Signature Lipids and Stable Carbon Isotope Analyses of Octopus Spring Hyperthermophilic Communities Compared with Those of Aquificales Representatives

The molecular and isotopic compositions of lipid biomarkers of cultured Aquificales genera have been used to study the community and trophic structure of the hyperthermophilic pink streamers and vent biofilm from Octopus Spring. Thermocrinis ruber, Thermocrinis sp. strain HI 11/12, Hydrogenobacter thermophilus TK-6, Aquifex pyrophilus, and Aquifex aeolicus all contained glycerol-ether phospholipids as well as acyl glycerides. The n-C_20:1 and cy-C₂₁ fatty acids dominated all of the Aquificales, while the alkyl glycerol ethers were mainly C_18:0. These Aquificales biomarkers were major constituents of the lipid extracts of two Octopus Spring samples, a biofilm associated with the siliceous vent walls, and the well-known pink streamer community (PSC). Both the biofilm and the PSC contained mono- and dialkyl glycerol ethers in which C₁₈ and C₂₀ alkyl groups were prevalent. Phospholipid fatty acids included both the Aquificales n-C_20:1 and cy-C₂₁, plus a series of iso-branched fatty acids (i-C_15:0 to i-C_21:0), indicating an additional bacterial component. Biomass and lipids from the PSC were depleted in ¹³C relative to source water CO₂ by 10.9 and 17.2‰, respectively. The C_20–21 fatty acids of the PSC were less depleted than the iso-branched fatty acids, 18.4 and 22.6‰, respectively. The biomass of T. ruber grown on CO₂ was depleted in ¹³C by only 3.3‰ relative to C source. In contrast, biomass was depleted by 19.7‰ when formate was the C source. Independent of carbon source, T. ruber lipids were heavier than biomass (+1.3‰). The depletion in the C_20–21 fatty acids from the PSC indicates that Thermocrinis biomass must be similarly depleted and too light to be explained by growth on CO₂. Accordingly, Thermocrinis in the PSC is likely to have utilized formate, presumably generated in the spring source region.     

Linking aboveground and belowground food webs through carbon and nitrogen stable isotope analyses

Carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) have been used for more than two decades in analyses of food web structure. The utility of isotope ratio measurements is based on the observation that consumer δ¹³C values are similar (<1‰ difference) to those of their diet, while consumer δ¹⁵N values are about 3‰ higher than those of their diet. The technique has been applied most often to aquatic and aboveground terrestrial food webs. However, few isotope studies have examined terrestrial food web structure that includes both above- and belowground (detrital) components. Here, we review factors that may influence isotopic signatures of terrestrial consumers in above- and belowground systems. In particular, we emphasize variations in δ¹³C and δ¹⁵N in belowground systems, e.g., enrichment of ¹³C and ¹⁵N in soil organic matter (likely related to soil microbial metabolism). These enrichments should be associated with the high ¹³C (~3‰) enrichment in belowground consumers relative to litter and soil organic matter and with the large variation in δ¹⁵N (~6‰) of the consumers. Because such enrichment and variation are much greater than the trophic enrichment generally used to estimate consumer trophic positions, and because many general predators are considered dependent on energy and material flows from belowground, the isotopic variation in belowground systems should be taken into account in δ¹³C and δ¹⁵N analyses of terrestrial food webs. Meanwhile, by measuring the δ¹³C of key predators, the linkage between above- and belowground systems could be estimated based on observed differences in δ¹³C of primary producers, detritivores and predators. Furthermore, radiocarbon (¹⁴C) measurements will allow the direct estimation of the dependence of predators on the belowground systems.     

Phylogenetic analysis of the hyperthermophilic pink filament community in Octopus Spring, Yellowstone National Park.

The phylogenetic diversity of a well-known pink filament community associated with the 84 to 88 degrees C outflow from Octopus Spring, Yellowstone National Park, was examined. Three phylogenetic types ("phylotypes"), designated EM 3, EM 17, and EM 19, were identified by cloning and sequencing the small subunit rRNA genes (16S rDNA) obtained by PCR amplification of mixed-population DNA. All three phylotypes diverge deeply within the phylogenetic domain Bacteria sensu Woese (C. R. Woese, O. Kandler, and M. L. Wheelis, Proc. Natl. Acad. Sci. USA 87:4576-4579, 1990). No members of the Archaea or Eucarya were detected. EM 3 comprises a unique lineage within the Thermotogales group, and EM 17 and EM 19 are affiliated with the Aquificales. A total of 35 clones were examined, of which the majority (26 clones) were of a single sequence type (EM 17) closely related to Aquifex pyrophilus. In situ hybridization with clone-specific probes attributes the majority sequence, EM 17, to the pink filaments.     

