Molecular exploration of fossil eggshell uncovers hidden lineage of giant extinct bird.

The systematics of Madagascar's extinct elephant birds remains controversial due to large gaps in the fossil record and poor biomolecular preservation of skeletal specimens. Here, a molecular analysis of 1000-year-old fossil eggshells provides the first description of elephant bird phylogeography and offers insight into the ecology and evolution of these flightless giants. Mitochondrial genomes from across Madagascar reveal genetic variation that is correlated with eggshell morphology, stable isotope composition, and geographic distribution. The elephant bird crown is dated to ca. 30 Mya, when Madagascar is estimated to have become less arid as it moved northward. High levels of between-clade genetic variation support reclassifying Mullerornis into a separate family. Low levels of within-clade genetic variation suggest there were only two elephant bird genera existing in southern Madagascar during the Holocene. However, we find an eggshell collection from Madagascar's far north that represents a unique lineage of Aepyornis. Furthermore, divergence within Aepyornis coincides with the aridification of Madagascar during the early Pleistocene ca. 1.5 Ma, and is consistent with the fragmentation of populations in the highlands driving diversification and the evolution of extreme gigantism over shorts timescales. We advocate for a revision of their taxonomy that integrates palaeogenomic and palaeoecological perspectives.

Comparative metagenomics at Solfatara and Pisciarelli hydrothermal systems in Italy reveal that ecological differences across substrates are not ubiquitous.

Introduction: Continental hydrothermal systems (CHSs) are geochemically complex, and they support microbial communities that vary across substrates. However, our understanding of these variations across the complete range of substrates in CHS is limited because many previous studies have focused predominantly on aqueous settings. Methods: Here we used metagenomes in the context of their environmental geochemistry to investigate the ecology of different substrates (i.e., water, mud and fumarolic deposits) from Solfatara and Pisciarelli. Results and Discussion: Results indicate that both locations are lithologically similar with distinct fluid geochemistry. In particular, all substrates from Solfatara have similar chemistry whereas Pisciarelli substrates have varying chemistry; with water and mud from bubbling pools exhibiting high SO4 2- and NH4 + concentrations. Species alpha diversity was found to be different between locations but not across substrates, and pH was shown to be the most important driver of both diversity and microbial community composition. Based on cluster analysis, microbial community structure differed significantly between Pisciarelli substrates but not between Solfatara substrates. Pisciarelli mud pools, were dominated by (hyper)thermophilic archaea, and on average, bacteria dominated Pisciarelli fumarolic deposits and all investigated Solfatara environments. Carbon fixation and sulfur oxidation were the most important metabolic pathways fueled by volcanic outgassing at both locations. Together, results demonstrate that ecological differences across substrates are not a widespread phenomenon but specific to the system. Therefore, this study demonstrates the importance of analyzing different substrates of a CHS to understand the full range of microbial ecology to avoid biased ecological assessments.

Multiple trophic pathways support fish on floodplains of California's Central Valley.

We used compound-specific isotope analysis of carbon isotopes in amino acids (AAs) to determine the biosynthetic source of AAs in fish from major tributaries to California's Sacramento-San Joaquin river delta (i.e., the Sacramento, Cosumnes and Mokelumne rivers). Using samples collected in winter and spring between 2016 and 2019, we confirmed that algae are a critical component of floodplain food webs in California's Central Valley. Results from bulk stable isotope analysis of carbon and nitrogen in producers and consumers were adequate to characterize a general trophic structure and identify potential upstream and downstream migration into our study site by American shad Alosa sapidissima and rainbow trout Oncorhynchus mykiss, respectively. However, owing to overlap and variability in source isotope compositions, our bulk data were unsuitable for conventional bulk isotope mixing models. Our results from compound-specific carbon isotope analysis of AAs clearly indicate that algae are important sources of organic matter to fish of conservation concern, such as Chinook salmon Oncorhynchus tshawytscha in California's Central Valley. However, algae were not the exclusive source of energy to metazoan food webs. We also revealed that other sources of AAs, such as bacteria, fungi and higher plants, contributed to fish as well. While consistent with the well-supported notion that algae are critical to aquatic food webs, our results highlight the possibility that detrital subsidies might intermittently support metazoan food webs.

