Metabolic analysis as a driver for discovery, diagnosis, and therapy.

Metabolic anomalies contribute to tissue dysfunction. Current metabolism research spans from organelles to populations, and new technologies can accommodate investigation across these scales. Here, we review recent advancements in metabolic analysis, including small-scale metabolomics techniques amenable to organelles and rare cell types, functional screening to explore how cells respond to metabolic stress, and imaging approaches to non-invasively assess metabolic perturbations in diseases. We discuss how metabolomics provides an informative phenotypic dimension that complements genomic analysis in Mendelian and non-Mendelian disorders. We also outline pressing challenges and how addressing them may further clarify the biochemical basis of human disease.

Determining and interpreting protein lifetimes in mammalian tissues.

The orchestration of protein production and degradation, and the regulation of protein lifetimes, play a central role in the majority of biological processes. Recent advances in proteomics have enabled the estimation of protein half-lives for thousands of proteins in vivo. What is the utility of these measurements, and how can they be leveraged to interpret the proteome changes occurring during development, aging, and disease? This opinion article summarizes leading technical approaches and highlights their strengths and weaknesses. We also disambiguate frequently used terminology, illustrate recent mechanistic insights, and provide guidance for interpreting and validating protein turnover measurements. Overall, protein lifetimes, coupled to estimates of protein levels, are essential for obtaining a deep understanding of mammalian biology and the basic processes defining life itself.

Elucidating the sustainability of 700 y of Inuvialuit beluga whale hunting in the Mackenzie River Delta, Northwest Territories, Canada.

Beluga whales play a critical role in the subsistence economies and cultural heritage of Indigenous communities across the Arctic, yet the effects of Indigenous hunting on beluga whales remain unknown. Here, we integrate paleogenomics, genetic simulations, and stable δ13C and δ15N isotope analysis to investigate 700 y of beluga subsistence hunting in the Mackenzie Delta area of northwestern Canada. Genetic identification of the zooarchaeological remains, which is based on radiocarbon dating, span three time periods (1290 to 1440 CE; 1450 to 1650 CE; 1800 to 1870 CE), indicates shifts across time in the sex ratio of the harvested belugas. The equal number of females and males harvested in 1450 to 1650 CE versus more males harvested in the two other time periods may reflect changes in hunting practices or temporal shifts in beluga availability. We find temporal shifts and sex-based differences in δ13C of the harvested belugas across time, suggesting historical adaptability in the foraging ecology of the whales. We uncovered distinct mitochondrial diversity unique to the Mackenzie Delta belugas, but found no changes in nuclear genomic diversity nor any substructuring across time. Our findings indicate the genomic stability and continuity of the Mackenzie Delta beluga population across the 700 y surveyed, indicating the impact of Inuvialuit subsistence harvests on the genetic diversity of contemporary beluga individuals has been negligible.

Isotope and morphometrical evidence reveals the technological package associated with agriculture adoption in western Europe.

This study aimed to reconstruct the environmental conditions and the crop management practices and plant characteristics when agriculture appeared in western Europe. We analyzed oak charcoal and a large number of cereal caryopsides recovered from La Draga (Girona, Spain), an early (5300 to 4800 cal. BC) agricultural site from the Iberian Peninsula. The carbon isotope discrimination (Δ13C) values of oak, the dominant forest species in the region, indicates prevalence of a wet climate at the site. Further, we reconstructed crop management conditions, achievable yield, and crop characteristics through the analysis of Δ13C, nitrogen isotope composition (δ15N), nitrogen content, and the reconstructed weight of wheat and barley caryopsides, following protocols developed by our team [Araus et al., Nat. Commun. 5, 3953 (2014)] and comparison of these parameters with present-day organic agriculture in the region. In parallel, a regional perspective was achieved through the study of wheat and barley grains of seventeen Neolithic sites from the western Mediterranean. The results suggest that rather than small-garden cultivation, a more extensive agriculture was practiced under good water availability and moderate manuring. Moreover, results from La Draga evidence that grain weight and spike morphology were comparable to contemporary cereals. Growing conditions and the prevalence of improved crop traits indicate that agriculture was fairly consolidated at the time it reached the western edge of Europe.

