Ruptured sinus of Valsalva aneurysm is rare and dangerous in parturients. Few cases of ruptured SVA in pregnancy are reported, and the anesthetic management for cesarean delivery has scarcely been described.A parturient at 37-week gestation complained of a sore throat and cough that started 3 days before admission, followed 1 day later by fever, dizziness, breathlessness, and palpitation on exertion. Case two at 36-week gestation complained of a 1-day history of bloating in the lower abdomen.Full term and preterm parturients with ruptured sinus of Valsalva aneurysm.Cesarean deliveries were performed with incremental epidural anesthesia technique under invasive monitoring. Surgical correction of the ruptured sinus of Valsalva aneurysms and ventricular septal defect were performed uneventfully 13 days and 7 days postpartum, respectively, for the 2 cases.No complications were observed in the intra- or postoperative period for both mothers and babies.We reviewed the pertinent literature and reached the following conclusions: use of a multidisciplinary team to guide anesthetic management is helpful and necessary; and both general anesthesia and incremental epidural anesthesia can be safely used in parturients with ruptured sinus of Valsalva aneurysm. Although SVA is rare in the Western world, it occurs much more commonly in male patients of Asian origin and comprises ∼0.1% to 3.5% of all congenital cardiac anomalies. Patients are often asymptomatic before the rupture of SVA, which is a life-threatening event and requires immediate recognition and intervention.,5 Few cases of ruptured SVA in pregnancy are reported, and the anesthetic management for cesarean delivery has scarcely been described.Cardiac disease is a major cause of maternal mortality, and about 25% of maternal cardiac deaths in the past 30 years have been due to congenital heart diseases.We present our successful anesthetic management of cesarean delivery of 2 recent cases. The reporting of these cases was approved by the Institutional Review Board and both patients gave written informed consent for the report.2To identify studies for inclusion in this review, 2 authors independently searched PubMed for relevant studies published up to May 2016. The search was limited to studies conducted in humans. Only cases in English language were included. Search terms used included [“ruptured sinus of Valsalva aneurysm” OR “sinus of Valsalva aneurysm”] and [“pregnant” OR “delivery” OR “parturient” OR “cesarean”].2.12.1.12 and a grade 4/6 systolic murmur over the precordial region, best heard in the pulmonary area. Breath sounds were low in both lungs, without rales or rhonchi. Her functional status of New York Heart Association (NYHA) was class II–III.A 27-year-old primigravida  was referred to our hospital on August 2010 with a 37-week gestation due to shortness of breath after a 3-day history of sore throat. She was asymptomatic before 36 weeks’ gestation. The patient complained of a sore throat and cough that started 3 days before admission, followed 1 day later by fever, dizziness, breathlessness, and palpitation on exertion. She reported no medical history except for a fully-recovered acute nephritis 15 years ago. On physical examination, the patient appeared acutely ill, and vital signs were temperature 37.8°C, heart rate 94 beats/min, respiratory rate 28 breathes/min, blood pressure 107/70 mm Hg, and oxygen saturation by pulse oximetry 98% on room air. Auscultation revealed a loud PLaboratory tests on the admission day were normal. Transthoracic color Doppler echocardiography revealed an 8 mm × 9 mm aneurysm in the right coronary sinus of Valsalva, protruding into the right ventricular outflow tract (RVOT), with a 4 mm-wide perforation. A high pressure left-to-right jet started from a laceration of aneurysm, the peak flow velocity (PFV) is 5.8 m/s and pressure gradient (PG) is 136 mm Hg. A mild tricuspid regurgitation was present. A 3 mm intracristal ventricular septal defect (VSD) was also present on echocardiography. The left atrium (37 mm) and left ventricle (55 mm) were enlarged. Of note, the size of right atrium (45 mm) and right ventricular (51 mm) were also enlarged. In addition, there was evidence of mild pulmonary hypertension, with an estimated pulmonary artery systolic pressure of 42 mm Hg and an estimated left ventricular ejection fraction of 69% at rest. The functional capacity of patient could not be determined due to bed rest requirement.The patient asked for cesarean delivery without maternal or fetal indication. Four days after admission, the patient received cesarean delivery under epidural anesthesia.2.1.22P0, weight 57 kg, height 163 cm) with a 36-week gestation was admitted for a 1-day history of bloating in the lower abdomen on August 2010. She was diagnosed with VSD in the childhood but remained asymptomatic without treatment. There was no other significant past medical history. On physical examination, the patient showed no acute distress. Vital signs were temperature 36.5°C, heart rate 105 beats/min, respiratory rate 20 breathes/min, blood pressure 145/57 mm Hg, and oxygen saturation by pulse oximetry 98% on room air. Auscultation revealed a grade 5/6 continuous murmur along the lower left sternal border with thrill. Breath sounds were normal in both lungs. Her functional capacity was NYHA II.A 22-year-old woman  and left ventricle (58 mm) and an estimated left ventricular ejection fraction of 63% at rest.Because ultrasonography revealed a complete placenta previa, an elective cesarean delivery under epidural anesthesia was planned.Both patients were managed by a multidisciplinary team of obstetricians, cardiologists, and anesthesiologists, and the cesarean deliveries were planned in a cardiac operating room with cardiopulmonary bypass capabilities on standby. Intravenous infusion of metoclopramide 10 mg and ranitidine 40 mg was used as for aspiration prophylaxis 30 minutes before the operations. Antibiotic prophylaxis of surgical infection with intravenous penicillin was started 30 minutes prior to skin incision.1–L2 intervertebral space on the left lateral decubitus position. After a 3 mL epidural test dose (2% lidocaine without epinephrine), incremental doses of 3 to 5 mL of 2% plain lidocaine were administered every 5 minutes, to avoid systemic hypotension. A total of 19 mL and 18 mL of 2% lidocaine was used for the 2 patients, respectively, including the test dose. An upper sensory level of T6 was achieved, tested by sharp pinprick. A prophylactic low-dose phenylephrine infusion was part of a standard protocol for preventing hemodynamic instability during epidural anesthesia.,8 The surgeries proceeded uneventfully without pain or discomfort, with delivery of newborns with satisfactory Apgar scores . Patient-controlled epidural analgesia of 0.2% ropivacaine was started with a background dose of 6 mL/h and 4 mL as a bolus with a 20-minute lockout interval, which was removed 48 hours later, achieving a 0 to 30 visual analog scale of pain for both patients.For case 