What’s wrong with a little sex?

In many species, most (or all) offspring are produced by sexual means. However, theory suggests that selection should often favour the evolution of species in which a small fraction of offspring are produced sexually, and the rest are produced asexually. Here, we present the analysis of a model that may help to resolve this paradox. We show that, when heterozygote advantage is in force, members of species in which sex is rare will tend to produce poorly adapted offspring when they mate. This problem should be less severe in species where most offspring are produced by sexual means. As a consequence, once the rate of sexual reproduction becomes sufficiently rare, the benefits of sex may vanish, leading to the evolution of obligate asexuality. Substantial benefits of sexual reproduction may tend to accrue only if a large proportion of offspring are produced sexually. We suggest that similar findings are likely in the case of epistatic interactions between loci.     

Is histoaspartic protease a serine protease with a pepsin‐like fold?

The primary structure of the so-called histoaspartic protease from Plasmodium falciparum has a very high percentage of identity and homology with the pepsin-like enzyme plasmepsin II. A homology modeling approach was used to calculate the three-dimensional structure of the enzyme. Molecular dynamics (MD) simulations were applied to find those structural properties of the histoaspartic protease that had a tendency to remain stable during all runs. The results have shown that hydrogen-bonded residues Ser37-His34-Asp214 are arranged without any strain, in a manner that resembles the active site of a serine protease, while Ser38 and Asn39 take up positions appropriate to formation of an oxyanion hole. Although there are several important differences between the enzyme and plasmepsin II, all of the structural features associated with a typical pepsin-like aspartic protease are present in the final model of the histoaspartic protease. A possibility that this enzyme may function as a serine protease is discussed.     

Nutrient cycling relative to δ<Superscript>15</Superscript>N and δ<Superscript>13</Superscript>C natural abundance in a coastal wetland with long-term nutrient additions

Because nitrogen and phosphorus are primary resources for plant, algal, and microbial production, increases in nutrient inputs can markedly alter aquatic ecosystems. Coastal wetland plots at Belle W. Baruch Marine Field Laboratory (South Carolina, USA) have been amended with nitrogen and phosphorus for ~20 years to determine the effects of nutrient loading on coastal wetlands. We conducted a survey of δ¹⁵N and δ¹³C natural abundance in coastal wetland organic pools (sediment, vegetation) with long-term nutrient amendments (control, no addition; nitrogen; phosphorus; and nitrogen + phosphorus additions). Additionally, we conducted laboratory assays to quantify pore water nutrient availability and nitrification rates. Marsh vegetation (Spartina alterniflora) had enriched δ¹³C values (mean −14‰) relative to bulk sediment samples (mean −18‰). Nitrogen-amended plots (alone and in combination with phosphorus) had enriched δ¹³C values in the surface sediment (0–5 cm; mean −16.1‰) relative to control (mean −16.5‰) and phosphorus-amended plots (mean −16.8‰). Nitrogen-amended plots also had depleted δ¹⁵N in S. alterniflora leaf tissues (−3.3‰) and surface sediment samples (mean 2.1‰) relative to leaf tissues (mean 2.1‰) or sediment samples (mean 5.8‰) from control or phosphorus-only amended plots. Nitrate availability (as increased pore water concentration) was higher in N-amended plots although ammonium availability did not differ. Phosphorus availability was higher only in phosphorus-only amended plots. Overall, we found that long-term nutrient amendments to coastal wetlands significantly altered nutrient availability and uptake rates as well as natural abundance of δ¹³C and δ¹⁵N in multiple organic matter sources.     

Can a mixed damage interfere with DNA‐prottein cross‐links repair?

Some photochemical and photobiological properties of 4,5',8-trimethylpsoralen (TMP) have been studied in comparison with 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2 one (FQ) and 8-methoxypsoralen (8-MOP). TMP and FQ can photobind to mammalian cell DNA in vivo , by UVA irradiation, forming DNA-protein cross-links (DPC), but only TMP shows a strong capacity of inducing interstrand cross-links (ISC). The mechanism of DPC formation was studied using the double irradiation method in Chinese hamster ovary (CHO) cells, and DPC were detected by alkaline elution. Both TMP and FQ induce covalent diadducts linking together DNA and proteins. Studying the formation of double strand breaks (DSB) in CHO cells we observed that TMP induced a low amount of DSB, similar to 8-MOP. TMP and 8-MOP induced chromosomal aberrations in CHO cells to the same extent, while FQ appeared to be more active. Our data suggest that the ISC induced by TMP could trap enzymes involved in DPC repair.     