Isotope effects on radical pair performance in cryptochrome: A new hypothesis for the evolution of animal migration: The quantum biology of migration.

Mechanisms occurring at the atomic level are now known to drive processes essential for life, as revealed by quantum effects on biochemical reactions. Some macroscopic characteristics of organisms may thus show an atomic imprint, which may be transferred across organisms and affect their evolution. This possibility is considered here for the first time, with the aim of elucidating the appearance of an animal innovation with an unclear evolutionary origin: migratory behaviour. This trait may be mediated by a radical pair (RP) mechanism in the retinal flavoprotein cryptochrome, providing essential magnetic orientation for migration. Isotopes may affect the performance of quantum processes through their nuclear spin. Here, we consider a simple model and then apply the standard open quantum system approach to the spin dynamics of cryptochrome RP. We changed the spin quantum number (I) and g-factor of hydrogen and nitrogen isotopes to investigate their effect on RP's yield and magnetic sensitivity. Strong differences arose between isotopes with I = 1 and I = 1/2 in their contribution to cryptochrome magnetic sensitivity, particularly regarding Earth's magnetic field strengths (25-65 µT). In most cases, isotopic substitution improved RP's magnetic sensitivity. Migratory behaviour may thus have been favoured in animals with certain isotopic compositions of cryptochrome.

Mobility and kinship in the world's first village societies.

Around 10,000 y ago in southwest Asia, the cessation of a mobile lifestyle and the emergence of the first village communities during the Neolithic marked a fundamental change in human history. The first communities were small (tens to hundreds of individuals) but remained semisedentary. So-called megasites appeared soon after, occupied by thousands of more sedentary inhabitants. Accompanying this shift, the material culture and ancient ecological data indicate profound changes in economic and social behavior. A shift from residential to logistical mobility and increasing population size are clear and can be explained by either changes in fertility and/or aggregation of local groups. However, as sedentism increased, small early communities likely risked inbreeding without maintaining or establishing exogamous relationships typical of hunter-gatherers. Megasites, where large populations would have made endogamy sustainable, could have avoided this risk. To examine the role of kinship practices in the rise of megasites, we measured strontium and oxygen isotopes in tooth enamel from 99 individuals buried at Pinarbasi, Boncuklu, and Çatalhöyük (Turkey) over 7,000 y. These sites are geographically proximate and, critically, span both early sedentary behaviors (Pinarbasi and Boncuklu) and the rise of a local megasite (Çatalhöyük). Our data are consistent with the presence of only local individuals at Pinarbasi and Boncuklu, whereas at Çatalhöyük, several nonlocals are present. The Çatalhöyük data stand in contrast to other megasites where bioarchaeological evidence has pointed to strict endogamy. These different kinship behaviors suggest that megasites may have arisen by employing unique, community-specific kinship practices.

FAMetA: a mass isotopologue-based tool for the comprehensive analysis of fatty acid metabolism.

The use of stable isotope tracers and mass spectrometry (MS) is the gold standard method for the analysis of fatty acid (FA) metabolism. Yet, current state-of-the-art tools provide limited and difficult-to-interpret information about FA biosynthetic routes. Here we present FAMetA, an R package and a web-based application (www.fameta.es) that uses 13C mass isotopologue profiles to estimate FA import, de novo lipogenesis, elongation and desaturation in a user-friendly platform. The FAMetA workflow covers the required functionalities needed for MS data analyses. To illustrate its utility, different in vitro and in vivo experimental settings are used in which FA metabolism is modified. Thanks to the comprehensive characterization of FA biosynthesis and the easy-to-interpret graphical representations compared to previous tools, FAMetA discloses unnoticed insights into how cells reprogram their FA metabolism and, when combined with FASN, SCD1 and FADS2 inhibitors, it enables the identification of new FAs by the metabolic reconstruction of their synthesis route.

Late Pleistocene megafauna extinction leads to missing pieces of ecological space in a North American mammal community.