Physiological and metabolic responses of chemolithoautotrophic NO 3 - reducers to high hydrostatic pressure.

We investigated the impact of pressure on thermophilic, chemolithoautotrophic NO 3 - reducing bacteria of the phyla Campylobacterota and Aquificota isolated from deep-sea hydrothermal vents. Batch incubations at 5 and 20 MPa resulted in decreased NO 3 - consumption, lower cell concentrations, and overall slower growth in Caminibacter mediatlanticus (Campylobacterota) and Thermovibrio ammonificans (Aquificota), relative to batch incubations near standard pressure (0.2 MPa) conditions. Nitrogen isotope fractionation effects from chemolithoautotrophic NO 3 - reduction by both microorganisms were, on the contrary, maintained under all pressure conditions. Comparable chemolithoautotrophic NO 3 - reducing activities between previously reported natural hydrothermal vent fluid microbial communities dominated by Campylobacterota at 25 MPa and Campylobacterota laboratory isolates at 0.2 MPa, suggest robust similarities in cell-specific NO 3 - reduction rates and doubling times between microbial populations and communities growing maximally under similar temperature conditions. Physiological and metabolic comparisons of our results with other studies of pressure effects on anaerobic chemolithoautotrophic processes (i.e., microbial S0 -oxidation coupled to Fe(III) reduction and hydrogenotrophic methanogenesis) suggest that anaerobic chemolithoautotrophs relying on oxidation-reduction (redox) reactions that yield higher Gibbs energies experience larger shifts in cell-specific respiration rates and doubling times at increased pressures. Overall, our results advance understanding of the role of pressure, its relationship with temperature and redox conditions, and their effects on seafloor chemolithoautotrophic NO 3 - reduction and other anaerobic chemolithoautotrophic processes.

Central Metabolism and Growth Rate Impacts on Hydrogen and Carbon Isotope Fractionation During Amino Acid Synthesis in E. coli.

Compound specific stable isotope analysis (CSIA) of amino acids from bacterial biomass is a newly emerging powerful tool for exploring central carbon metabolism pathways and fluxes. By comparing isotopic values and fractionations relative to water and growth substrate, the impact of variable flow path for metabolites through different central metabolic pathways, perturbations of these paths, and their resultant consequences on intracellular pools and resultant biomass may be elucidated. Here, we explore the effects that central carbon metabolism and growth rate can have on stable hydrogen (δ2H) and carbon (δ13C) compound specific isotopic values of amino acids, and whether diagnostic isotopic fingerprints are revealed by these paired analyses. We measured δ2H and δ13C in amino acids in the wild type Escherichia coli (MG1655) across a range of growth rates in chemostat cultures to address the unknown isotopic consequences as metabolic fluxes are shuffled between catabolic and anabolic metabolisms. Additionally, two E. coli knockout mutants, one with deficiency in glycolysis -pgi (LC1888) and another inhibiting the oxidative pentose phosphate pathway (OPPP) -zwf (LC1889), were grown on glucose and used as a comparison against the wild type E. coli (MG1655) to address the isotopic changes of amino acids produced in these perturbed metabolic pathways. Amino acid δ2H values, which collectively vary in composition by more than 400‰, are altered along with δ13C values demonstrating fundamental shifts in central metabolic pathways and/or fluxes. Within our linear discriminant analysis with a simple model organism to examine potential amino acid fingerprinting, our knockout strains and variable growth rate samples plot across a wider array of organism classification than merely within the boundaries of other bacterial data.

Compound-specific δ2H analysis highlights the relationship between direct assimilation and de novo synthesis of amino acids from food and water in a terrestrial mammalian omnivore.