Sources of variation in consumer-diet δ<Superscript>15</Superscript>N enrichment: a meta-analysis

Measurements of ¹⁵N of consumers are usually higher than those of their diet. This general pattern is widely used to make inferences about trophic relationships in ecological studies, although the underlying mechanisms causing the pattern are poorly understood. However, there can be substantial variation in consumer-diet ¹⁵N enrichment within this general pattern. We conducted an extensive literature review, which yielded 134 estimates from controlled studies of consumer-diet ¹⁵N enrichment, to test the significance of several potential sources of variation by means of meta-analyses. We found patterns related to processes of nitrogen assimilation and excretion. There was a significant effect of the main biochemical form of nitrogenous waste: ammonotelic organisms show lower ¹⁵N enrichment than ureotelic or uricotelic organisms. There were no significant differences between animals feeding on plant food, animal food, or manufactured mixtures, but detritivores yielded significantly lower estimates of enrichment. ¹⁵N enrichment was found to increase significantly with the C:N ratio of the diet, suggesting that a nitrogen-poor diet can have an effect similar to that already documented for fasting organisms. There were also differences among taxonomic classes: molluscs and crustaceans generally yielded lower ¹⁵N enrichment. The lower ¹⁵N enrichment might be related to the fact that molluscs and crustaceans excrete mainly ammonia, or to the fact that many were detritivores. Organisms inhabiting marine environments yielded significantly lower estimates of ¹⁵N enrichment than organisms inhabiting terrestrial or freshwater environments, a pattern that was influenced by the number of marine, ammonotelic, crustaceans and molluscs. Overall, our analyses point to several important sources of variation in ¹⁵N enrichment and suggest that the most important of them are the main biochemical form of nitrogen excretion and nutritional status. The variance of estimates of ¹⁵N enrichment, as well as the fact that enrichment may be different in certain groups of organisms should be taken into account in statistical approaches for studying diet and trophic relationships.     

Fabrication of carbon films with ∼500 nm holes for cryo-EM with a direct detector device

Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500 nm holes spaced 4 μm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently.     

Can tree-ring δ<Superscript>15</Superscript>N be used as a proxy for foliar δ<Superscript>15</Superscript>N in European beech and Norway spruce?

Key message

For long-term environmental investigations, tree-ring δ ¹⁵ N values are inappropriate proxies for foliar δ ¹⁵ N for both Fagus sylvatica and Picea abies under moderate N loads.

Abstract

Currently it is unclear whether stable nitrogen isotope signals of tree-rings are related to those in foliage, and whether they can be used to infer tree responses to environmental changes. We studied foliar and tree-ring nitrogen (δ¹⁵N) and carbon (δ¹³C) isotope ratios in European beech (Fagus sylvatica L.) and Norway spruce (Picea abies L.) from six long-term forest monitoring sites in Switzerland together with data on N deposition and soil N availability, as well as a drought response index over the last two decades. For both species, tree-ring δ¹⁵N and δ¹³C values were less negative compared to foliar δ¹⁵N and δ¹³C values, most likely due to recycling and reallocation of N within the tree and fractionation processes associated with the transport of sucrose and the formation of tree-rings, respectively. Temporal trends recorded in foliar δ¹⁵N were not reflected in tree-ring δ¹⁵N, with much higher variations in tree-rings compared to foliage. Soil N availability and N deposition were partially able to explain changes in foliar δ¹³C, while there were no significant correlations between environmental variables and either tree-ring or foliar δ¹⁵N. Our results suggest an uncoupling between the N isotopic composition of tree-rings and foliage. Consequently, tree-ring δ¹⁵N values are inappropriate proxies of foliar δ¹⁵N values under low-to-moderate N deposition loads. Furthermore, at such low levels of deposition, tree-ring δ¹⁵N values are not recommended as archives of tree responses to soil C/N or bulk N deposition.