1, the invasive blood pressure (IBP) abruptly decreased to 90/55 mm Hg following slow intravenous bolus injection of 5-unit oxytocin for prophylaxis against uterine atony, and marked bradycardia developed after a higher dose of phenylephrine infusion. So norepinephrine infusion was started instead immediately, at a rate of 0.05 to 0.08 μg kgAfter the operation, both patients were doing well without any complications, and surgical closure of VSD and correction of ruptured SVA under general anesthesia with cardiopulmonary bypass was planned. The procedures proceeded uneventfully 13 days and 7 days postpartum, respectively, for the 2 cases. Postoperatively, no residual shunt or aortic regurgitation was noted by transthoracic echocardiography at regular follow-ups. Both mothers and babies were doing well 12 months later, without any complications.3We present 2 Chinese parturients with ruptured SVA. Both of them gave birth to a healthy baby under cesarean delivery with well-managed epidural anesthesia. Both patients received repaired surgery and they were alive with complete remission on a 1-year follow-up. SVA frequently co-exist with other cardiac abnormalities, including VSD in 30% to 60% cases and aortic regurgitation in 44% to 50% cases, which may present a diagnostic conundrum because the diverse clinical presentations.,10 Sakakibara and Konno proposed the first formal classification system for SVA in 1962. Then, Xin-jin and Xuan proposed a modified classification system in 2013. This modified classification system described 5 types of aneurysms. Type I protrusion and rupture into right ventricle beneath pulmonary valve; type II penetration and rupture into or just beneath crista supraventricularis of right ventricle; type III penetration and rupture into right heart adjacent to or at tricuspid annulus; type IV protrusion and rupture into right atrium; type V other rare conditions .The underlying histopathological etiology of SVA is usually thought to be an inherited deficiency of connective tissue and muscular tissue in the aortic sinus. However, it can also be acquired from infection, trauma, surgery, inflammatory diseases or degenerative diseases, which usually affects more than 1 sinus of Valsalva. About 65% to 85% of SVA originate from the right sinus of Valsalva, and those originating from the noncoronary (10% to 30%) or left sinuses (<5%) are relatively rare. Associated symptoms may include dyspnea, chest pain, syncope, or congestive heart failure. Usually, color Doppler echocardiography is a simple and accurate noninvasive way of diagnosing ruptured SVA, and it was valuable in the correct diagnosis of our patients in spite of the VSD which complicated the hemodynamic change. Coronary angiography, computed tomography, magnetic resonance imaging is also valuable in the diagnosis of SVA. However, potential radiation risks must be considered.Clinical manifestation of an unruptured SVA may range from an asymptomatic murmur to sudden death as a consequence of right ventricular outflow obstruction, infective endocarditis, arrhythmias, myocardial infarction, and ischemia due to distortion of the coronary ostia or compression of the coronary trunk. Progressive dilatation of the SVA can lead to aortic regurgitation secondary to displacement of valve leaflets by the aneurysm. Chest pain and dyspnea may precede heart failure if rupture is abrupt, but the size and the location of the shunt, rapidity of onset, and the chamber into which the rupture occurs are the major determinants of the exact hemodynamic consequences and prognosis. In a Texas Heart Institute study, most SVAs ruptured into the right ventricle (60%) or the right atrium (29%), only a few ruptured into left atrium (6%), the left ventricle (4%), or the pericardium (1%). Right-heart overload, right ventricular failure, and significant tricuspid regurgitation are frequent sequela of rupture into the right-sided chambers. Rupture of SVA into the left heart may induce acute left heart failure, which may be clinically indistinguishable from acute aortic insufficiency. An extracardiac rupture of the aneurysm into the pericardium might lead to a fatal cardiac tamponade.Acute symptomatic ruptures can proceed spontaneously or are precipitated by exertion, trauma, endocarditis, or cardiac catheterization. Histopathologic findings in human aortic media associated with pregnancy includes fragmentation of the reticulum fibers, diminished amount of acid mucopolysaccharides, and loss of the corrugation of elastic fibers in the vessel. These changes begin early in gestation and are most pronounced in the third trimester and the peripartum period.,19 In our patients, perhaps the rupture of SVA was precipitated by the hyperdynamic state of late pregnancy coupled with hormonally induced changes in the mechanical properties of connective tissue.During pregnancy, the cardiovascular system is progressively stressed due to increased blood volume, increased heart rate, and stroke volume, and pregnancy is extraordinarily risky for women with underlying cardiac diseases.Previously reported cases were identified by searches conducted on PubMed (1960 to May 2016) using the following key words: “ruptured sinus of Valsalva aneurysm” or “sinus of Valsalva aneurysm” and “pregnant” or “delivery” or “parturient” or “cesarean” or “pregnancy.” Only cases in English language were included. Twelve articles published between 1960 and 2016 were retrieved, and peripartum management of 13 deliveries in 12 patients was described. Time of publication, diagnosis and severity of ruptured SVA, mode of delivery, peripartum care, time of corrective surgery, and maternal and fetal outcome are shown in Table ,21 Cesarean delivery is usually performed for obstetrical indications only. However, if the patient was already hemodynamically compromised, cesarean delivery was the preferred method. A prospective study demonstrated that pregnant women with cardiac disease may be safe when received cesarean delivery under regional anesthesia. And successful outcomes have been reported with regional anesthesia for every type of cardiac pathology.,23 For the high-risk parturients, various successful regional anesthetic management options have been reported: combined spinal epidural,,24 spinal catheter insertion, or incremental epidural anesthesia.,27 Epidural anesthesia with incremental doses is the most often advocated as the best regional technique. There is now an increasing number of case reports highlighting the use of incremental epidural anesthesia with good outcome,–28 including in patients with ruptured SVA.Multiple studies have shown that vaginal delivery is well tolerated in most patients with cardiac disease. Also, the reduced SVR can alleviate the left-to-right shunting and consequent overload of the right ventricle and pulmonary vasculature due to the ruptured SVA and VSD in our patients. Incremental small boluses of 2% lidocaine without epinephrine were used to produce a T6 block without substantial circulatory changes. For patients with