The conservation status of large-bodied mammals is dire. Their decline has serious consequences because they have unique ecological roles not replicated by smaller-bodied animals. Here, we use the fossil record of the megafauna extinction at the terminal Pleistocene to explore the consequences of past biodiversity loss. We characterize the isotopic and body-size niche of a mammal community in Texas before and after the event to assess the influence on the ecology and ecological interactions of surviving species (>1 kg). Preextinction, a variety of C4 grazers, C3 browsers, and mixed feeders existed, similar to modern African savannas, with likely specialization among the two sabertooth species for juvenile grazers. Postextinction, body size and isotopic niche space were lost, and the δ13C and δ15N values of some survivors shifted. We see mesocarnivore release within the Felidae: the jaguar, now an apex carnivore, moved into the specialized isotopic niche previously occupied by extinct cats. Puma, previously absent, became common and lynx shifted toward consuming more C4-based resources. Lagomorphs were the only herbivores to shift toward C4 resources. Body size changes from the Pleistocene to Holocene were species-specific, with some animals (deer, hare) becoming significantly larger and others smaller (bison, rabbits) or exhibiting no change to climate shifts or biodiversity loss. Overall, the Holocene body-size-isotopic niche was drastically reduced and considerable ecological complexity lost. We conclude biodiversity loss led to reorganization of survivors and many "missing pieces" within our community; without intervention, the loss of Earth's remaining ecosystems that support megafauna will likely suffer the same fate.

Fine-scaled climate variation in equatorial Africa revealed by modern and fossil primate teeth.

Variability in resource availability is hypothesized to be a significant driver of primate adaptation and evolution, but most paleoclimate proxies cannot recover environmental seasonality on the scale of an individual lifespan. Oxygen isotope compositions (δ18O values) sampled at high spatial resolution in the dentitions of modern African primates (n = 2,352 near weekly measurements from 26 teeth) track concurrent seasonal precipitation, regional climatic patterns, discrete meteorological events, and niche partitioning. We leverage these data to contextualize the first δ18O values of two 17 Ma Afropithecus turkanensis individuals from Kalodirr, Kenya, from which we infer variably bimodal wet seasons, supported by rainfall reconstructions in a global Earth system model. Afropithecus' δ18O fluctuations are intermediate in magnitude between those measured at high resolution in baboons (Papio spp.) living across a gradient of aridity and modern forest-dwelling chimpanzees (Pan troglodytes verus). This large-bodied Miocene ape consumed seasonally variable food and water sources enriched in 18O compared to contemporaneous terrestrial fauna (n = 66 fossil specimens). Reliance on fallback foods during documented dry seasons potentially contributed to novel dental features long considered adaptations to hard-object feeding. Developmentally informed microsampling recovers greater ecological complexity than conventional isotope sampling; the two Miocene apes (n = 248 near weekly measurements) evince as great a range of seasonal δ18O variation as more time-averaged bulk measurements from 101 eastern African Plio-Pleistocene hominins and 42 papionins spanning 4 million y. These results reveal unprecedented environmental histories in primate teeth and suggest a framework for evaluating climate change and primate paleoecology throughout the Cenozoic.

Novel sulfur isotope analyses constrain sulfurized porewater fluxes as a minor component of marine dissolved organic matter.