     

δ<Superscript>15</Superscript>N dynamics of ammonium and particulate nitrogen in a temperate eutrophic estuary

We monitored the stable nitrogen isotopic composition (δ¹⁵N) of suspended matter and ammonium in the freshwater stretch of the Scheldt estuary (Belgium) over a full year to investigate for seasonal evolution and possible co-variation between isotopic signatures. The δ¹⁵N value of ammonium remained rather constant during winter (average = +11.4‰) but increased significantly with the spring and summer bloom, reaching values as high as +70‰. This enrichment of the ammonium pool in ¹⁵N coincided with significant ammonium depletion during summer period, suggesting a close causal relationship. Based on a semi-closed system approach we deduced an apparent fractionation factor associated with NH₄⁺ utilization (i.e. combining effects of uptake and nitrification) of 18.4‰ (SE = 2.0‰), which is similar to values reported in literature. Observed variations of ammonium δ¹⁵N could account for about 69% of δ¹⁵N variation in suspended matter.     

Meiotic Chromosome Behavior in a Human Male t（8;15） Carrier

Reciprocal translocation is one of the most common structural chromosomal rearrangements in human beings; it is widely recognized to be associated with male infertility. This association is mainly based on the abnormal chromosome behavior of the translocated chromosomes and sex chromosomes during meiosis prophase I in reciprocal translocation carriers. However, the underlying mechanisms are not completely known. Here we report a reciprocal translocation carrier of t（8;15）, who is oligozoospermic due to apoptosis of primary spermatocytes and to premature germ cell desquamation from seminiferous tubules. Further analysis showed abnormal synapsis and recombination frequency in this patient, indicating a connection between chromosome behavior and apoptosis of primary spermatocytes. We also compared these observations with recently reported findings on spermatogenesis defects in reciprocal translocation carriers, and discuss the possible mechanisms underlying both common and unique phenotypes of reciprocal translocations involving different chromosomes with the aim of further understanding the regulation of human spermatogenesis.     

Does a multi‐plant diet benefit a polyphagous herbivore? A case study with Bemisia tabaci

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a highly polyphagous herbivore. This research was conducted to compare the development of B. tabaci reared in a multi‐plant treatment (polyculture) with those in single‐plant treatments (monocultures). Adult B. tabaci females fed on a mixture of tomato, cabbage, cotton, cucumber, and kidney bean survived longer and laid more eggs than those fed exclusively on one of these plant species. Egg numbers per plant laid in the polyculture treatment were positively correlated with those laid on the same plant species in the monoculture treatments, and egg numbers per plant laid on tomato, cotton, and cucumber in the polyculture were significantly higher than those laid on the same plants in the monocultures. Concentrations of total protein and trehalose in B. tabaci were not significantly different after 7 days of feeding in the respective treatments, but activities of superoxide dismutases (SOD) and alkaline phosphatase (AKP) of B. tabaci in polyculture were lower than those in monoculture. Conversely, activities of trehalase, sucrase, and amylase in B. tabaci kept in polyculture were higher than those of insects from the monoculture. In each of the monoculture treatments, there was a negative correlation between AKP in B. tabaci and oviposition, and also between AKP and amylase. SOD and sucrase activities in B. tabaci were positively correlated with polyphenol oxidase (PPO) and peroxidase (POD) activities in plants. In the plants damaged by whiteflies in the polyculture treatment, activities of SOD in cucumber, PPO in cotton and kidney bean, and POD in tomato and cucumber were lower than those in the monoculture treatments, whereas SOD in cabbage and catalase (CAT) in tomato in the polyculture treatment were higher than those in the monoculture treatments.     

Robotic total gastrectomy with <Emphasis Type="Italic">π</Emphasis>-shaped esophagojejunostomy using a linear stapler as a novel technique

Objective

To evaluate the intraoperative and short-term postoperative outcomes of a novel robotic intracorporeal π-shaped esophagojejunostomy (EJS) after D2 total gastrectomy (TG) using the Da Vinci robotic surgical system for intracorporeal anastomosis after TG.