Hydrogen isotope (δ2H) analysis has been routinely used as an ecological tracer for animal movement and migration, yet a biochemical understanding of how animals incorporate this element in the synthesis of tissues is poorly resolved. Here, we apply a new analytical tool, amino acid (AA) δ2H analysis, in a controlled setting to trace the influence of drinking water and dietary macromolecules on the hydrogen in muscle tissue. We varied the δ2H of drinking water and the proportions of dietary protein and carbohydrates with distinct hydrogen and carbon isotope compositions fed to house mice among nine treatments. Our results show that hydrogen in the non-essential (AANESS) and essential (AAESS) AAs of mouse muscle is not readily exchanged with body water, but rather patterns among these compounds can be described through consideration of the major biochemical pathway(s) used by organisms to synthesize or route them from available sources. Dietary carbohydrates contributed more hydrogen than drinking water to the synthesis of AANESS in muscle. While neither drinking water nor dietary carbohydrates directly contributed to muscle AAESS, we did find that a minor but measurable proportion (10-30%) of the AAESS in muscle was synthesized by the gut microbiome using hydrogen and carbon from dietary carbohydrates. δ2H patterns among individual AAs in mice muscle are similar to those we previously reported for bacteria, which provides additional support that this approach may allow for the simultaneous analysis of different AAs that are more influenced by drinking water (AANESS) versus dietary (AAESS) sources of hydrogen.

Isotopic and genetic methods reveal the role of the gut microbiome in mammalian host essential amino acid metabolism.

Intestinal microbiota perform many functions for their host, but among the most important is their role in metabolism, especially the conversion of recalcitrant biomass that the host is unable to digest into bioavailable compounds. Most studies have focused on the assistance gut microbiota provide in the metabolism of carbohydrates, however, their role in host amino acid metabolism is poorly understood. We conducted an experiment on Mus musculus using 16S rRNA gene sequencing and carbon isotope analysis of essential amino acids (AAESS) to quantify the community composition of gut microbiota and the contribution of carbohydrate carbon used by the gut microbiome to synthesize AAESS that are assimilated by mice to build skeletal muscle tissue. The relative abundances of Firmicutes and Bacteroidetes inversely varied as a function of dietary macromolecular content, with Firmicutes dominating when mice were fed low-protein diets that contained the highest proportions of simple carbohydrates (sucrose). Mixing models estimated that the microbial contribution of AAESS to mouse muscle varied from less than 5% (threonine, lysine, and phenylalanine) to approximately 60% (valine) across diet treatments, with the Firmicute-dominated microbiome associated with the greatest contribution. Our results show that intestinal microbes can provide a significant source of the AAESS their host uses to synthesize structural tissues. The role that gut microbiota play in the amino acid metabolism of animals that consume protein-deficient diets is likely a significant but under-recognized aspect of foraging ecology and physiology.

Assimilation and discrimination of hydrogen isotopes in a terrestrial mammal.

Stable isotope analysis has revolutionized the way ecologists study animal resource use from the individual to the community level. Recent interest has emerged in using hydrogen isotopes (2H/1H) as ecological tracers, because they integrate information from both abiotic and biotic processes. A better physiological understanding of how animals assimilate hydrogen and use it to synthesize tissues is needed to further refine this tool and broaden its use in animal ecology. We conducted a controlled-feeding experiment using laboratory mice (Mus musculus) in which we varied the hydrogen isotope (δ2H) values of water and the proportions of dietary protein and carbohydrates among nine experimental treatments. For each tissue, we calculated the percent of hydrogen derived from water and the percent hydrogen derived from dietary protein versus carbohydrates using linear relationships and isotope mixing models based on accompanying carbon isotope (δ13C) data. The net discrimination (∆2HNet) between mice tissues and potential water and dietary sources of hydrogen differed among tissues. ∆2HNet was positively correlated with dietary protein content in red blood cells (RBC) and muscle, but negatively correlated in liver and plasma. We also report the first estimates for hydrogen isotope discrimination factors (∆2H) for different sources of hydrogen (∆2HWater, ∆2HProtein, and ∆2HCarbs) available for tissue synthesis. This research provides a foundation for understanding how diet quality (e.g., protein content) influences hydrogen isotope assimilation and discrimination in different tissues of a terrestrial mammal, which is a first step towards using δ2H as a tracer of resource use in free-ranging mammals.