Marine dissolved organic matter (DOM) is a major reservoir that links global carbon, nitrogen, and phosphorus. DOM is also important for marine sulfur biogeochemistry as the largest water column reservoir of organic sulfur. Dissolved organic sulfur (DOS) can originate from phytoplankton-derived biomolecules in the surface ocean or from abiotically "sulfurized" organic matter diffusing from sulfidic sediments. These sources differ in 34S/32S isotope ratios (δ34S values), with phytoplankton-produced DOS tracking marine sulfate (21‰) and sulfurized DOS mirroring sedimentary porewater sulfide (∼0 to -10‰). We measured the δ34S values of solid-phase extracted (SPE) DOM from marine water columns and porewater from sulfidic sediments. Marine DOMSPE δ34S values ranged from 14.9‰ to 19.9‰ and C:S ratios from 153 to 303, with lower δ34S values corresponding to higher C:S ratios. Marine DOMSPE samples showed consistent trends with depth: δ34S values decreased, C:S ratios increased, and δ13C values were constant. Porewater DOMSPE was 34S-depleted (∼-0.6‰) and sulfur-rich (C:S ∼37) compared with water column samples. We interpret these trends as reflecting at most 20% (and on average ∼8%) contribution of abiotic sulfurized sources to marine DOSSPE and conclude that sulfurized porewater is not a main component of oceanic DOS and DOM. We hypothesize that heterogeneity in δ34S values and C:S ratios reflects the combination of sulfurized porewater inputs and preferential microbial scavenging of sulfur relative to carbon without isotope fractionation. Our findings strengthen links between oceanic sulfur and carbon cycling, supporting a realization that organic sulfur, not just sulfate, is important to marine biogeochemistry.

Use of δ18Oatm in dating a Tibetan ice core record of Holocene/Late Glacial climate.

Ice cores from the northwestern Tibetan Plateau (NWTP) contain long records of regional climate variability, but refrozen meltwater and dust in these cores has hampered development of robust timescales. Here, we introduce an approach to dating the ice via the isotopic composition of atmospheric O2 in air bubbles (δ18Oatm), along with annual layer counting and radiocarbon dating. We provide a robust chronology for water isotope records (δ18Oice and d-excess) from three ice cores from the Guliya ice cap in the NWTP. The measurement of δ18Oatm, although common in polar ice core timescales, has rarely been used on ice cores from low-latitude, high-altitude glaciers due to (1) low air pressure, (2) the common presence of refrozen melt that adds dissolved gases and reduces the amount of air available for analysis, and (3) the respiratory consumption of molecular oxygen (O2) by micro-organisms in the ice, which fractionates the δ18O of O2 from the atmospheric value. Here, we make corrections for melt and respiration to address these complications. The resulting records of water isotopes from the Guliya ice cores reveal climatic variations over the last 15,000 y, the timings of which correspond to those observed in independently dated lake and speleothem records and confirm that the Guliya ice cap existed before the Holocene. The millennial-scale drivers of δ18Oice are complex and temporally variable; however, Guliya δ18Oice values since the mid-20th century are the highest since the beginning of the Holocene and have increased with regional air temperature.

Co-habiting ants and silverfish display a converging feeding ecology.

BACKGROUND: Various animal taxa have specialized to living with social hosts. Depending on their level of specialization, these symbiotic animals are characterized by distinct behavioural, chemical, and morphological traits that enable close heterospecific interactions. Despite its functional importance, our understanding of the feeding ecology of animals living with social hosts remains limited. We examined how host specialization of silverfish co-habiting with ants affects several components of their feeding ecology. We combined stable isotope profiling, feeding assays, phylogenetic reconstruction, and microbial community characterization of the Neoasterolepisma silverfish genus and a wider nicoletiid and lepismatid silverfish panel where divergent myrmecophilous lifestyles are observed. RESULTS: Stable isotope profiling (δ13C and δ15N) showed that the isotopic niches of granivorous Messor ants and Messor-specialized Neoasterolepisma exhibit a remarkable overlap within an ant nest. Trophic experiments and gut dissections further supported that these specialized Neoasterolepisma silverfish transitioned to a diet that includes plant seeds. In contrast, the isotopic niches of generalist Neoasterolepisma silverfish and generalist nicoletiid silverfish were clearly different from their ant hosts within the shared nest environment. The impact of the myrmecophilous lifestyle on feeding ecology was also evident in the internal silverfish microbiome. Compared to generalists, Messor-specialists exhibited a higher bacterial density and a higher proportion of heterofermentative lactic acid bacteria. Moreover, the nest environment explained the infection profile (or the 16S rRNA genotypes) of Weissella bacteria in Messor-specialized silverfish and the ant hosts. CONCLUSIONS: Together, we show that social hosts are important determinants for the feeding ecology of symbiotic animals and can induce diet convergence.

