Continuous sterane and phytane δ13C record reveals a substantial pCO2 decline since the mid-Miocene.

Constraining the relationship between temperature and atmospheric concentrations of carbon dioxide (pCO2) is essential to model near-future climate. Here, we reconstruct pCO2 values over the past 15 million years (Myr), providing a series of analogues for possible near-future temperatures and pCO2, from a single continuous site (DSDP Site 467, California coast). We reconstruct pCO2 values using sterane and phytane, compounds that many phytoplankton produce and then become fossilised in sediment. From 15.0-0.3 Myr ago, our reconstructed pCO2 values steadily decline from 650 ± 150 to 280 ± 75 ppmv, mirroring global temperature decline. Using our new range of pCO2 values, we calculate average Earth system sensitivity and equilibrium climate sensitivity, resulting in 13.9 °C and 7.2 °C per doubling of pCO2, respectively. These values are significantly higher than IPCC global warming estimations, consistent or higher than some recent state-of-the-art climate models, and consistent with other proxy-based estimates.

Parasite effects on host's trophic and isotopic niches.

Wild animals are usually infected with parasites that can alter their hosts' trophic niches in food webs as can be seen from stable isotope analyses of infected versus uninfected individuals. The mechanisms influencing these effects of parasites on host isotopic values are not fully understood. Here, we develop a conceptual model to describe how the alteration of the resource intake or the internal resource use of hosts by parasites can lead to differences of trophic and isotopic niches of infected versus uninfected individuals and ultimately alter resource flows through food webs. We therefore highlight that stable isotope studies inferring trophic positions of wild organisms in food webs would benefit from routine identification of their infection status.

A stable isotope assay with 13C-labeled polyethylene to investigate plastic mineralization mediated by Rhodococcus ruber.

Methods that unambiguously prove microbial plastic degradation and allow for quantification of degradation rates are necessary to constrain the influence of microbial degradation on the marine plastic budget. We developed an assay based on stable isotope tracer techniques to determine microbial plastic mineralization rates in liquid medium on a lab scale. For the experiments, 13C-labeled polyethylene (13C-PE) particles (irradiated with UV-light to mimic exposure of floating plastic to sunlight) were incubated in liquid medium with Rhodococcus ruber as a model organism for proof of principle. The transfer of 13C from 13C-PE into the gaseous and dissolved CO2 pools translated to microbially mediated mineralization rates of up to 1.2 % yr-1 of the added PE. After incubation, we also found highly 13C-enriched membrane fatty acids of R. ruber including compounds involved in cellular stress responses. We demonstrated that isotope tracer techniques are a valuable tool to detect and quantify microbial plastic degradation.

Exploration speed in captivity predicts foraging tactics and diet in free-living red knots.

Variation in foraging tactics and diet is usually attributed to differences in morphology, experience and prey availability. Recently, consistent individual differences in behaviour (personality) have been shown to be associated with foraging strategies. Bolder or more exploratory individuals are predicted to have a faster pace-of-life and offset the costs of moving more or in risky areas, with higher energetic gains by encountering profitable foraging opportunities and prey. However, the relationship between personality, foraging and diet is poorly understood. We investigated how exploratory behaviour in red knots Calidris canutus is associated with foraging tactics and diet by combining laboratory experiments, field observations and stable isotope analysis. First, we developed a mobile experimental arena to measure exploration speed in controlled settings. We validated the method by repeated testing of individuals over time and contexts. This setup allowed us to measure exploratory personality at the field site, eliminating the need to bring birds into captivity for long periods of time. After releasing birds within days of their capture, we asked whether exploration speed was associated with differences in foraging tactics and diet in the wild. We found that tactile foraging red knots mainly caught hard-shelled prey that are buried in the sediment, whereas visual foraging knots only captured soft preys located close to or on the surface. We also found that faster explorers showed a higher percentage of visual foraging than slower explorers. By contrast, morphology (bill length and gizzard size) had no significant effect on foraging tactics. Diet analysis based on δ15 N and δ13 C stable isotope values of plasma and red blood cells confirmed our field observations with slower explorers mainly consumed hard-shelled prey while faster explorers consumed more soft than hard-shelled prey. Our results show that foraging tactics and diet are associated with a personality trait, independent of morphological differences. We discuss how consistent behaviour might develop early in life through positive feedbacks between foraging tactics, prey type and foraging efficiency.