Biogeochemical probing of microbial communities in a basalt-hosted hot spring at Kverkfjöll volcano, Iceland.

We investigated bacterial and archaeal communities along an ice-fed surficial hot spring at Kverkfjöll volcano-a partially ice-covered basaltic volcano at Vatnajökull glacier, Iceland, using biomolecular (16S rRNA, apsA, mcrA, amoA, nifH genes) and stable isotope techniques. The hot spring environment is characterized by high temperatures and low dissolved oxygen concentrations at the source (68°C and <1 mg/L (±0.1%)) changing to lower temperatures and higher dissolved oxygen downstream (34.7°C and 5.9 mg/L), with sulfate the dominant anion (225 mg/L at the source). Sediments are comprised of detrital basalt, low-temperature alteration phases and pyrite, with <0.4 wt. % total organic carbon (TOC). 16S rRNA gene profiles reveal that organisms affiliated with Hydrogenobaculum (54%-87% bacterial population) and Thermoproteales (35%-63% archaeal population) dominate the micro-oxic hot spring source, while sulfur-oxidizing archaea (Sulfolobales, 57%-82%), and putative sulfur-oxidizing and heterotrophic bacterial groups dominate oxic downstream environments. The δ13 Corg (‰ V-PDB) values for sediment TOC and microbial biomass range from -9.4‰ at the spring's source decreasing to -12.6‰ downstream. A reverse effect isotope fractionation of ~3‰ between sediment sulfide (δ34 S ~0‰) and dissolved water sulfate (δ34 S +3.2‰), and δ18 O values of ~ -5.3‰ suggest pyrite forms abiogenically from volcanic sulfide, followed by abiogenic and microbial oxidation. These environments represent an unexplored surficial geothermal environment analogous to transient volcanogenic habitats during putative "snowball Earth" scenarios and volcano-ice geothermal environments on Mars.

Stable hydrogen isotope variability within and among plumage tracts (δ2HF) of a migratory wood warbler.

Hydrogen isotope analysis of feather keratin (δ2HF) has become an essential tool for tracking movements between breeding and wintering populations of migratory birds. In particular, δ2HF has been used to create δ2HF isoscapes that can be used to assign the geographic origins of molt. The majority of past studies have sampled a portion of a single feather as an isotopic proxy for the entire plumage although surprisingly little is known about variation of stable isotopes within and between feather tracts of individuals in local populations. Here we examine δ2HF variation in 24 pterylographic variables (9 primaries, 6 secondaries, 6 rectrices, and 3 patches of ventral contour feathers) in individual specimens of black-throated blue warbler (Setophaga caerulescens) breeding in the Big Santeetlah Creek watershed (5350 ha), southern Appalachian Mountains. By restricting our study to territorial ASY males (after second year) inhabiting a small watershed, we could focus on δ2HF variation generated during the complete prebasic annual molt in a circumscribed population while factoring out age and sexual differences in foraging behavior, isotopic incorporation, and post-breeding dispersal. Summed within-individual variation (δ2HF) across 24 pterylographic variables ranged from 12 to 60‰ (= 21.8 ± 9.4‰), with 81% of the individuals exhibiting variation ≥ 16‰ (reproducibility of analyses was ≤ 4 ‰). Adjacent feathers in feather tracts tend to have more similar δ2HF values than feathers grown weeks apart, consistent with the stepwise replacement of flight feathers. The pooled population sample exhibited significant δ2HF variation in primaries (-78 to -21‰), secondaries (-80 to -17‰), rectrices (-78 to -23‰), and ventral contour feathers (-92 to -32‰). Strong year effects in δ2HF variation were observed in each of the 24 pterylographic variables. Altitudinal effects were observed only in ventral contour feathers. The current findings demonstrate that within-individual variation (δ2HF) may be much greater than previously thought in migratory species that molt on or near breeding territories. Our study also highlights the need for greater pterylographic precision in research design of isotope-based studies of avian movement. Within-individual and within-population δ2HF variation should be incorporated in geographic assignment models. In a broader context, the staggered Staffelmauser pattern of molt in wood warblers provides an exceptional view of the seasonal variation of hydrogen isotopes circulating in blood plasma during the six-week period of annual molt.