Background

Intracorporeal π-shaped EJS, using a linear stapler, was recently reported for laparoscopic total gastrectomy in patients with gastric cancer. However, robotic intracorporeal π-shaped EJS using a linear stapler has not been reported. This report aimed to describe the use of a novel technique for π-shaped EJS using the Da Vinci robotic system.

Methods

Robotic intracorporeal π-shaped esophagojejunostomy after total gastrectomy was performed in 11 consecutive patients diagnosed with early gastric cancer, and their perioperative outcomes were analyzed.

Results

All the operations were successful without conversion to open or laparoscopic surgery and postoperative complications. The total number of patients was 11 (7 males and 4 females). The mean age of the patients was 63.36?±?10.56?years old. Seven patients were diagnosed with cardia cancer, 3 patients were diagnosed with gastric body cancer, and 1 patient was diagnosed with gastric antrum cancer. The patients’ mean proximal resection margin was 3.18?±?1.17?cm, the distal resection margin was 6.18?±?1.40?cm, the mean length of the incision was 4.55?±?0.69?cm, the mean operative time was 287.27?±?30.69?min, the mean day of first flatus was 3.27?±?0.79?days, the mean day of the start of diet was 2.91?±?0.94?days, the mean postoperative hospital stay was 11.45?±?5.13?days, and the mean operative blood loss was 47.27?±?31.33?ml. No complications were observed during anastomosis, and the median anastomosis time was 19.5?min. The mean number of lymph node dissections was 17.91?±?4.59, the mean number of positive lymph nodes was 0.45?±?0.69, all patients were diagnosed with stage I–II gastric cancer, and the mean maximum diameter of the tumor was 2.67?±?1.30?cm. All the patients had a smooth hospital discharge.

Conclusion

A novel robotic gastrectomy with intracorporeal π-shaped EJS for esophagojejunal anastomosis described and shows acceptable resulted. This technique has the potential to offer better short-term surgical outcomes and overcomes the drawbacks of laparoscopy with a decreased risk of complications during and after surgery.

     

Plasma visfatin,associated with a genetic polymorphism −1535C > T,is correlated with C-reactive protein in Chinese Han patients with traumatic brain injury

Visfatin is a newly identified pro-inflammatory adipokine and a genetic polymorphism −1535 C > T located in the visfatin gene promoter has been suggested to be associated with the regulation of visfatin expression in some inflammatory illness. However, there were some conflicting results regarding whether this variant is functional or not. This study aimed to examine the relations of the −1535 C > T single nucleotide polymorphism (SNP) of visfatin gene to the plasma visfatin and C-reactive protein concentrations in traumatic brain injury (TBI). 318 Chinese Han patients with TBI were recruited in this study. Plasma visfatin and C-reactive protein levels were significantly different between the genotypes in the SNP-1535 C > T even after adjustment for age, sex and body mass index. The genotype C–C had the highest plasma visfatin and C-reactive protein concentrations. The plasma visfatin and C-reactive protein concentrations between the variant genotypes C–T and T–T did not differ significantly. Plasma visfatin level was significantly associated with plasma C-reactive protein level using multivariate linear regression. Thus, the SNP-1535 C > T of visfatin gene seemed to be potentially involved in the inflammatory component of TBI through a decreased production of visfatin.     

The <Emphasis Type="Italic">MTR</Emphasis> 2756A&gt;G polymorphism and maternal risk of birth of a child with Down syndrome: a case–control study and a meta-analysis

Methionine synthase (MTR) is required for the conversion of homocysteine (hcy) to methionine in the one-carbon metabolic pathway. Previous studies investigating a common MTR 2756A>G polymorphism as a maternal risk factor for the birth of a child with Down syndrome (DS) are conflicting and limited by small case–control cohorts, and its contribution to circulating hcy levels is still debated. We performed a large case–control study and a meta-analysis of the literature to further address the role of MTR 2756A>G as a maternal risk factor for the birth of a child with DS. 286 mothers of a DS child (MDS) and 305 control mothers of Italian origin were included in the case–control study. Genotyping was performed by means of PCR/RFLP technique. Data on circulating levels of hcy, folates, and vitamin B12 were available for 189 MDS and 194 control mothers. The meta analysis of previous and present data involved a total of 8 studies (1,171 MDS and 1,402 control mothers). Both the case–control study and the meta-analysis showed no association of MTR 2756A>G with the maternal risk of birth of a child with DS (OR = 1.15; 95 % CI 0.85–1.55, and OR = 1.08; 95 % CI 0.93–1.25, respectively), even after stratification of the overall data available for the meta-analysis into ethnic groups. No association of the studied polymorphism with circulating levels of hcy, folates, and vitamin B12 was observed. Present data do not support a role for MTR 2756A>G as independent maternal risk factor for a DS birth.     