ruptured SVA, the level of neuraxial blockade involves an equilibrium between safety and comfort. Traditional T4 level blockade can cause bradycardia due to sympathetic blockade of cardio-accelerator fibers, which is dangerous for these high-risk patients. We aim to control the sensory blockade level to T6 and the procedure proceeded without significant pain or discomfort. Even though, the circulation side-effects of epidural anesthesia may still occur, which can be managed with carefully selected vasopressors. Phenylephrine is the first vasopressor of choice during routine cesarean delivery, and for people with cardiac disease. It is highly effective, easily titratable, and safe for the baby. Norepinephrine has been shown to have similar efficacy for maintaining blood pressure, with smaller incidence of bradycardia.Well-managed epidural anesthesia has the advantage of avoiding myocardial depression associated with volatile anesthetics and the stress of endotracheal intubation in general anesthesia, and causes minimal hemodynamic and respiratory disturbance. The mildly decreased SVR due to sympathetic blockade can be expected to favor forward blood flow in patients with aortic insufficiency.In our hospital, general anesthesia is used when neuraxial anesthesia is contraindicated or in cases of life-threatening emergency with inadequate time for neuraxial anesthesia. The choice of general versus epidural-spinal anesthesia must be made after considering the patient's unique pathophysiology and in consultation with cardiologists, obstetricians, obstetric anesthesiologists, and cardiac anesthesiologists. The usefulness of a pulmonary artery catheterization in patients with ruptured SVA is not clear. It has been reported that ventricular tachycardia occurred repeatedly during attempted pulmonary artery catheterization due to a sinus of Valsalva aneurysm protruding into the right ventricle and obstructing advancement of the pulmonary artery catheter. Transesophageal echocardiography can provide information about intracardiac shunting and valve function, but it may not be tolerated in conscious patients. The use of transthoracic echocardiography is usually more effective and meaningful.In these 2 cases, regurgitation of multiple valves and pericardial effusion were present, and we think vigilant monitoring of the hemodynamic status, heart sounds, and electrocardiography is essential for these patients. Continuous arterial and CVP monitoring were valuable in guiding our fluid therapy and infusion of vasopressors. Pulmonary artery catheterization is a helpful technique for intensive bedside hemodynamic monitoring, which has been believed to be necessary in patients with cardiogenic shock, for the differential diagnosis of pulmonary arterial hypertension, and for diagnosis and treatment of uncommon causes and complications of heart failure. However, its routine use is not necessary and may be associated with increased complication. It is not clear when corrective surgery should be performed in a pregnant woman with ruptured SVA, which involves the safety of both the mother and the fetus. Both our patients were close to full-term, so decision of cesarean delivery before ruptured SVA repair was appropriate. Had the patients presented earlier in pregnancy, the decision to terminate pregnancy would involve the balance between the risk of early delivery to the fetus and the hemodynamic consequences with continuing gestation to the mother. In situations in which the baby is viable, it may be in the best interest of the mother and baby for the obstetrician to perform a cesarean delivery before the cardiac surgery, with the parents’ informed consent. Percutaneous transcatheter closure is an effective minimally invasive alternative for ruptured SVA correction, and may be a good alternative for patients of earlier pregnancy. However, it was unavailable at the time in our hospital.For patients with uncorrected ruptured SVA, the average survival time after diagnosis is 3.9 years, supporting the indication for surgical repair.For our patients, close observation and early mobilization without anticoagulants for several days after delivery was allowed before surgical correction of the aneurysms, to minimize the risk of hemorrhage from the placental site. To date, there is no consensus on the timing of subsequent surgical correction of SVA. On the one hand, at very short interval, there is the risk of more complicated perioperative course and outcome of the second surgery , whereas on the other hand, there is always a risk of worsening of the underlying disease in the postpartum period.4Use of a multidisciplinary team to guide anesthetic management is helpful and necessary, and both general anesthesia and incremental epidural anesthesia can be safely used for parturients with ruptured sinus of Valsalva aneurysm.


Four lines from a segregating doubled haploid population derived of a cross of the wheat elite cvs. Avalon and Cadenza were chosen as they showed pairwise differences in either N utilization efficiency (NUtE) or senescence timing. 108 annotated metabolites of primary metabolism and ions were determined. The analysis did not provide genotype specific markers because of a remarkable stability of the metabolome between lines. We speculate that the reason for failing to identify genotypic markers might be due to insufficient genetic diversity of the wheat parents and/or the known tendency of plants to keep metabolome homeostasis even under adverse conditions through multiple adaptations and rescue mechanism. The data, however, provided a consistent catalogue of metabolites and their respective responses to environmental and developmental factors and may bode well for future systems biology approaches, and support plant breeding and crop improvement.Progress in improving crop growth is an absolute goal despite the influence multifactorial components have on crop yield and quality. An Avalon × Cadenza doubled-haploid wheat mapping population was used to study the leaf metabolome of field grown wheat at weekly intervals during the time in which the canopy contributes to grain filling, i.e., from anthesis to 5 weeks post-anthesis. Wheat was grown under four different nitrogen supplies reaching from residual soil N to a luxury over-fertilization (0, 100, 200, and 350 kg N ha Among the abiotic parameters necessary for plant growth and development a balanced nutrient supply is essential , Harpenden, U.K.) in 2010/11. The population of doubled-haploid individuals was developed by Clare Ellerbrook, Liz Sayers and the late Tony Worland (John Innes Centre), as part of a Department for Environment, Food and Rural Affairs (Defra)-funded project led by ADAS . The four lines, 100, 116, 122, and 181 were selected and chosen for this experiment based on their pairwise contrasting responses to nitrogen  and post-anthesis senescence  in previous field trials (unpublished). For this experiment, wheat was grown at four N fertilizer levels of 0, 100, 200, and 350 kg N ha−1 supplied as