Climate change promotes parasitism in a coral symbiosis.

Coastal oceans are increasingly eutrophic, warm and acidic through the addition of anthropogenic nitrogen and carbon, respectively. Among the most sensitive taxa to these changes are scleractinian corals, which engineer the most biodiverse ecosystems on Earth. Corals' sensitivity is a consequence of their evolutionary investment in symbiosis with the dinoflagellate alga, Symbiodinium. Together, the coral holobiont has dominated oligotrophic tropical marine habitats. However, warming destabilizes this association and reduces coral fitness. It has been theorized that, when reefs become warm and eutrophic, mutualistic Symbiodinium sequester more resources for their own growth, thus parasitizing their hosts of nutrition. Here, we tested the hypothesis that sub-bleaching temperature and excess nitrogen promotes symbiont parasitism by measuring respiration (costs) and the assimilation and translocation of both carbon (energy) and nitrogen (growth; both benefits) within Orbicella faveolata hosting one of two Symbiodinium phylotypes using a dual stable isotope tracer incubation at ambient (26 °C) and sub-bleaching (31 °C) temperatures under elevated nitrate. Warming to 31 °C reduced holobiont net primary productivity (NPP) by 60% due to increased respiration which decreased host %carbon by 15% with no apparent cost to the symbiont. Concurrently, Symbiodinium carbon and nitrogen assimilation increased by 14 and 32%, respectively while increasing their mitotic index by 15%, whereas hosts did not gain a proportional increase in translocated photosynthates. We conclude that the disparity in benefits and costs to both partners is evidence of symbiont parasitism in the coral symbiosis and has major implications for the resilience of coral reefs under threat of global change.

Canopy gradients in leaf functional traits for species that differ in growth strategies and shade tolerance.

In temperate deciduous forests, vertical gradients in leaf mass per area (LMA) and area-based leaf nitrogen (Narea) are strongly controlled by gradients in light availability. While there is evidence that hydrostatic constraints on leaf development may diminish LMA and Narea responses to light, inherent differences among tree species may also influence leaf developmental and morphological response to light. We investigated vertical gradients in LMA, Narea and leaf carbon isotope composition (δ13C) for three temperate deciduous species (Carpinus caroliniana Walter, Fagus grandifolia Ehrh., Liriodendron tulipifera L.) that differed in growth strategy (e.g., indeterminate and determinate growth), shade tolerance and leaf area to sapwood ratio (Al:As). Leaves were sampled across a broad range of light conditions within three vertical layers of tree crowns to maximize variation in light availability at each height and to minimize collinearity between light and height. All species displayed similar responses to light with respect to Narea and δ13C, but not for LMA. Light was more important for gradients in LMA for the shade-tolerant (C. caroliniana) and -intolerant (L. tulipifera) species with indeterminate growth, and height (e.g., hydrostatic gradients) and light were equally important for the shade-tolerant (F. grandifolia) species with determinate growth. Fagus grandifolia had a higher morphological plasticity in response to light, which may offer a competitive advantage in occupying a broader range of light conditions throughout the canopy. Differences in responses to light and height for the taller tree species, L. tulipifera and F. grandifolia, may be attributed to differences in growth strategy or Al:As, which may alter morphological and functional responses to light availability. While height was important in F. grandifolia, height was no more robust in predicting LMA than light in any of the species, confirming the strong role of light availability in determining LMA for temperate deciduous species.

Investigating Bermuda's pollution history through stable isotope analyses of modern and museum-held gorgonian corals.