Getting to the fat of the matter: models,methods and assumptions for dealing with lipids in stable isotope analyses

Within an organism, lipids are depleted in ¹³C relative to proteins and carbohydrates (more negative δ¹³C), and variation in lipid content among organisms or among tissue types has the potential to introduce considerable bias into stable isotope analyses that use δ¹³C. Despite the potential for introduced error, there is no consensus on the need to account for lipids in stable isotope analyses. Here we address two questions: (1) If and when is it important to account for the effects of variation in lipid content on δ¹³C? (2) If it is important, which method(s) are reliable and robust for dealing with lipid variation? We evaluated the reliability of direct chemical extraction, which physically removes lipids from samples, and mathematical normalization, which uses the carbon-to-nitrogen (C:N) ratio of a sample to normalize δ¹³C after analysis by measuring the lipid content, the C:N ratio, and the effect of lipid content on δ¹³C (Δδ¹³C) of plants and animals with a wide range of lipid contents. For animals, we found strong relationships between C:N and lipid content, between lipid content and Δδ¹³C, and between C:N and Δδ¹³C. For plants, C:N was not a good predictor of lipid content or Δδ¹³C, but we found a strong relationship between carbon content and lipid content, lipid content and Δδ¹³C, and between and carbon content and Δδ¹³C. Our results indicate that lipid extraction or normalization is most important when lipid content is variable among consumers of interest or between consumers and end members, and when differences in δ¹³C between end members is <10–12‰. The vast majority of studies using natural variation in δ¹³C fall within these criteria. Both direct lipid extraction and mathematical normalization reduce biases in δ¹³C, but mathematical normalization simplifies sample preparation and better preserves the integrity of samples for δ¹⁵N analysis.     

Lipid corrections in carbon and nitrogen stable isotope analyses: comparison of chemical extraction and modelling methods

1. Lipids have more negative delta(13)C values relative to other major biochemical compounds in plant and animal tissues. Although variable lipid content in biological tissues alters results and conclusions of delta(13)C analyses in aquatic food web and migration studies, no standard correction protocol exists. 2. We compared chemical extraction and mathematical correction methods for freshwater and marine fishes and aquatic invertebrates to better understand impacts of correction approaches on carbon (delta(13)C) and nitrogen (delta(15)N) stable isotope data. 3. Fish and aquatic invertebrate tissue delta(13)C values increased significantly following extraction for almost all species and tissue types relative to nonextracted samples. In contrast, delta(15)N was affected for muscle and whole body samples from only a few freshwater and marine species and had a limited effect for the entire data set. 4. Lipid normalization models, using C : N as a proxy for lipid content, predicted lipid-corrected delta(13)C for paired data sets more closely with parameters specific to the tissue type and species to which they were applied. 5. We present species- and tissue-specific models based on bulk C : N as a reliable alternative to chemical extraction corrections. By analysing a subset of samples before and after lipid extraction, models can be applied to the species and tissues of interest that will improve estimates of dietary sources using stable isotopes.     

Diets of crustacean zooplankton,inferred from stable carbon and nitrogen isotope analyses,in lakes with varying allochthonous dissolved organic carbon content

We used stable carbon and nitrogen isotope analyses to estimate the relative proportions of three putative food sources (1) algae, (2) allochthonous organic matter (but including also heterotrophic bacteria and green-sulphur bacteria having similar isotopic values) and (3) methane-oxidizing bacteria (MOB) in the diets of crustacean zooplankton in five small boreal lakes representing a gradient of dissolved organic carbon (DOC) concentration from ca. 5 to 40 mg C l⁻¹. The lakes were sampled in May, after establishment of stratification, and again in October during autumnal mixing of the water column. IsoSource mixing model outputs indicated that the proportion of algae in the diets of zooplankton was generally higher in May than in October, and that bacteria contributed to the diets of both cladocerans and copepods. Our results indicate that bacteria, especially MOB, can make an appreciable contribution to zooplankton diets in these small lakes, even in those with relatively low DOC concentrations.     