ammonium nitrate in two applications and in three randomized replicate block/split-plot design at the Black Horse and Bylands fields  at Rothamsted Research. The plot size was 15 × 3 m non-destructive for yield measurement and 1.5 × 3 m destructive for sampling. Under UK conditions, N0  would be considered deficient, and N200 sufficient for average yields (8–10 t/ha). Plant growth was monitored for the onset of anthesis. For metabolome and chlorophyll analyses, 10 2nd leaf (from top) samples per replicate were sampled immediately in liquid nitrogen at anthesis and consecutively on a weekly basis thereafter until 5 weeks post-anthesis with nearly complete senescence. Grain yield, straw yield and %N in the grain and the straw were recorded at final harvest at the end of the growing season.Among in total 25 wheat varieties four selected Avalon × Cadenza doubled-haploid lines were grown in a field trial at Rothamsted Research (grown in the Defra WGIN project (The concentration of chlorophyll (Chl) content per unit area was estimated in attached leaves by taking SPAD-502  meter readings weekly on the second fully expanded leaf, midway along the leaf blade and half way between the central vein and the leaf edge. Measurements were made on leaves which were subsequently harvested for the metabolite analyses. Leaf greenness is an accepted proxy for leaf nitrogen contents Lawlor, .665 – 2.07A645, Chl b = 9.29A645 – 2.74A665, where A645 and A665 were absorbances at 645 nm and 665 nm, respectively . Extracts were combined. The Chl contents of the combined extracts were measured with a Gemini XPS Fluorescence Micro plate Reader . The contents of chlorophyll a (Chl a) and chlorophyll b (Chl b) were calculated with the following formulas: Chl a = 5.21Ay Arnon, .2O) used for quantitative internal standardisation of the polar phase. The extract was heated for 15 min at 70°C before 200 μl chloroform was added to the polar phase. 400 μl H2O was added to the mixture after shake incubation at 37°C for 5 more minutes, vortexed vigorously before centrifugation to separate the polar and non-polar phases. Aliquots of 100 μl each were dried in a speed-vac. Primary metabolites, amino acid and ion contents were measured from dried extracts.The extraction of metabolites was performed by methanol/chloroform extraction as described previously  pH 6.2. The samples were centrifuged at 20,000 g for 30 min at 4°C. Amino acids were measured by precolumn derivatisation with ortho-phthaldialdehyde in combination with fluorescence detection   was performed as described previously  was used for most of the statistical analyses in this study. In the metabolite correlation analysis, each metabolite was normalized by dividing its average from all conditions . The R “stats” package was used to implement the Pearson correlation analysis. For the principal component analysis (PCA), the biological replicates of each metabolite were firstly averaged as its metabolite expression level, and then each metabolite was normalized by dividing its average from all conditions. PCA was implemented using R package “pcaMethods”  and higher, significantly increased grain yield in all four lines. N application levels higher than 100 kg N ha−1 (N200 and N350) did not further improve grain yield significantly for lines 100 and 181, while lines 116 (higher NUtE) and 112 (delayed senescence) achieved slight but significantly higher grain yields at N200 and N350. It is noteworthy that no yield increase could be achieved with applications beyond 200 kg N ha−1 as an application of 350 kg N ha−1 did not further affect performance under field conditions. Although no increase of grain yield in kg ha−1, the high N supply increased nitrogen contents in the grain  different levels of N-supply, (ii) developmental series during grain filling, and (iii) reveal genotype specific differences i.e., markers.in vivo on site .−1 the components tend to be spread quite far from one another without any clear tendency for any of the two lines which indicates a high variation within the data set. The calculation of the Euclidian distance of the data set in relation to N supply and time point  showed that principal component 1  separates data mainly with respect to N levels while PC2 (25.4% of variance) separates data to due time point of harvest and thus developmental stages, essentially plant age Figure . It is nt Figure  insert at Figure .−1 the components tend to be spread quite far from one another. Here, the slow senescing line 112 is clearly much stronger separated by PC2 (N-supply) from its corresponding data point of line 181. This indeed indicates a correlation between delayed senescence and effect of N application on line 112. The calculation of the Euclidian distances of the data set in relation to N supply and time point  shows that principal component  separates data due to N levels while PC2 (30.3%) separates data mainly with respect to time and thus developmental stages Figure . Also fot Figure  insert dt Figure .The PCs are determined by the contribution of all metabolites. The impact of a distinct metabolite on the principal component is captured in its loading value. The plots of the PCA loadings Figures , 4B dispBased on a three-way ANOVA analysis including as factors (i) genotype, (ii) time , and (iii) treatment (N-supply environment), the factors time and treatment were identified as influencing metabolite levels most significantly. By comparison, genotype had a negligible effect Figure  and as aTo assess metabolic signatures of distinct metabolites a hierarchical cluster analysis was employed at all 4 N levels and within respective N-levels the six samplings are depicted Figure . At leas2-fold transformed values for each metabolite were clustered by computing the Euclidean distance on average linkage distance which defines an unweighted pair-group method using arrhythmic averages, i.e., the average distance between any two members  decrease at all N levels over time and show higher abundances the less N is supplied, i.e., stress compound accumulation occurs under reduced N-supply. Actually, “Cluster F” consisting of putrescine, citrate, and phosphate could be added to this group. Cluster F is strongly induced under N0 only, thus, being markers for nitrogen starvation in wheat leaves. Clusters B and C  display metabolites which are senescence related  of N100, N200, and N350. N200 is the common amount applied in the UK, while N350 is an oversupply. The second leaf from top was sampled and a metabolome analysis, mainly targeting primary metabolites and ions, was performed which yielded 108 annotated compounds. Non-annotatable peaks were not taken into consideration. The second leaf was chosen as a representative of wheat canopy supply to the grain not ignoring the fact that also other tissues contribute to grain filling  and a doubled haploid population derived of this cross previously scored for agronomical parameters. The lines, pairwise varying in NUtE  and senescence timing , were exposed to four different N