For centuries, Bermuda has been challenged with wastewater management for the protection of human and environmental health. By quantifying the δ15N of the common sea fan Gorgonia ventalina sampled from 30 sites throughout Bermuda we show that sewage-derived nitrogen is detectable on nearshore coral reefs and declines across the lagoon to the outer rim. We also sampled gorgonians from two museum collections representing a 50y time-series (1958-2008). These samples revealed an increase in δ15N of >4.0‰ until the mid-1970s, after which δ15N values slowly declined by ~2.0‰. A δ15N chronology from a gorgonian skeleton exhibited a similar decline over the last 30-40years of approximately 0.6‰. We conclude that policies have been effective in reducing sewage impacts to Bermudian reefs. However, significant sources of sewage pollution persist and are likely have a strong impact on harbor and nearshore coral communities and human health.

Fractionation of Hydrogen Isotopes by Sulfate- and Nitrate-Reducing Bacteria.

Hydrogen atoms from water and food are incorporated into biomass during cellular metabolism and biosynthesis, fractionating the isotopes of hydrogen-protium and deuterium-that are recorded in biomolecules. While these fractionations are often relatively constant in plants, large variations in the magnitude of fractionation are observed for many heterotrophic microbes utilizing different central metabolic pathways. The correlation between metabolism and lipid δ(2)H provides a potential basis for reconstructing environmental and ecological parameters, but the calibration dataset has thus far been limited mainly to aerobes. Here we report on the hydrogen isotopic fractionations of lipids produced by nitrate-respiring and sulfate-reducing bacteria. We observe only small differences in fractionation between oxygen- and nitrate-respiring growth conditions, with a typical pattern of variation between substrates that is broadly consistent with previously described trends. In contrast, fractionation by sulfate-reducing bacteria does not vary significantly between different substrates, even when autotrophic and heterotrophic growth conditions are compared. This result is in marked contrast to previously published observations and has significant implications for the interpretation of environmental hydrogen isotope data. We evaluate these trends in light of metabolic gene content of each strain, growth rate, and potential flux and reservoir-size effects of cellular hydrogen, but find no single variable that can account for the differences between nitrate- and sulfate-respiring bacteria. The emerging picture of bacterial hydrogen isotope fractionation is therefore more complex than the simple correspondence between δ(2)H and metabolic pathway previously understood from aerobes. Despite the complexity, the large signals and rich variability of observed lipid δ(2)H suggest much potential as an environmental recorder of metabolism.

Hydrogen isotopes in individual amino acids reflect differentiated pools of hydrogen from food and water in Escherichia coli.

Hydrogen isotope (δ(2)H) analysis is widely used in animal ecology to study continental-scale movement because δ(2)H can trace precipitation and climate. To understand the biochemical underpinnings of how hydrogen is incorporated into biomolecules, we measured the δ(2)H of individual amino acids (AAs) in Escherichia coli cultured in glucose-based or complex tryptone-based media in waters with δ(2)H values ranging from -55‰ to +1,070‰. The δ(2)H values of AAs in tryptone spanned a range of ∼250‰. In E. coli grown on glucose, the range of δ(2)H among AAs was nearly 200‰. The relative distributions of δ(2)H of AAs were upheld in cultures grown in enriched waters. In E. coli grown on tryptone, the δ(2)H of nonessential AAs varied linearly with the δ(2)H of media water, whereas δ(2)H of essential AAs was nearly identical to δ(2)H in diet. Model calculations determined that as much as 46% of hydrogen in some nonessential AAs originated from water, whereas no more than 12% of hydrogen in essential AAs originated from water. These findings demonstrate that δ(2)H can route directly at the molecular level. We conclude that the patterns and distributions in δ(2)H of AAs are determined through biosynthetic reactions, suggesting that δ(2)H could become a new biosignature for studying novel microbial pathways. Our results also show that δ(2)H of AAs in an organism's tissues provides a dual tracer for food and environmental (e.g., drinking) water.

Human predation contributed to the extinction of the Australian megafaunal bird Genyornis newtoni ∼47 ka.