木荷稳定碳同位素分辨率的种源差异

利用设置在福建建瓯、浙江淳安和浙江庆元3个区试点的5年生木荷种源试验林,选取18个代表性种源测定叶片的稳定碳同位素分辨率(Δ值),研究其在种源间的差异和地理变异模式,以及造林立地环境和种源生长对其的影响.结果表明：木荷种源叶片稳定碳同位素分辨率存在很大差异,福建建瓯、浙江淳安和庆元3个区试点的叶片Δ值最高和最低种源分别相差6.9％、3.0％和3.7％.种源叶片Δ值与其产地纬度呈显著负相关,而与产地经度的相关性较小,表现为典型的纬向渐变模式,来自木荷分布区南部的种源叶片Δ值较大,说明其长期水分利用效率较低.随着造林立地环境的改善和年降雨量的增多,木荷叶片Δ值显著增加.种源叶片Δ值与其树高、胸径、一级侧枝总数和最大侧枝长等皆呈显著正相关,树冠浓密的速生种源具有较高的Δ值.此外, 在木荷中心产区(福建建瓯)和边缘产区(浙江淳安)分别初选出2个和4个水分利用效率高的速生优良种源,供推广应用.     

稳定同位素分析在鸟类食性及营养级结构中的应用

稳定同位素分析(stable isotope analysis,SIA)自20世纪70年代末引入鸟类生态学领域以来,在研究鸟类食性和营养级结构方面展现了强大的发展潜力和广泛的应用前景。总结了该技术在鸟类食源组成和营养级结构方面研究的前期准备、实验流程、数据分析和研究进展等,重点阐述了其在鸟类保护与管理方面的应用。鉴于稳定同位素在生物体内存在分馏现象,从鸟类不同组织和部位对营养级富集因子、转化周期、同位素印记的差异,提出了完善该方法的建议和应用前景。     

Trophic structure of a coastal fish community determined with diet and stable isotope analyses

A combination of dietary guild analysis and nitrogen (δ¹⁵N) and carbon (δ¹³C) stable‐isotope analysis was used to assess the trophic structure of the fish community in Rhode Island and Block Island Sounds, an area off southern New England identified for offshore wind energy development. In the autumn of 2009, 2010 and 2011, stomach and tissue samples were taken from 20 fish and invertebrate species for analysis of diet composition and δ¹⁵N and δ¹³C signatures. The food chain in Rhode Island and Block Island Sounds comprises approximately four trophic levels within which the fish community is divided into distinct dietary guilds, including planktivores, benthivores, crustacivores and piscivores. Within these guilds, inter‐species isotopic and dietary overlap is high, suggesting that resource partitioning or competitive interactions play a major role in structuring the fish community. Carbon isotopes indicate that most fishes are supported by pelagic phytoplankton, although there is evidence that benthic production also plays a role, particularly for obligate benthivores such as skates Leucoraja spp. This type of analysis is useful for developing an ecosystem‐based approach to management, as it identifies species that act as direct links to basal resources as well as species groups that share trophic roles.     

沙棘属植物叶片碳稳定同位素含量与气候的关系

本试验以131个沙棘属植物种群为研究对象,通过测定其叶片碳稳定同位素(δ13C)值,分析了碳稳定同位素特征与环境因子之间的关系.结果 表明:沙棘属植物叶片的δ13C值介于-24.65‰～-29.11‰‰,平均值为-26.97‰,属于C3植物,叶片δ13C值变异系数为种内大于种间,表明环境因子是影响沙棘属植物叶片δ13C...     

Tracking activity and function of microorganisms by stable isotope probing of membrane lipids

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稳定碳同位素技术在土壤-植物系统碳循环中的应用

碳作为重要的生命元素,在土壤 植物系统物质循环中发挥重要作用.作为一种天然的示踪物,稳定碳同位素（¹³C）较放射性同位素具有安全、无污染、易控制的优点,在土壤 植物生态系统碳循环研究中得到广泛应用.通过检测土壤 植物体系中稳定碳同位素的自然丰度或采用稳定碳同位素标记有机材料,能够较真实地了解植物的光合特性、光合产物在土壤 植物体系中的运转及其在土壤中的分解、转化等过程.本文概述了稳定碳同位素技术在植物光合作用及光合产物运转、古气候重建、土壤有机质周转以及植物 根际微生物相互作用等方面的研究进展,并针对当前研究中存在的问题提出了今后的研究展望.     