Reconstructing the diet, trophic level and migration pattern of mysticete whales based on baleen isotopic composition.

Baleen from mysticete whales is a well-preserved proteinaceous material that can be used to identify migrations and feeding habits for species whose migration pathways are unknown. Analysis of δ13C and δ15N values from bulk baleen have been used to infer migration patterns for individuals. However, this approach has fallen short of identifying migrations between regions as it is difficult to determine variations in isotopic shifts without temporal sampling of prey items. Here, we apply analysis of δ15N values of amino acids to five baleen plates belonging to three species, revealing novel insights on trophic position, metabolic state and migration between regions. Humpback and minke whales had higher reconstructed trophic levels than fin whales (3.7-3.8 versus 3-3.2, respectively) as expected due to different feeding specialization. Isotopic niche areas between baleen minima and maxima were well separated, indicating regional resource use for individuals during migration that aligned with isotopic gradients in Atlantic Ocean particulate organic matter. Phenylanine δ15N values confirmed regional separation between the niche areas for two fin whales as migrations occurred and elevated glycine and threonine δ15N values suggested physiological changes due to fasting. Simultaneous resolution of trophic level and physiological changes allow for identification of regional migrations in mysticetes.

Combining isotopic analysis of bulk-skin and individual amino acids to investigate the trophic position and foraging areas of multiple cetacean species in the western South Atlantic.

We investigated the trophic structure and habitat use of ten cetacean species occurring in the oceanic waters of the western South Atlantic using naturally-occurring stable isotopes. We analysed δ15N in individual amino acids (AA) to estimate cetacean trophic position (TP) and to evaluate the spatial differences in baseline δ15N (source AAs). We adjusted cetacean bulk-skin δ13C and δ15N for the effect of trophic level using their estimated TPs, obtaining δ13CAdjusted and δ15NAdjusted, respectively. These values were applied to estimate the overlap in the niche areas of cetacean baseline sources. Our analyses showed spatial segregation between Steno bredanensis and the remaining odontocetes, and the high δ15N in this species reflects its occurrence in neritic waters of the southern region. The highest TPs were observed in Physeter macrocephalus, Stenella attenuata and Globicephala melas, while the lowest TPs were reported for S. longirostris, S. clymene and Orcinus orca. Overall, source AA-δ15N showed similar patterns as those of baseline-δ15N (zooplankton) and were higher in species sampled in the southernmost region of the study area (e.g., Delphinus delphis). Isotopic niche areas estimated using δ13CAdjusted and δ15NAdjusted suggested high overlap in foraging area between S. frontalis and Tursiops truncatus, with the latter occupying a higher TP. Our analyses of δ15N in AAs provide a unique insight into the trophic ecology, forage areas and spatial segregation in resource use among these cetacean populations. Additionally, our work provides AA-δ15N baseline for future studies on the trophic ecology and habitat use of marine organisms in the western South Atlantic.

Organic Matter Type Defines the Composition of Active Microbial Communities Originating From Anoxic Baltic Sea Sediments.