fertilization regimes providing no (N0) additional N allowing the plant to only grow on residual remaining soil nitrogen which essentially corresponds to nutrient depletion and successively increased N  vs. 100 (high)] and delayed senescence [line 181 (early) vs. 112 (late)] which have been determined phenotypically in previous investigations of the separating population from the Avalon × Cadenza cross. Though metabolic signatures could be assigned for either varied N supply (environment) or sampling time point (development) metabolites which were valid enough to define them as metabolic markers describing the trait (genotype) and potentially serving as marker for future plant breeding, could not be identified. There were differences of metabolite contents detectable between lines; however, these were mainly gradual in nature and not decisive enough to provide a basis for a high throughput measuring system necessary for plant breeding.Metabolite determination has previously been shown to have the potential to provide such markers , nitrogen supply (E), and development (D) could be reduced to the two factors environment (E) and development (D) by fusing the dataset of all determined lines. Even though this dataset could not provide genotypic markers indicative of the traits under study; it provided a quite exhaustive catalogue of primary metabolites and ions which will be helpful in future for understanding wheat physiology in general and under field  conditions in response to nutrient availability.Individual metabolite signatures over time and in response to N supply were plotted next to a pathway map depicting the core primary metabolism Figure . The colMost of the metabolites associated with sugar metabolism, such as the monosaccharides fructose, glucose, and galactose displayed a response pattern strongly dependent on the N-treatment increasing strongly up to 2 weeks post-anthesis (wpa). The decreases in glucose and fructose levels occurred from 2 wpa onwards and were more apparent in N fertilised plants. Furthermore, glucose and fructose peaked earlier in plants grown at the high N (N200 and N350) than in those grown at low N (N0 and N100) levels. This response of glucose was absent in the N0 plants suggesting that these plants have impaired photosynthesis rates, as also chlorophyll levels are low, incapable of building up glucose and fructose reserves. However, even under these conditions (N0) sucrose accumulated, though total biomass was impaired and grain yield reduced. Such biomass reduction due to insufficient nutrient availability is a common response (Sieh et al., A tight interconnection exists between carbon and nitrogen metabolism (Stitt et al., While the early TCA cycle intermediates citrate, cis-aconitate, and isocitrate are gradually reduced over time, succinate, fumarate and malate accumulate which could be a result of catabolism of amino acids and other compounds, since their break down products may be fed into the TCA cycle through their conversion to the key C-skeleton, 2-oxoglutarate (Araújo et al., Nitrate contents in the leaves decreased at 2 wpa and parallel to this the first decreases in chlorophyll contents announced the start of senescence and transport of solutes to the developing grain. This highlights the tight regulation of C/N ratio with the onset of senescence (Foyer et al., Throughout the post-anthesis time-course, the amino acid response patterns can be assigned to three groups. Glu was the major amino acid pool in leaves of all plants at anthesis which is in accordance with other studies (Lopes et al., Phosphate was one of the metabolites displaying the strongest increases in N0 treated plants Figures , 9, and Hakea prostrata (Kuppusamy et al., It has also been documented that the transport of sucrose from the shoot to the root stimulates phosphate transporters to further enhance phosphate uptake (Liu et al., This investigation demonstrated that nitrogen starvation has wide-ranging effects on primary metabolism that are further influenced by processes occurring during post-anthesis development and defines grain yield. This study provides a metabolic map of those primary metabolites and nutrient ions that could be annotated in this study in response to N level during grain filling which will be useful for breeding for generating genetic diversity for improving nutrient use efficiency in general, and more specifically NUtE.The major findings in this study can be summarised as follows:This analysis could identify metabolites which are either responsive to nitrate availability, development or both. We could not identify genotypic markers for the agronomically identified traits high and low NUtE and early and late senescence probably due to the narrow genetic composition of the breeding lines or due to general processes of wheat metabolism thriving at keeping metabolome homeostasis.The nitrogen fertiliser level was a strong determinant of the %N in the grain and straw. Plants grown on nitrogen poor soil delivered very poor yields, while N levels of 100 kg/ha doubled the yield increase in comparison to the unfertilised plants but with low pervcentage of N content in both, straw and grain. Grain yields of plants that received more than 200 kg/ha nitrogen did not increase any further. It was possible to identify metabolites positively correlating to yield increase or %N in the grain or straw. Amino acids were expectedly affected strongest by perturbations in N level and displayed a strong positive correlation to yield attributes.RH and MH: designed the research; EH, PB, MW, and AE: performed the research; EH, PB, MW, GD, AE, JK, and DW: analysed the data; EH and RH: wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Numerous studies have investigated utility of esophageal intraluminal baseline impedance levels (BILs) in gastroesophageal reflux disease (GERD). However, effect of BILs in refractory GERD (RGERD) has not been well investigated. The aim of this study is to evaluate role of BILs in RGERD patients. Total 62 subjects with refractory gastroesophageal reflux symptoms underwent 24-hour impedance-pH monitoring and gastroendoscopy. Distal BILs in acid reflux type were significantly lower than those in nonacid reflux type and functional heartburn (FH) group. Distal BILs of reflux esophagitis (RE) patients were lower than those of nonerosive reflux disease (NERD) patients, while there were no statistical significance between 2 groups. Patients with severe esophagitis had lower distal BILs than those with mild esophagitis and NERD patients, and patients with severe esophagitis in acid reflux type had the lowest distal BILs. Distal BILs were significantly negatively correlated with DeMeester score, episodes of acid reflux, and acid exposure time, but no correlated with episodes of nonacid reflux. Characteristics of BILs in RGERD patients were similar with those in GERD patients, but might be more complicated. Evaluating BILs in RGERD patients could achieve a better understanding of pathophysiology in RGERD. Multiple elements have been proposed for play in the genesis of PPI-refractory symptoms: insufficient suppression of acid reflux; association with nonacid reflux; abnormal esophageal motility; esophageal hypersensitivity; and psychological factors.,3Gastroesophageal reflux disease (GERD) is considered as refractory when the improvement of symptoms with a standard proton pump inhibitors (PPIs) treatment for 12 weeks is <50%.