Predator-driven behavioural shifts in a common lizard shape resource-flow from marine to terrestrial ecosystems.

Foraging decisions shape the structure of food webs. Therefore, a behavioural shift in a single species can potentially modify resource-flow dynamics of entire ecosystems. To examine this, we conducted a field experiment to assess foraging niche dynamics of semi-arboreal brown anole lizards in the presence/absence of predatory ground-dwelling curly-tailed lizards in a replicated set of island ecosystems. One year after experimental translocation, brown anoles exposed to these predators had drastically increased perch height and reduced consumption of marine-derived food resources. This foraging niche shift altered marine-to-terrestrial resource-flow dynamics and persisted in the diets of the first-generation offspring. Furthermore, female lizards that displayed more risk-taking behaviours consumed more marine prey on islands with predators present. Our results show how predator-driven rapid behavioural shifts can alter food-web connectivity between oceanic and terrestrial ecosystems and underscore the importance of studying behaviour-mediated niche shifts to understand ecosystem functioning in rapidly changing environments.

A bout of aerobic exercise in the heat increases carbohydrate use but does not enhance the disposal of an oral glucose load, in healthy active individuals.

We investigated if a bout of exercise in a hot environment (HEAT) would reduce the postprandial hyperglycemia induced by glucose ingestion. The hypothesis was that HEAT stimulating carbohydrate oxidation and glycogen use would increase the disposal of an ingested glucose load [i.e., oral glucose tolerance test (OGTT); 75 g of glucose]. Separated by at least 1 wk, nine young healthy individuals underwent three trials after an overnight fast in a randomized order. Two trials included 50 min of pedaling at 58 ± 5% V̇o2max either in a thermoneutral (21 ± 1°C; NEUTRAL) or in a hot environment (33 ± 1°C; HEAT) eliciting similar energy expenditure (503 ± 101 kcal). These two trials were compared with a no-exercise trial (NO EXER). Twenty minutes after exercise (or rest), subjects underwent an OGTT, while carbohydrate oxidation (CHOxid, using indirect calorimetry) plasma blood glucose, insulin concentrations (i.e., [glucose], [insulin]), and double tracer glucose kinetics ([U-13C] glucose ingestion and [6,6-2H2] glucose infusion) were monitored for 120 min. At rest, [glucose], [insulin], and rates of appearance/disappearance of glucose in plasma (glucose Ra/Rd) were similar among trials. During exercise, heart rate, tympanic temperature, [glucose], glycogen oxidation, and total CHOxid were higher during HEAT than NEUTRAL (i.e., 149 ± 35 vs. 124 ± 31 µmol·kg-1·min-1, P = 0.010). However, during the following OGTT, glucose Rd was similar in HEAT and NEUTRAL trials (i.e., 25.1 ± 3.6 vs. 25.2 ± 5.3 µmol·kg-1·min-1, P = 0.981). Insulin sensitivity (i.e., ISIndexMATSUDA) only improved in NEUTRAL compared with NO EXER (10.1 ± 4.6 vs. 8.8 ± 3.7 au; P = 0.044). In summary, stimulating carbohydrate use with exercise in a hot environment does not improve postprandial plasma glucose disposal or insulin sensitivity in a subsequent OGTT.NEW & NOTEWORTHY Exercise in the heat increases estimated muscle glycogen use. Reduced muscle glycogen after exercise in the heat could increase insulin-mediated glucose uptake during a subsequent oral glucose tolerance test (OGTT). However, plasma glucose kinetics are not improved during the OGTT in response to a bout of exercise in the heat, and insulin sensitivity worsens. Heat stress activates glucose counterregulatory hormones whose actions may linger during the OGTT, preventing increased glucose uptake.

Ethnic differences in postprandial fatty acid trafficking and utilisation between overweight and obese White European and Black African-Caribbean men.