Carbon cycling in anoxic marine sediments is dependent on uncultured microbial communities. Niches of heterotrophic microorganisms are defined by organic matter (OM) type and the different phases in OM degradation. We investigated how OM type defines microbial communities originating from organic-rich, anoxic sediments from the Baltic Sea. We compared changes in the sediment microbial community, after incubation with different stable isotope labeled OM types [i.e., particulate algal organic matter (PAOM), protein, and acetate], by using DNA stable isotope probing (DNA-SIP). Incorporation of 13C and/or 15N label was predominantly detected in members of the phyla Planctomycetes and Chloroflexi, which also formed the majority (>50%) of the original sediment community. While these phylum-level lineages incorporated label from all OM types, phylogenetic analyses revealed a niche separation at the order level. Members of the MSBL9 (Planctomycetes), the Anaerolineales (Chloroflexi), and the class Bathyarchaeota, were identified as initial degraders of carbohydrate-rich OM, while other uncultured orders, like the CCM11a and Phycisphaerales (Planctomycetes), Dehalococcoidia, and JG30-KF-CM66 (Chloroflexi), incorporated label also from protein and acetate. Our study highlights the importance of initial fermentation of complex carbon pools in shaping anoxic sediment microbial communities and reveals niche specialization at the order level for the most important initial degraders in anoxic sediments.

Isotopic discrimination in helminths infecting coral reef fishes depends on parasite group, habitat within host, and host stable isotope value.

Stable isotopes of carbon and nitrogen characterize trophic relationships in predator-prey relationships, with clear differences between consumer and diet (discrimination factor Δ13C and Δ15N). However, parasite-host isotopic relationships remain unclear, with Δ13C and Δ15N remaining incompletely characterized, especially for helminths. In this study, we used stable isotopes to determine discrimination factors for 13 parasite-host pairings of helminths in coral reef fish. Differences in Δ15N values grouped according to parasite groups and habitat within the host with positive Δ15N values observed for trematodes and nematodes from the digestive tract and variable Δ15N values observed for cestodes and nematodes from the general cavity. Furthermore, Δ13C values showed more complex patterns with no effect of parasite group or habitat within host. A negative relationship was observed between Δ15N and host δ15N values among different host-parasite pairings as well as within 7 out of the 13 pairings, indicating that host metabolic processing affects host-parasite discrimination values. In contrast, no relationships were observed for Δ13C values. Our results indicate that parasite group, habitat within host, and host stable isotope value drive Δ15N of helminths in coral reef fish while their effect on Δ13C is more idiosyncratic. These results call for use of taxon- or species-specific and scaled framework for bulk stable isotopes in the trophic ecology of parasites.

A late Pleistocene dataset of Agulhas Current variability.

The interocean transfer of thermocline water between the Indian and the Atlantic Oceans known as 'Agulhas leakage' is of global significance as it influences the Atlantic Meridional Overturning Circulation (AMOC) on different time scales. Variability in the Agulhas Current regime is key in shaping hydroclimate on the adjacent coastal areas of the African continent today as well as during past climates. However, the lack of long, continuous records from the proximal Agulhas Current region dating beyond the last glacial cycle prevents elucidation of its role in regional and wider global climate changes. This is the first continuous record of hydrographic variability (SST; δ18Osw) from the Agulhas Current core region spanning the past 270,000 years. The data set is analytical sound and provides a solid age model. As such, it can be used by paleoclimate scientists, archaeologists, and climate modelers to evaluate, for example, linkages between the Agulhas Current system and AMOC dynamics, as well as connections between ocean heat transport and Southern African climate change in the past and its impact on human evolution.

Trophic position, elemental ratios and nitrogen transfer in a planktonic host-parasite-consumer food chain including a fungal parasite.

Parasitism is arguably the most commonly occurring consumer strategy. However, only a few food web studies assess how well stable isotopes reflect the trophic position of parasitic consumers and results are variable. Even fewer studies have measured the nutrient transfer by parasitic consumers, hindering an assessment of their role in nutrient transfer through food webs. Here we used a food chain consisting of a diatom as host, a chytrid as its parasitic consumer and a rotifer as the predatory consumer of the chytrid, to assess the trophic position of all three food-chain components using their natural 13C and 15N isotope signatures, and to measure the nitrogen transfer from the host via the chytrid to the rotifer by tracing 15N of a labelled host up the food chain. Additionally, we measured the carbon to nitrogen (C:N) ratios of all food-chain components. Natural isotope abundance results showed no clear 15N enrichment in the chytrid or rotifer relative to the primary producer. However, estimates of nitrogen transfer indicated that about 14% of host nitrogen was transferred per day from host to chytrid during infection epidemics and that some of this nitrogen was also transferred onward to the rotifer. Moreover, C:N ratios decreased with trophic level, suggesting that the chytrid provided a high-quality food source to the rotifer. In conclusion, our results support the "mycoloop". The mycooloop proposes that chytrid infections allow the transfer of nutrients bound in large, inedible phytoplankton to zooplankton through the production of edible transmission spores, thereby rerouting nutrients back into the food web.