–6 and enables classification of RGERD into reflux-related disease, including acid reflux and nonacid reflux types, and including functional heartburn (FH).,7 Although when there are no swallows or episodes of reflux, the resulting intraluminal baseline impedance level (BIL) reflects the primary electrical conductivity of the esophagus based on that the esophageal wall contacts directly with the MII-pH sensor catheter.Twenty four hour multichannel intraluminal impedance and pH monitoring (MII-pH monitoring) plays a very important role in detection of reflux episodes in GERD patients,–12 However, role of BILs in the pathogenesis of RGERD has not been adequately investigated until now. Recent study in Japan demonstrated that among patients with PPI-refractory nonerosive reflux disease (NERD), acid reflux group had lower BILs compared with nonacid reflux group and FH group. Martinucci et al showed that BILs of FH patients negative response to PPIs were lower than those of responders.Previous studies, which focused on GERD and FH diseases, have revealed that BILs in GERD patients were lower than FH patients and healthy people, and demonstrated that BILs are correlated with acid exposure time (AET) and mucosal injury of the esophagus, and suggested that BILs are considered as an indicator of the esophageal mucosal integrity.We hypothesized that BILs are altered in certain segments of the esophagus in RGERD patients such as GERD patients, and that the alterations are associated with reflux events and esophageal function. The aims of this study were: to evaluate the composition of BILs from proximal to distal esophagus in RGERD patients and compare the BILs among patients with different reflux type; to evaluate their relation to different reflux events and acid-related parameters in RGERD patients; and to assess whether BILs are associated to erosive esophagitis.22.1 lasting >6 months, and their reflux disease questionnaire scores were not less than 12. They met the criteria of RGERD which improvement of symptoms was <50% after they were treated with omeprazole 20 mg or rabeprazole 10 mg bid for at least 12 weeks. All subjects underwent endoscopy in 1 month before analysis, the degree of esophageal mucosal injury was graded according to Los Angeles classification (LA A–D). Patients were excluded if they had a history of peptic ulcer, gastrointestinal tumor or surgery, or severe esophageal motility disorders. In addition, subjects were also excluded if they were found to have abnormalities other than erosive esophagitis or chronic superficial gastritis by gastroendoscopy. The study was approved by the Ethical Committee of First Affiliated Hospital of Nanjing Medical University.All patients referred to the First Affiliated Hospital of Nanjing Medical University outpatient clinics from January 2014 to December 2015 in this retrospective study. Inclusion criteria included age of 18 years or older and any gender. All patients had typical heartburn or regurgitation according to the Montreal consensus,2.22-antagonist, or prokinetic drugs therapy which could influence esophageal motor function or gastric acid secretion at least 1 week underwent MII-pH monitoring using an ambulatory monitoring system . The catheter was inserted into distal esophagus through nasal cavity. The catheter contained 6 impedance segments and 1 pH electrode sensor. The pH sensor was placed at 5 cm above the lower esophageal sphincter (LES) , and 6 impedance values  were recorded at 6 sites .Subjects of PPIs, H2.3The data of reflux events and parameters were measured by automatic analysis software of monitoring system. Two investigators (LJ and BY) blinded for the diagnosis of patients reviewed the tracings manually to ensure accurate detection and classification of reflux episodes and BILs. The meal periods were excluded from the analysis.; DeMeester score; and AET. Reflux type is usually divided into 3 categories: acid, weakly acid, and alkaline reflux. Therefore, in our study we characterized the reflux episodes as acid or nonacid , respectively, if the nadir pH reached <4.0 or was constantly ≥4.0. AET was defined as the distal esophageal total time with pH below 4, divided by the total time of monitoring. BILs were assessed in a manner blinded to the diagnostic results at 3 time points  between meals, avoiding close to any period of swallowing or reflux. Each single BIL represents the average level of 3 suitable baseline levels around each time point. We chose BILs from the 5th impedance site  to analyze their correction with reflux parameters, where there was a pH sensor to ensure that BILs were selected just when pH > 6.The following reflux parameters were analyzed in our study: the number and type of reflux episodes2.4,19,20In our study, the patients would be classified as having RGERD if reflux parameters were positive according to pH monitoring. On the basis of endoscopy results, RGERD patients were divided into reflux esophagitis (RE) and NERD. We subclassified RGERD patients into acid reflux and nonacid reflux types according to the level of AET and acid reflux episodes. Patients with RGERD were considered as acid reflux type if there were abnormal AET (>4.2%) and acid reflux episodes. Other RGERD patients were considered as nonacid reflux type. Patients with typical heartburn, negative endoscopy, and negative pH monitoring are classified as FH.2.5t test when there were 2 groups being compared and analysis of variance for difference in mean values. Post hoc comparisons were performed using the LSD correction in the case of significant analysis of variance (ANOVA) results. Correlation between BILs from z5 and reflux parameters were performed with Spearman's rank test 2-tailed). A P < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS version 19.0 for windows .Data are expressed as means ± SD. We used Student 33.1Total 62 patients with refractory gastroesophageal reflux symptoms were enrolled . All patients tolerated MII-pH monitoring and had no adverse events. Before MII-pH monitoring, 48 patients completed esophageal manometry to locate LES. RGERD was identified in 52 patients and was classified as acid reflux type (24 patients) and nonacid reflux type (28 patients). A total of 10(16.1%) patients were diagnosed as FH, 27 patients were diagnosed as RE (7 LA C/D and 20 LA A/B), and 25 patients were diagnosed as NERD. Detailed demographic data and MII-pH monitoring parameters are summarized in Table 3.2P < 0.01). Acid reflux type's BILs decreased from z1 to z6, BILs from z5 were lower than those from z1 to z4 , and BILs from z6 were lower those from z1 to z5 , while there