions of sedimentary organic materials should reflect both variations in the input of marine algae relative to terrestrial plants and in the proportions of C3 and C4 terrestrial plant inputs. Many previous studies have estimated the relative contribution between two biological sources (marine algae vs. terrestrial plants or terrestrial C3 vs. C4 plants) for sedimentary organic materials from riverine (or coastal) to marine environments based on biomarker assessments (e.g., Laureillard and Saliot 1993; Mannino and Harvey 1999). Carbon isotopic compositions of bulk organic carbon (e.g., Wada et al. 1987; Carreira et al. 2002) and specific biomarkers including lignin-derived com1

Sterols are key components of eukaryotic cellular membranes that are synthesized by multi-enzyme pathways that require molecular oxygen. Because prokaryotes fundamentally lack sterols, it is unclear how the vast diversity of bacterivorous eukaryotes that inhabit hypoxic environments obtain, or synthesize, sterols. Here we show that tetrahymanol, a triterpenoid that does not require molecular oxygen for its biosynthesis, likely functions as a surrogate of sterol in eukaryotes inhabiting oxygen-poor environments. Genes encoding the tetrahymanol synthesizing enzyme squalene-tetrahymanol cyclase were found from several phylogenetically diverged eukaryotes that live in oxygen-poor environments and appear to have been laterally transferred among such eukaryotes. Reviewers This article was reviewed by Eric Bapteste and Eugene Koonin.