Testing algal-based pCO2 proxies at a modern CO2 seep (Vulcano, Italy).

Understanding long-term trends in atmospheric concentrations of carbon dioxide (pCO2) has become increasingly relevant as modern concentrations surpass recent historic trends. One method for estimating past pCO2, the stable carbon isotopic fractionation associated with photosynthesis (Ɛp) has shown promise over the past several decades, in particular using species-specific biomarker lipids such as alkenones. Recently, the Ɛp of more general biomarker lipids, organic compounds derived from a multitude of species, have been applied to generate longer-spanning, more ubiquitous records than those of alkenones but the sensitivity of this proxy to changes in pCO2 has not been constrained in modern settings. Here, we test Ɛp using a variety of general biomarkers along a transect taken from a naturally occurring marine CO2 seep in Levante Bay of the Aeolian island of Vulcano in Italy. The studied general biomarkers, loliolide, cholesterol, and phytol, all show increasing depletion in 13C over the transect from the control site towards the seep, suggesting that CO2 exerts a strong control on isotopic fractionation in natural phytoplankton communities. The strongest shift in fractionation was seen in phytol, and pCO2 estimates derived from phytol confirm the utility of this biomarker as a proxy for pCO2 reconstruction.

Seasonal variation in the diet of estuarine bivalves.

Estuarine food webs are generally considered to be supported by marine pelagic and benthic primary producers and by the import of dead organic matter from the open sea. Although estuaries receive considerable amounts of freshwater phytoplankton and organic compounds from adjacent rivers, the potential contribution of these living and dead matter to estuarine food webs is often assumed to be negligible and, therefore, not examined. Based on stable isotope analyses, we report the importance of freshwater suspended particulate organic matter (FW-SPOM) for fuelling estuarine food webs in comparison to estuarine SPOM and microphytobenthos. This previously neglected food source contributed 50-60% (annual average) of food intake of suspension-feeding bivalves such as cockles (Cerastoderma edule), mussels (Mytilus edulis) and Pacific oysters (Magallana gigas) at the Balgzand tidal flats, an estuarine site in the western Wadden Sea (12-32 psu). For these species, this proportion was particularly high in autumn during strong run-off of SPOM-rich freshwater, whilst estuarine SPOM (20%-25%) and microphytobenthos (15%-30%) were relatively important in summer when the freshwater run-off was very low. These findings have implications for our understanding of the trophic interactions within coastal food webs and for freshwater management of estuarine ecosystems.

Response of the North Atlantic surface and intermediate ocean structure to climate warming of MIS 11.

Investigating past interglacial climates not only help to understand how the climate system operates in general, it also forms a vital basis for climate predictions. We reconstructed vertical stratification changes in temperature and salinity in the North Atlantic for a period some 400 ka ago (MIS11), an interglacial time analogue of a future climate. As inferred from a unique set of biogeochemical, geochemical, and faunal data, the internal upper ocean stratification across MIS 11 shows distinct depth-dependent dynamical changes related to vertical as well as lateral shifts in the upper Atlantic meridional circulation system. Importantly, transient cold events are recognized near the end of the long phase of postglacial warming at surface, subsurface, mid, and deeper water layers. These data demonstrate that MIS 11 coolings over the North Atlantic were initially triggered by freshwater input at the surface and expansion of cold polar waters into the Subpolar Gyre. The cooling signal was then transmitted downwards into mid-water depths. Since the cold events occurred after the main deglacial phase we suggest that their cause might be related to continuous melting of the Greenland ice sheet, a mechanism that might also be relevant for the present and upcoming climate.