Rubisco activase: an enzyme with a temperature‐dependent dual function?

Heat treatment of intact spinach leaves was found to induce a unique thylakoid membrane association of an approximately 40 kDa stromal protein. This protein was identified as rubisco activase. Most of the rubisco activase was sequestered to the thylakoid membrane, particularly to the stroma-exposed regions, during the first 10 min of heat treatment at 42 degrees C. At lower temperatures (38-40 degrees C) the association of rubisco activase with the thylakoid membrane occurred more slowly. The temperature-dependent association of rubisco activase with the thylakoid membrane was due to a conformational change in the rubisco activase itself, not to heat-induced alterations in the thylakoid membrane. Association of the 41 kDa isoform of rubisco activase occurred first, followed by the binding of the 45 kDa isoform to the thylakoid membrane. Fractionation of thylakoid membranes revealed a specific association of rubisco activase with thylakoid-bound polysomes. Our results suggest a temperature-dependent dual function for rubisco activase. At optimal temperatures it functions in releasing inhibitory sugar phosphates from the active site of Rubisco. During a sudden and unexpected exposure of plants to heat stress, rubisco activase is likely to manifest a second role as a chaperone in association with thylakoid-bound ribosomes, possibly protecting, as a first aid, the thylakoid associated protein synthesis machinery against heat inactivation.     

Discovery of olodaterol,a novel inhaled β2-adrenoceptor agonist with a 24 h bronchodilatory efficacy

Compound 4p was identified from a series of 6-hydroxy-4H-benzo[1,4]oxazin-3-ones as potent agonist of the human β₂-adrenoceptor with a high β₁/β₂-selectivity. A complete reversal of acetylcholine-induced bronchoconstriction which lasted over the whole study period of 5 h was demonstrated for 4p in a guinea pig in vivo model without any signs of cardiovascular effects up to 10-fold above the first dose reaching 100% bronchoprotection. The enantiomerically pure (R)-form of 4p exerted a bronchodilatory efficacy over 24 h in dogs and guinea pigs in the absence of systemic pharmacodynamic effects. Formoterol which was tested as comparator in the same in vivo models of acetylcholine-induced bronchoconstriction did not retain efficacy after 24 h. In summary, the preclinical profile of compound (R)-4p (olodaterol, also known as BI 1744 CL) suggests a potential for once-daily dosing in man accompanied with an improved safety profile.     

Flow of Deposited Inorganic N in Two Gleysol-dominated Mountain Catchments Traced with <Superscript>15</Superscript>NO<Subscript>3</Subscript><Superscript>−</Superscript> and <Superscript>15</Superscript>NH<Subscript>4</Subscript><Superscript>+</Superscript>

In two mountain ecosystems at the Alptal research site in central Switzerland, pulses of ¹⁵NO₃ and ¹⁵NH₄ were separately applied to trace deposited inorganic N. One forested and one litter meadow catchment, each approximately 1600 m², were delimited by trenches in the Gleysols. K¹⁵NO₃ was applied weekly or fortnightly over one year with a backpack sprayer, thus labelling the atmospheric nitrate deposition. After the sampling and a one-year break, ¹⁵NH₄Cl was applied as a second one-year pulse, followed by a second sampling campaign. Trees (needles, branches and bole wood), ground vegetation, litter layer and soil (LF, A and B horizon) were sampled at the end of each labelling period. Extractable inorganic N, microbial N, and immobilised soil N were analysed in the LF and A horizons. During the whole labelling period, the runoff water was sampled as well. Most of the added tracer remained in both ecosystems. More NO₃⁻ than NH₄⁺ tracer was retained, especially in the forest. The highest recovery was in the soil, mainly in the organic horizon, and in the ground vegetation, especially in the mosses. Event-based runoff analyses showed an immediate response of ¹⁵NO₃⁻ in runoff, with sharp ¹⁵N peaks corresponding to discharge peaks. NO₃⁻ leaching showed a clear seasonal pattern, being highest in spring during snowmelt. The high capacity of N retention in these ecosystems leads to the assumption that deposited N accumulates in the soil organic matter, causing a progressive decline of its C:N ratio.     