were no difference among BILs of each site in nonacid reflux type   Table .P < 0.01). The lower BILs from z5 to z6 compared with FH group were consistent in acid reflux type . BILs from z6 in acid reflux type were the lowest value among all groups.Figure 3.3P < 0.05). Detailed characteristics of each group were summarized in Table BILs were lower in RE group than those in NERD group, but there was no statistical significance between 2 groups. DeMeester score, AET, and acid reflux episodes were significantly higher in LA C/D subgroup than those in LA A/B subgroup and NERD group. BILs from z4 to z6 were significantly lower in LA C/D subgroup than those in LA A/B subgroup and NERD group  and 10 LA A/B patients (LA A/B + acid reflux); within nonacid reflux type, there were 2 LA C/D patients (LA C/D + nonacid reflux) and 10 LA A/B patients (LA A/B + nonacid reflux). BILs with different degree esophagitis in different reflux type were summarized in Table 3.4r = −0.507, P = 0.000, n = 62)     Fig. A, with e4,3,21 and that those patients diagnosed as GERD were more related with nonacid reflux .–27 Consistent with the above studies, our findings showed 45.2% patients with refractory gastroesophageal reflux symptoms were associated with nonacid reflux and 16.1% patients were considered as FH. Our study specially aimed to determine role of BILs in RGERD patients.We acknowledged the possibility that some patients with refractory gastroesophageal reflux symptoms may have been misclassified owing to clinical examination limitations. Previous studies reported that patients with refractory gastroesophageal reflux symptoms often did not have GERD,,9,28 while in distal esophagus of patients with acid reflux or esophagitis were in the range of several hundreds of Ohms, and that distal esophageal BILs were significantly lower than proximal esophageal BILs.,29 In our study, we found that there was a decreasing tendency in BILs from proximal esophagus to distal esophagus in RGERD patients and patients with acid reflux type. But the lowest distal BILs of RGERD patients were nearly 2 thousands of Ohms, which were higher than those from above-mentioned studies. We have yet to figure out a clear explanation for our findings above. Anyway, we believe that composition of BILs in RGERD patients was more complex than that in regular GERD patients, because long-term PPIs or other medicines usage could lead to mucosal inflammatory improvement or recovery and esophageal mucosal injury may be just one of pathogenic factors of RGERD but not the most important one. Furthermore, the design of this retrospective study and small sample size might be related with this result.Previous investigations demonstrated that BILs by using MII-pH monitoring in healthy subjects were in the range of thousands of Ohms,,9 Zhong et al revealed that distal BILs in GERD patients with acid reflux were lowest, and followed by those with weakly acid reflux, alkali reflux, and normal population. Kandulski et al found that distal BILs in GERD patients were lower than those in FH patients. In our study, there were no difference in BILs between nonacid reflux group and FH group. We found total episodes of abnormal reflux in nonacid reflux group were significantly less than those in acid reflux group and similar with FH group. We think that less episodes of abnormal reflux could lead to relatively mild injury, and that a lot of medicines usage in RGERD patients could improve or even cure esophageal mucosal injury.Several studies showed that distal BILs of GERD patients with pathological acid reflux were markedly lower than those of healthy volunteers.–11,29,30 Overall, it is reasonable to hypothesize that BILs decrease in parallel with the severity of GERD, from healthy to NERD, and then RE. Consistent with above studies, our data revealed that patients with severe esophagitis had lower distal BILs than those with mild esophagitis and NERD, and that patients with severe esophagitis in acid reflux type had lowest distal BILs. However, our findings showed that there were no statistical significance in distal BILs between RE patients and NERD patients. Small sample size may be related with the results. Furthermore, long-term usage of PPIs might reduce difference of mucosal injury between 2 groups.Numerous studies reported that BILs were associated to esophageal inflammation in GERD patients, and distal BILs in RE patients were significantly lower than those in NERD patients.,31,32 suggested a possible role of acid, although at physiological levels, in GERD. The mechanisms responsible for reflux perception are not yet fully understood. In our data, we also showed there was no correlation between BILs and episodes of nonacid reflux. In this regard, acid reflux remains more important determinant in the generation of symptoms, although MII-pH studies have shown that both acid and nonacid reflux can generate symptoms.,24,33,34 We think BILs are related to the chemical clearance of the esophagus and concomitant with physiological levels of esophageal acid or nonacid exposure, but to our knowledge, we have no idea that how many episodes of nonacid reflux will be beyond the capacity of chemical clearance of esophagus. On the other hand, this finding maybe could explain partly why there was no response to PPI therapy in RGERD patients, possibly other factors such as visceral hypersensitivity or psychological disturbances may be more important than the severity of esophageal mucosal damage.We found that distal BILs were negatively correlated to acid-related parameters, such as episodes of acid reflux, DeMeester score, and AET. These data, in keeping with previous findings,According to our results, we believe that a more in-depth pathophysiological evaluation of MII-pH tracings by adding of BILs could be of help to better investigate and identify patients with RGERD. However, there still were limitations of our study were: our study was retrospective, we could not collect enough information such as smoking, drinking, BMI, severity of reflux symptoms, and detailed information about other medications; all patients came from our single clinical center, the numbers were limited, and we lack of regular GERD patients and normal population.In conclusion, based on our data, BILs were related with acid exposure and severity of esophagitis in RGERD patients, which was similar to that of regular GERD patients but may be more complex. Moreover, we believe that the assessment of esophageal BILs could represent a marker for acid or nonacid reflux induced changes to the esophageal mucosa. However, the results from our study warrant further research in RGERD patients to validate the measurement of BILs.