Impact of metabolism and growth phase on the hydrogen isotopic composition of microbial fatty acids.

Microorganisms are involved in all elemental cycles and therefore it is important to study their metabolism in the natural environment. A recent technique to investigate this is the hydrogen isotopic composition of microbial fatty acids, i.e., heterotrophic microorganisms produce fatty acids enriched in deuterium (D) while photoautotrophic and chemoautotrophic microorganisms produce fatty acids depleted in D compared to the water in the culture medium (growth water). However, the impact of factors other than metabolism have not been investigated. Here, we evaluate the impact of growth phase compared to metabolism on the hydrogen isotopic composition of fatty acids of different environmentally relevant microorganisms with heterotrophic, photoautotrophic and chemoautotrophic metabolisms. Fatty acids produced by heterotrophs are enriched in D compared to growth water with εlipid/water between 82 and 359‰ when grown on glucose or acetate, respectively. Photoautotrophs (εlipid/water between -149 and -264‰) and chemoautotrophs (εlipid/water between -217 and -275‰) produce fatty acids depleted in D. Fatty acids become, in general, enriched by between 4 and 46‰ with growth phase which is minor compared to the influence of metabolisms. Therefore, the D/H ratio of fatty acids is a promising tool to investigate community metabolisms in nature.

Comparison of the effect of salinity on the D/H ratio of fatty acids of heterotrophic and photoautotrophic microorganisms.

The core metabolism of microorganisms has a major influence on the hydrogen isotopic composition of their fatty acids. Heterotrophic microorganisms produce fatty acids with a deuterium to hydrogen (D/H) ratio either slightly depleted or enriched in D compared to the growth water, while photo- and chemoautotrophic microorganisms produce fatty acids which are heavily depleted in D. However, besides metabolism other biochemical and environmental factors (i.e. biosynthetic pathways, growth phase and temperature) have been shown to affect the D/H ratio of fatty acids, and it is necessary to evaluate the magnitude of these effects compared to that of metabolism. Here, we show that the effect of salinity on the D/H ratio of fatty acids depends on the core metabolism of the microorganism. While fatty acids of the photoautotroph Isochrysis galbana become more enriched in D with increasing salinity (enrichment of 30-40‰ over a range of 25 salinity units), no effect of salinity on the D/H ratio of fatty acids of the heterotrophic Pseudomonas str. LFY10 was observed ((ε)lipid/water of the C16:0 fatty acid of ~120‰ over a range of 10 salinity units). This can likely be explained by the relative contributions of different H and nicotinamide adenine dinucleotide phosphate sources during fatty acid biosynthesis.

Critical assessment of glyco- and phospholipid separation by using silica chromatography.

Phospholipid-derived fatty acids (PLFAs) are commonly used to characterize microbial communities in situ and the phylogenetic positions of newly isolated microorganisms. PLFAs are obtained through separation of phospholipids from glycolipids and neutral lipids using silica column chromatography. We evaluated the performance of this separation method for the first time using direct detection of intact polar lipids (IPLs) with high-performance liquid chromatography-mass spectrometry (HPLC-MS). We show that under either standard or modified conditions, the phospholipid fraction contains not only phospholipids but also other lipid classes such as glycolipids, betaine lipids, and sulfoquinovosyldiacylglycerols. Thus, commonly reported PLFA compositions likely are not derived purely from phospholipids and perhaps may not be representative of fatty acids present in living microbes.

Thermodesulfobacterium geofontis sp. nov., a hyperthermophilic, sulfate-reducing bacterium isolated from Obsidian Pool, Yellowstone National Park.