Can we disentangle predator–prey interactions from species distributions at a macro‐scale? A case study with a raptor species

There is a debate on whether an influence of biotic interactions on species distributions can be reflected at macro‐scale levels. Whereas the influence of biotic interactions on spatial arrangements is beginning to be studied at local scales, similar studies at macro‐scale levels are scarce. There is no example disentangling, from other similarities with related species, the influence of predator–prey interactions on species distributions at macro‐scale levels. In this study we aimed to disentangle predator–prey interactions from species distribution data following an experimental approach including a factorial design. As a case of study we selected the short‐toed eagle because of its known specialization on certain prey reptiles. We used presence–absence data at a 100 km2 spatial resolution to extract the explanatory capacity of different environmental predictors (five abiotic and two biotic predictors) on the short‐toed eagle species distribution in peninsular Spain. Abiotic predictors were relevant climatic and topographic variables, and relevant biotic predictors were prey richness and forest density. In addition to the short‐toed eagle, we also obtained the predictor's explanatory capacities for 1) species of the same family Accipitridae (as a reference), 2) for other birds of different families (as controls) and 3) artificial species with randomly selected presences (as null models). We run 650 models to test for similarities of the short‐toed eagle, controls and null models with reference species, assessed by regressions of explanatory capacities. We found higher similarities between the short‐toed eagle and other species of the family Accipitridae than for the other two groups. Once corrected by the family effect, our analyses revealed a signal of predator–prey interaction embedded in species distribution data. This result was corroborated with additional analyses testing for differences in the concordance between the distributions of different bird categories and the distributions of either prey or non‐prey species of the short‐toed eagle. Our analyses were useful to disentangle a signal of predator–prey interactions from species distribution data at a macro‐scale. This study highlights the importance of disentangling specific features from the variation shared with a given taxonomic level.     

Ecosystem N Distribution and δ<Superscript>15</Superscript>N during a Century of Forest Regrowth after Agricultural Abandonment

Abstract Stable isotope ratios of terrestrial ecosystem nitrogen (N) pools reflect internal processes and input–output balances. Disturbance generally increases N cycling and loss, yet few studies have examined ecosystem δ¹⁵N over a disturbance-recovery sequence. We used a chronosequence approach to examine N distribution and δ¹⁵N during forest regrowth after agricultural abandonment. Site ages ranged from 10 to 115 years, with similar soils, climate, land-use history, and overstory vegetation (white pine Pinus strobus). Foliar N and δ¹⁵N decreased as stands aged, consistent with a progressive tightening of the N cycle during forest regrowth on agricultural lands. Over time, foliar δ¹⁵N became more negative, indicating increased fractionation along the mineralization–mycorrhizal–plant uptake pathway. Total ecosystem N was constant across the chronosequence, but substantial internal N redistribution occurred from the mineral soil to plants and litter over 115 years (>25% of ecosystem N or 1,610 kg ha⁻¹). Temporal trends in soil δ¹⁵N generally reflected a redistribution of depleted N from the mineral soil to the developing O horizon. Although plants and soil δ¹⁵N are coupled over millennial time scales of ecosystem development, our observed divergence between plants and soil suggests that they can be uncoupled during the disturbance-regrowth sequence. The approximate 2‰ decrease in ecosystem δ¹⁵N over the century scale suggests significant incorporation of atmospheric N, which was not detected by traditional ecosystem N accounting. Consideration of temporal trends and disturbance legacies can improve our understanding of the influence of broader factors such as climate or N deposition on ecosystem N balances and δ¹⁵N. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.