Black African-Caribbean (BAC) populations are at greater risk of cardiometabolic disease than White Europeans (WE), despite lower fasting triacylglycerol (TAG) concentrations. However, limited data exist regarding postprandial fatty acid metabolism in BAC populations. This study determined the ethnic differences in postprandial fatty acid metabolism between overweight and obese WE and BAC men. WE (n=10) and BAC (n=9) men consumed two consecutive moderate-to-high fat meals; the first labelled with U-13C palmitate. The plasma concentration and appearance of meal-derived fatty acids in very-low density lipoprotein (VLDL)-TAG, chylomicron-TAG, and NEFA were determined over 8-hours. Indirect calorimetry with 13CO2 enrichment determined total and meal-derived fatty acid oxidation rates, and plasma b-hydroxybutyrate (3-OHB) concentration was measured to assess ketogenesis. BAC exhibited lower postprandial TAG (P=0.006) and VLDL-TAG (P=0.002) concentrations than WE. The appearance of meal-derived fatty acids in VLDL-TAG was lower in BAC than WE (P=0.004). Following the second meal, BAC showed a trend for lower chylomicron-TAG concentration (P=0.057). There were no ethnic differences in the appearance of meal-derived fatty acids in chylomicron-TAG. Cumulative fatty acid oxidation and the NEFA:3-OHB ratio were similar in WE and BAC. In conclusion, BAC exhibit lower postprandial TAG concentrations compared with WE men, driven by lower VLDL-TAG concentrations and possibly lower chylomicron-TAG in the late postprandial period. In BAC, the lower VLDL-TAG concentration was partially driven by a lower appearance of meal-derived fatty acids in VLDL-TAG. These findings suggest that postprandial fatty acid trafficking may be a less important determinant of cardiometabolic risk in BAC than WE men.

Sedimentary biomarkers and bone specimens reveal a history of prehistoric occupation on Somerset Island (Arctic Canada).

Archaeological studies of pre-historic Arctic cultures are often limited to artefacts and architecture; such records may be incomplete and often do not provide a continuous record of past occupation. Here, we used lake sediment archives to supplement archaeological evidence to explore the history of Thule and Dorset populations on Somerset Island, Nunavut (Canada). We examined biomarkers in dated sediment cores from two ponds adjacent to abandoned Thule settlements (PaJs-3 and PaJs-13) and compared these to sediment cores from two ponds without past human occupation. Coprostanol and epicoprostanol, δ15N measurements, sedimentary chlorophyll a and the ratio of diatom valves to chrysophyte cysts were elevated in the dated sediment profiles at both sites during Thule and Dorset occupations. Periods of pronounced human impact during the Thule occupation of the site were corroborated by 14C-dated caribou bones found at both sites that identified intense caribou hunting between ca 1185 and 1510 CE. Notably, these sediment core data show evidence of the Dorset occupation from ca 200 to 500 CE at sites where archaeological evidence was heretofore lacking. We highlight the utility of lake sediments in assisting archaeological studies to better establish the timings, peak occupations and even lifestyle practices of the Dorset and Thule Arctic peoples.

Mesophotic corals in Hawai'i maintain autotrophy to survive low-light conditions.

In mesophotic coral ecosystems, reef-building corals and their photosynthetic symbionts can survive with less than 1% of surface irradiance. How depth-specialist corals rely upon autotrophically and heterotrophically derived energy sources across the mesophotic zone remains unclear. We analysed the stable carbon (δ13C) and nitrogen (δ15N) isotope values of a Leptoseris community from the 'Au'au Channel, Maui, Hawai'i (65-125 m) including four coral host species living symbiotically with three algal haplotypes. We characterized the isotope values of hosts and symbionts across species and depth to compare trophic strategies. Symbiont δ13C was consistently 0.5‰ higher than host δ13C at all depths. Mean colony host and symbiont δ15N differed by up to 3.7‰ at shallow depths and converged at deeper depths. These results suggest that both heterotrophy and autotrophy remained integral to colony survival across depth. The increasing similarity between host and symbiont δ15N at deeper depths suggests that nitrogen is more efficiently shared between mesophotic coral hosts and their algal symbionts to sustain autotrophy. Isotopic trends across depth did not generally vary by host species or algal haplotype, suggesting that photosynthesis remains essential to Leptoseris survival and growth despite low light availability in the mesophotic zone.