Although the temporal overlap between human dispersal across Australia and the disappearance of its largest animals is well established, the lack of unambiguous evidence for human-megafauna interactions has led some to question a human role in megafaunal extinction. Here we show that diagnostic burn patterns on eggshell fragments of the megafaunal bird Genyornis newtoni, found at >200 sites across Australia, were created by humans discarding eggshell in and around transient fires, presumably made to cook the eggs. Dating by three methods restricts their occurrence to between 53.9 and 43.4 ka, and likely before 47 ka. Dromaius (emu) eggshell occur frequently in deposits from >100 ka to present; burnt Dromaius eggshell first appear in deposits the same age as those with burnt Genyornis eggshell, and then continually to modern time. Harvesting of their eggs by humans would have decreased Genyornis reproductive success, contributing to the bird's extinction by ∼47 ka.

Variability in the routing of dietary proteins and lipids to consumer tissues influences tissue-specific isotopic discrimination.

RATIONALE: The eco-physiological mechanisms that govern the incorporation and routing of macronutrients from dietary sources into consumer tissues determine the efficacy of stable isotope analysis (SIA) for studying animal foraging ecology. We document how changes in the relative amounts of dietary proteins and lipids affect the metabolic routing of these macronutrients and the consequent effects on tissue-specific discrimination factors in domestic mice using SIA. We also examine the effects of dietary macromolecular content on a commonly used methodological approach: lipid extraction of potential food sources. METHODS: We used carbon ((13) C) and nitrogen ((15) N) isotopes to examine the routing of carbon from dietary proteins and lipids that were used by mice to biosynthesize hair, blood, muscle, and liver. Growing mice were fed one of four diet treatments in which the total dietary content of C4 -based lipids (δ(13) C = -14.5‰) and C(3) -based proteins (δ(13) C = -27‰) varied inversely between 5% and 40%. RESULTS: The δ(13) C values of mouse tissues increased by approximately 2-6‰ with increasing dietary lipid content. The difference in δ(13) C values between mouse tissues and bulk diet ranged from 0.1 ± 1.5‰ to 2.3 ± 0.6‰ for all diet treatments. The mean (±SD) difference between the δ(13) C values of mouse tissues and dietary protein varied systematically among tissues and ranged from 3.1 ± 0.1‰ to 4.5 ± 0.6‰ for low fat diets and from 5.4 ± 0.4‰ to 10.5 ± 7.3‰ for high fat diets. CONCLUSIONS: Mice used some fraction of their dietary lipid carbon to synthesize tissue proteins, suggesting flexibility in the routing of dietary macromolecules to consumer tissues based on dietary macromolecular availability. Consequently, all constituent dietary macromolecules, not just protein, should be considered when determining the relationship between diets and consumer tissues using SIA. In addition, in cases where animals consume diets with high lipid contents, non lipid-extracted prey samples should be analyzed to estimate diets using SIA.

Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses.

Many cnidarians host endosymbiotic dinoflagellates from the genus Symbiodinium. It is generally assumed that the symbiosis is mutualistic, where the host benefits from symbiont photosynthesis while providing protection and photosynthetic substrates. Diverse assemblages of symbiotic gorgonian octocorals can be found in hard bottom communities throughout the Caribbean. While current research has focused on the phylo- and population genetics of gorgonian symbiont types and their photo-physiology, relatively less work has focused on biogeochemical benefits conferred to the host and how these benefits vary across host species. Here we examine this symbiosis among 11 gorgonian species collected in Bocas del Toro, Panama. By coupling light and dark bottle incubations (P/R) with (13)C-bicarbonate tracers, we quantified the link between holobiont oxygen metabolism with carbon assimilation and translocation from symbiont to host. Our data show that P/R varied among species, and was correlated with colony morphology and polyp size. Sea fans and sea plumes were net autotrophs (P/R>1.5), while nine species of sea rods were net heterotrophs with most below compensation (P/R<1.0). (13)C assimilation corroborated the P/R results, and maximum δ(13)Chost values were strongly correlated with polyp size, indicating higher productivity by colonies with high polyp SA:V. A survey of gorgonian-Symbiodinium associations revealed that productive species maintain specialized, obligate symbioses and are more resistant to coral bleaching, whereas generalist and facultative associations are common among sea rods that have higher bleaching sensitivities. Overall, productivity and polyp size had strong phylogenetic signals with carbon fixation and polyp size showing evidence of trait covariance.