Conservation of biotrophy in Hygrophoraceae inferred from combined stable isotope and phylogenetic analyses

The nutritional modes of genera in Hygrophoraceae (Basidiomycota: Agaricales), apart from the ectomycorrhizal Hygrophorus and lichen-forming taxa, are uncertain. New δ(15)N and δ(13)C values were obtained from 15 taxa under Hygrophoraceae collected in central Massachusetts and combined with isotopic datasets from five prior studies including a further 12 species using a data standardization method to allow cross-site comparison. Based on these data, we inferred the probable nutritional modes for species of Hygrophorus, Hygrocybe, Humidicutis, Cuphophyllus and Gliophorus. A phylogeny of Hygrophoraceae was constructed by maximum likelihood analysis of nuclear ribosomal 28S and 5.8S sequences and standardized δ(15)N and δ(13)C values were used for parsimony optimization on this phylogeny. Our results supported a mode of biotrophy in Hygrocybe, Humidicutis, Cuphophyllus and Gliophorus quantitatively unlike that in more than 450 other fungal taxa sampled in the present and prior studies. Parsimony optimization of stable isotope data suggests moderate conservation of nutritional strategies in Hygrophoraceae and a single switch to a predominantly ectomycorrhizal life strategy in the lineage leading to Hygrophorus. We conclude that Hygrophoraceae of previously unknown nutritional status are unlikely to be saprotrophs and are probably in symbiosis with bryophytes or other understory plants.     

Comparing pellet and stable isotope analyses of nestling Bonelli's Eagle Aquila fasciata diet

Diet analyses are central to the study of avian trophic ecology, and stable isotope analyses have made an increasing contribution in the last two decades. Few isotopic studies have assessed the diet of raptor species, which are more frequently analysed by conventional diet methods such as pellet analysis. In this study, we compare prey consumption estimates of nestling Bonelli's Eagles Aquila fasciata from conventional pellet analysis (in terms of items and biomass) and stable isotopic mixing models (SIAR) using δ¹³C, δ¹⁵N and δ³⁴S of feathers. The pellet analysis showed that European Rabbits Oryctolagus cuniculus, pigeons (mainly Common Wood Pigeons Columba palumbus and Domestic Pigeons Columba livia dom.), Red‐legged Partridges Alectoris rufa, passerines, Yellow‐legged Gulls Larus michahellis and Eurasian Red Squirrels Sciurus vulgaris were the main prey, so they were selected for diet reconstructions in SIAR. At the population level, mean prey consumption estimates were similar for pellets (both items and biomass) and SIAR. At the territory level, the weighted kappa statistic showed good ordinal scale agreement in main prey consumption between items or biomass and SIAR. Although the intraclass correlation coefficient showed poor method agreement when considering all prey in the same analysis, the intraclass correlation coefficients for each prey category showed significant agreement between pellets and SIAR when estimating the consumption of Rabbits, pigeons and Gulls, with lower agreement for passerines and Squirrels. Lastly, there was poor method agreement for estimates of Partridges. Our results suggest an overall agreement between the pellet analysis and SIAR when estimating nestling Bonelli's Eagle diet at both the population and, to a lesser extent, the territory level, supporting the usefulness of isotopic mixing models when identifying the terrestrial and marine components of raptor diets.     

Effects of preservation method on stable carbon and nitrogen isotope values

Some methods of tissue preservation have significant effects on values of stable isotopes of carbon (delta(13)C) and nitrogen (delta(15)N), but studies on this topic are scattered in the literature. The goals of this study were to (1) summarize the results from studies of preservation effects in the literature and (2) test the effects of four common preservatives on delta(13)C and delta(15)N in epidermis tissue of three turtle species. Turtle tissue samples were subjected to up to five time intervals in five methods of preservation: drying at 60 degrees C for 24 h (the control), immersion in a 70% ethanol solution, immersion in a saturated NaCl aqueous solution, freezing at -10 degrees C in a frost-free freezer, and immersion in a dimethyl sulfoxide (DMSO)-ethylenediaminetetraacetic acid buffer. The delta(13)C and delta(15)N values for tissues preserved in 70% ethanol and NaCl aqueous solution were not significantly different from those of tissues dried at 60 degrees C, but samples preserved in DMSO were significantly different from dried samples. Freezing preservation had a significant effect on delta(13)C and delta(15)N at 60 d, which may have resulted from the use of a frost-free freezer. The effects of 20 different preservative methods on delta(13)C and delta(15)N in different tissues are summarized.     