Warming influences carbon and nitrogen assimilation between a widespread Ericaceous shrub and root-associated fungi.

High-latitude ecosystems are warming faster than other biomes and are often dominated by a ground layer of Ericaceous shrubs, which can respond positively to warming. The carbon-for-nitrogen (C-for-N) exchange between Ericaceous shrubs and root-associated fungi may underlie shrub responses to warming, but has been understudied. In a glasshouse setting, we examined the effects of warming on the C-for-N exchange between the Ericaceous shrub Empetrum nigrum ssp. hermaphroditum and its root-associated fungi. We applied different 13 C and 15 N isotope labels, including a simple organic N form (glycine) and a complex organic N form (moss litter) and quantified their assimilation into soil, plant biomass, and root fungal biomass pools. We found that warming lowered the amount of 13 C partitioned to root-associated fungi per unit of glycine 15 N assimilated by E. nigrum, but only in the short term. By contrast, warming increased the amount of 13 C partitioned to root-associated fungi per unit of moss 15 N assimilated by E. nigrum. Our study suggests that climate warming affects the short-term exchange of C and N between a widespread Ericaceous shrub and root-associated fungi. Furthermore, while most isotope tracing studies use labile N sources, we demonstrate that a ubiquitous recalcitrant N source may produce contrasting results.

Do exchangeable hydrogens affect the evaluation of partial mycoheterotrophy in orchids? Insights from δ2H analysis in bulk, α-cellulose, and cellulose nitrate samples.

To evaluate the nutritional modes of orchids associated with 'rhizoctonia' fungi, analyses of hydrogen (δ2H), carbon (δ13C), and nitrogen (δ15N) stable isotope ratios are usually adopted. However, previous studies have not fully accounted for exchangeable hydrogens, which could affect these evaluations. Here, we performed standard δ13C, δ15N, and δ2H analyses on bulk samples. Additionally, we conducted δ2H analysis on α-cellulose and cellulose nitrate samples to investigate whether the heterogeneity of exchangeable hydrogens among plant species influences the assessment of nutritional modes. The δ2H of orchids were consistently higher than those of surrounding autotrophic plants, irrespective of the three pretreatments. Although the rhizoctonia-associated orchid exhibited lower δ13C, its δ2H was higher than those of the autotrophs. Notably, among all response variables, δ15N and δ2H exhibited high abilities for discriminating the nutritional modes of rhizoctonia-associated orchids. These results indicate that a time-efficient bulk sample analysis is an effective method for evaluating plant nutritional modes, as the heterogeneity of exchangeable hydrogens does not significantly impact the estimation. Using δ15N and δ2H benefits the assessment of partial mycoheterotrophy among rhizoctonia-associated orchids.

Carbon budget at the individual-tree scale: dominant Eucalyptus trees partition less carbon belowground.

Large trees in plantations generally produce more wood per unit of resource use than small trees. Two processes may account for this pattern: greater photosynthetic resource use efficiency or greater partitioning of carbon to wood production. We estimated gross primary production (GPP) at the individual scale by combining transpiration with photosynthetic water-use efficiency of Eucalyptus trees. Aboveground production fluxes were estimated using allometric equations and modeled respiration; total belowground carbon fluxes (TBCF) were estimated by subtracting aboveground fluxes from GPP. Partitioning was estimated by dividing component fluxes by GPP. Dominant trees produced almost three times as much wood as suppressed trees. They used 25 ± 10% (mean ± SD) of their photosynthates for wood production, whereas suppressed trees only used 12 ± 2%. By contrast, dominant trees used 27 ± 19% of their photosynthate belowground, whereas suppressed trees used 58 ± 5%. Intermediate trees lay between these extremes. Photosynthetic water-use efficiency of dominant trees was c. 13% greater than the efficiency of suppressed trees. Suppressed trees used more than twice as much of their photosynthate belowground and less than half as much aboveground compared with dominant trees. Differences in carbon partitioning were much greater than differences in GPP or photosynthetic water-use efficiency.