Amino acid δ13C analysis shows flexibility in the routing of dietary protein and lipids to the tissue of an omnivore.

Stable-isotope analysis (SIA) has revolutionized animal ecology by providing time-integrated estimates of the use of resources and/or habitats. SIA is based on the premise that the isotopic composition of a consumer's tissues originates from its food, but is offset by trophic-discrimination (enrichment) factors controlled by metabolic processes associated with the assimilation of nutrients and the biosynthesis of tissues. Laboratory preparation protocols dictate that tissues both of consumers and of their potential prey be lipid-extracted prior to analysis, because (1) lipids have carbon isotope (δ(13)C) values that are lower by approximately 3-8‰ than associated proteins and (2) amino acids in consumers' proteinaceous tissues are assumed to be completely routed from dietary protein. In contrast, models of stable-isotope mixing assume that dietary macromolecules are broken into their elemental constituents from which non-essential amino acids are resynthesized to build tissues. Here, we show that carbon from non-protein dietary macromolecules, namely lipids, was used to synthesize muscle tissue in an omnivorous rodent (Mus musculus). We traced the influence of dietary lipids on the synthesis of consumers' tissues by inversely varying the dietary proportion of C4-based lipids and C3-based protein while keeping carbohydrate content constant in four dietary treatments, and analyzing the δ(13)C values of amino acids in mouse muscle after 4 months of feeding. The influence of dietary lipids on non-essential amino acids varied as function of biosynthetic pathway. The source of carbon in ketogenic amino acids synthesized through the Krebs cycle was highly sensitive to dietary lipid content, with significant increases of approximately 2-4‰ in Glutamate and Aspartate δ(13)C values from the 5% to 15% dietary lipid treatment. Glucogenic amino acids (Glycine and Serine) were less sensitive to dietary lipid, but increased by approximately 3-4‰ from the 25% to 40% lipid diet. As expected, the δ(13)C values of essential amino acids did not vary significantly among diets. Although lipids provide a calorie-rich resource that fuels energy requirements, our results show that they also can be an important elemental source of carbon that contributes to the non-essential amino acids used to build structural tissue like muscle. As such, the calculation of trophic-discrimination factors for animals that consume a lipid-rich diet should consider lipid carbon as a building block for proteinaceous tissues. Careful consideration of the macromolecular composition in the diet of the consumer of interest will help to further refine the use of SIA to study animal ecology and physiology.

Microbial community composition and endolith colonization at an Arctic thermal spring are driven by calcite precipitation.

Environmental conditions shape community composition. Arctic thermal springs provide an opportunity to study how environmental gradients can impose strong selective pressures on microbial communities and provide a continuum of niche opportunities. We use microscopic and molecular methods to conduct a survey of microbial community composition at Troll Springs on Svalbard, Norway, in the high Arctic. Microorganisms there exist under a wide range of environmental conditions: in warm water as periphyton, in moist granular materials, and in cold, dry rock as endoliths. Troll Springs has two distinct ecosystems, aquatic and terrestrial, together in close proximity, with different underlying environmental factors shaping each microbial community. Periphyton are entrapped during precipitation of calcium carbonate from the spring's waters, providing microbial populations that serve as precursors for the development of endolithic communities. This process differs from most endolith colonization, in which the rock predates the communities that colonize it. Community composition is modulated as environmental conditions change within the springs. At Troll, the aquatic environments show a small number of dominant operational taxonomic units (OTUs) that are specific to each sample. The terrestrial environments show a more even distribution of OTUs common to multiple samples.