Weaning age among foragers at Matjes river rock shelter, South Africa, from stable nitrogen and carbon isotope analyses

Matjes River Rock Shelter is a large shell midden on the southern coast of South Africa. Stable nitrogen (delta(15)N) and carbon (delta(13)C) isotope ratios were measured in bone collagen and dentine from human skeletons excavated from this site in order to establish a weaning curve in mid-Holocene hunter-gatherers. delta(15)N results show a progressive increase in individuals from birth to 1.5 years old. delta(13)C results are more tightly clustered and mirror the steady progressive change seen for delta(15)N. We deduce that children at Matjes River Rock Shelter were breastfed for at least the first 1.5 years after birth, and were weaned sometime between 2-4 years of age. A similar pattern was documented for historic-era Kalahari foraging people, where the interbirth spacing was approximately 3 years. This study provides the first direct evidence for an extended period of breastfeeding, and thus long interbirth intervals, among prehistoric foragers, even when those foragers lived in an environment with abundant food resources.     

Tracing prey origins,proportions and feeding periods for predatory beetles from agricultural systems using carbon and nitrogen stable isotope analyses

Predatory beetles are an important component of the natural enemy complex that preys on insect pests such as aphids within agroecosystems. Tracing diet origins and movement of natural enemies aids understanding their role in the food web and informs strategies for their effective conservation. Field sampling and laboratory experiments were carried out to examine the changes of carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) among crops (cotton and maize), pests (cotton and maize aphids), and between wing and abdomen of predatory beetles, Propylea japonica, and to test the hypothesis that prey origins, proportions and feeding periods of the predatory beetles can be deduced by this stable isotope analysis. Results showed that the δ¹³C values both in wing and abdomen of adult P. japonica were changing from a C₃- to a C₄-based diet of aphids reared on maize or cotton, respectively; the isotope ratio of their new C₄ substrates were detectable within 7 days and the δ¹⁵N values began to reflect their new C₄ substrates within 3 days. The relationship between δ¹³C and δ¹⁵N values of P. japonica adults in wing or abdomen and diets of aphids from a C₃-based resource transitioning to a C₄-based resource were described best in linear or quadratic equations. Results suggest that integrative analysis of δ¹³C and δ¹⁵N values can be regarded as a useful method for quantifying to trace prey origins, proportions of diets and feeding periods of natural enemies. The results can provide quantifying techniques for habitat management of natural enemies.     

Inverse carbon isotope patterns of lipids and kerogen record heterogeneous primary biomass

Throughout the Proterozoic δ(13)C values for preserved n-alkyl lipids are more positive than for syngenetic kerogen. This pattern is the inverse of biosynthetic expectations. It has been suggested that this isotopic inversion results from selective preservation of lipids from (13)C-enriched heterotrophic populations, while the bulk of kerogen derives from primary producers. Here, we formulate a degradation model to calculate the (13)C content of sedimentary total organic carbon and lipid. The model addresses two scenarios. The first scenario explores preferential preservation of heterotrophic lipid, thereby quantifying the existing hypothesis. In the second, we suggest that an inverse signature could be the result of prokaryotic phytoplankton contributing the majority of the total ecosystem biomass. Photosynthetic prokaryotes bearing a relative (13)C enrichment would contribute much of the resulting preserved lipids, while primary eukaryotic biomass would dominate the total organic carbon. We find that our hypothesis of a mixed primary producer community generates inverse isotopic patterns while placing far fewer requirements on specific degradation conditions. It also provides a possible explanation as to why there are large variations in the (13)C content of the isoprenoid lipids pristane and phytane relative to n-alkyl lipid, while the difference between n-alkyl lipid and kerogen is more constant. Our results suggest that the disappearance of the inverse (13)C signature in the late Ediacaran is a natural consequence of the fundamental shift to oceans in which export production has a higher ratio of eukaryotic biomass.