A novel sulfate-reducing bacterium designated OPF15(T) was isolated from Obsidian Pool, Yellowstone National Park, Wyoming. The phylogeny of 16S rRNA and functional genes (dsrAB) placed the organism within the family Thermodesulfobacteriaceae. The organism displayed hyperthermophilic temperature requirements for growth with a range of 70-90 °C and an optimum of 83 °C. Optimal pH was around 6.5-7.0 and the organism required the presence of H2 or formate as an electron donor and CO2 as a carbon source. Electron acceptors supporting growth included sulfate, thiosulfate, and elemental sulfur. Lactate, acetate, pyruvate, benzoate, oleic acid, and ethanol did not serve as electron donors. Membrane lipid analysis revealed diacyl glycerols and acyl/ether glycerols which ranged from C14:0 to C20:0. Alkyl chains present in acyl/ether and diether glycerol lipids ranged from C16:0 to C18:0. Straight, iso- and anteiso-configurations were found for all lipid types. The presence of OPF15(T) was also shown to increase cellulose consumption during co-cultivation with Caldicellulosiruptor obsidiansis, a fermentative, cellulolytic extreme thermophile isolated from the same environment. On the basis of phylogenetic, phenotypic, and structural analyses, Thermodesulfobacterium geofontis sp. nov. is proposed as a new species with OPF15(T) representing the type strain.

Cultivation and genomic, nutritional, and lipid biomarker characterization of Roseiflexus strains closely related to predominant in situ populations inhabiting Yellowstone hot spring microbial mats.

Roseiflexus sp. strains were cultivated from a microbial mat of an alkaline siliceous hot spring in Yellowstone National Park. These strains are closely related to predominant filamentous anoxygenic phototrophs found in the mat, as judged by the similarity of small-subunit rRNA, lipid distributions, and genomic and metagenomic sequences. Like a Japanese isolate, R. castenholzii, the Yellowstone isolates contain bacteriochlorophyll a, but not bacteriochlorophyll c or chlorosomes, and grow photoheterotrophically or chemoheterotrophically under dark aerobic conditions. The genome of one isolate, Roseiflexus sp. strain RS1, contains genes necessary to support these metabolisms. This genome also contains genes encoding the 3-hydroxypropionate pathway for CO(2) fixation and a hydrogenase, which might enable photoautotrophic metabolism, even though neither isolate could be grown photoautotrophically with H(2) or H(2)S as a possible electron donor. The isolates exhibit temperature, pH, and sulfide preferences typical of their habitat. Lipids produced by these isolates matched much better with mat lipids than do lipids produced by R. castenholzii or Chloroflexus isolates.

Archaeal and bacterial glycerol dialkyl glycerol tetraether lipids in hot springs of yellowstone national park.

Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids originally thought to be produced mainly by (hyper)thermophilic archaea. Environmental screening of low-temperature environments showed, however, the abundant presence of structurally diverse GDGTs from both bacterial and archaeal sources. In this study, we examined the occurrences and distribution of GDGTs in hot spring environments in Yellowstone National Park with high temperatures (47 to 83 degrees C) and mostly neutral to alkaline pHs. GDGTs with 0 to 4 cyclopentane moieties were dominant in all samples and are likely derived from both (hyper)thermophilic Crenarchaeota and Euryarchaeota. GDGTs with 4 to 8 cyclopentane moieties, likely derived from the crenarchaeotal order Sulfolobales and the euryarchaeotal order Thermoplasmatales, are usually present in much lower abundance, consistent with the relatively high pH values of the hot springs. The relative abundances of cyclopentane-containing GDGTs did not correlate with in situ temperature and pH, suggesting that other environmental and possibly genetic factors play a role as well. Crenarchaeol, a biomarker thought to be specific for nonthermophilic group I Crenarchaeota, was also found in most hot springs, though in relatively low concentrations, i.e., <5% of total GDGTs. Its abundance did not correlate with temperature, as has been reported previously. Instead, the cooccurrence of relatively abundant nonisoprenoid GDGTs thought to be derived from soil bacteria suggests a predominantly allochthonous source for crenarchaeol in these hot spring environments. Finally, the distribution of bacterial branched GDGTs suggests that they may be derived from the geothermally heated soils surrounding the hot springs.