Rhodiola species, beginning with the Last Interglacial  through to 2050. Distributions of Rhodiola species appear to be shaped by temperature-related variables. The southeastern QTP, and especially the Hengduan Mountains, were the origin and center of distribution for Rhodiola, and also served as refugia during the LGM. Under future climate scenario in 2050, Rhodiola species might have to migrate upward and northward, but many species would expand their ranges contra the prediction of the “nowhere to go” hypothesis, caused by the appearance of additional potential habitat concomitant with the reduction of permafrost with climate warming.Climate change profoundly influences species distributions. These effects are evident in poleward latitudinal range shifts for many taxa, and upward altitudinal range shifts for alpine species, that resulted from increased annual global temperatures since the Last Glacial Maximum . For the latter, the ultimate consequence of upward shifts may be extinction as species in the highest alpine ecosystems can migrate no further, a phenomenon often characterized as “nowhere to go”. To predict responses to climate change of the alpine plants on the Qinghai-Tibetan Plateau (QTP), we used ecological niche modelling (ENM) to estimate the range shifts of 14  Rising temperatures of the last few decades have shifted the latitudinal and altitudinal ranges of many species2. This is particularly concerning for alpine species, for which there may not be sufficient suitable alpine habitats at higher altitudes to facilitate their migration. This phenomenon has been termed the “nowhere to go” hypothesis4. Chen et al.5 conducted a meta-analysis of over 1,000 species and found that the median rate of increased altitudinal range shifting was 11 meters per decade, while latitudinal range shifts showed a median rate of 16.9 kilometers poleward per decade . Lenoir et al.6 investigated 171 plant species in European montane regions, and found that mean elevation of ranges had moved upward by 29 meters per decade, coincident with rapidly rising temperatures occurring after 1986. These altitudinal shifts were faster for species adapted to montane regions than for more broadly-distributed species and for herbaceous plants compared to woody plants7. These studies collectively suggest that, under future climate warming scenarios, montane species, especially herbaceous plants, may face the aforementioned “nowhere to go” predicament. However, other researchers predict that global climate warming may drive snowlines upward, exposing new areas of suitable habitat that could facilitate alpine species’ upward migration; thus the notion of “nowhere to go” may be an oversimplification of the future of alpine taxa8. For example, Holzinger et al.9 reported that species richness of vascular plants in montane communities increased by 11% per decade under climatic warming. Clearly more research is required to fully explore the effects of global warming on alpine plants.Climate change has profoundly impacted the distributions of species across the globe11. The QTP and environs (like Hengduan Mountains (HM), Fig. 12. Recent phylogeography studies of QTP plants reveal that Quaternary glaciations, especially the Last Glacial Maximum ), have profoundly influenced the distributions of these species14. These studies rarely discuss how QTP plants will respond to future climate change, a major focus of our paper.The Qinghai-Tibetan Plateau (QTP) is the highest alpine ecosystem in the world, and perhaps one of the most sensitive to climate change. The temperature increase in the QTP has been faster than the mean temperature increases in the Northern Hemisphere as well as in other regions at the same latitudeRhodiola is a perennial herb genus comprising nearly 70 species, which are primarily distributed in the QTP and neighboring mountains15. Molecular phylogenetic analyses suggest that Rhodiola originated in the QTP about 21.0 Mya and rapidly diversified beginning 12.1 Mya coincident with the uplift of the QTP15. The genus subsequently expanded into adjacent regions, with a handful of species (e.g. R. rosea) dispersing to other parts of the globe16. The evolutionary history and contemporary geographic ranges suggest that Rhodiola species are adapted to low temperatures, making them an ideal model for investigating the response of montane herbaceous species to climate change. To date, the genealogical patterns of three Rhodiola species  of the QTP have been investigated19 and these studies suggested that contemporary Rhodiola distributions have been highly influenced by the LGM temperature where they expanded from refugia in the southeastern QTP and HM19. A limitation of these studies is that they do not utilize ecological niche modelling (ENM) with their molecular data, and thus exhibit only a cursory understanding of how changing environments might have shaped contemporary distributions and phylogeographic patterns.20. These environmental variables  constrain the distribution and abundance of a species22. Using occurrence data of a particular species, ENM can be used to project its ecological niche back into palaeoclimatic scenarios, and also into future climate scenarios, predicting potential distributional patterns under the assumptions of niche conservatism25.ENM is based on the concept of the ecological niche, which associates a set of environmental variables to the ability of a species to persist in an environment, and is related to the fitness of a speciesRhodiola15, we focus on 14 species with sufficient occurrence records for ENM analysis  test which climatic factors underlie the distributions and range shifts of Rhodiola species in different periods  ca. 120,000–140,000 years BP, LGM ca. 22,000 years BP, current, and future (2050), and 2) test the predictions of the “nowhere to go” hypothesis under future climate change scenarios.In this study, framing our research questions using a molecular phylogeny of Rhodiola species: (1) MEAN ENSEMBLE: using each Global Climate Model  to do ensemble ENM, and then calculated the mean and standard deviation of all predictions; (2) MMM ENSEMBLE: using the multi-model mean (MMM) of all the GCMs to do ensemble ENM; (3) The maximum entropy algorithm, Maxent: using the MMM of all the GCMs to do Maxent modelling. The Area Under the Receiver Operator Curve (AUC) of ensemble models (MEAN ENSEMBLE and MMM ENSEMBLE) ranged from 0.968 to 0.995 for individual species with our designated groups   values from 0.902 to 0.993, both statistics being significantly higher than would be expected at random  (Supplementary Table Rhodiola species), for each species group, and for constituent species of each species group revealed no significant differences , indicating that all three modelling approaches performed well irrespective of the grouping.We used three modelling approaches to predict the distributions of Rhodiola species were temperature-related  , and GQTP (species mainly confined to the QTP); whereas Mean Temperature of Wettest Quarter (BIO8) was the most important variable for the distributions of R. alsia, R. dumulosa, R. himalensi, R. kirilowii, R. quadrifida and Gwide (species with relatively wide distributions). Mean Temperature of Driest Quarter (BIO9) was the top performing variable for predicting the distributions of R. chryanthemifolia, R. forrestii, R. henryi, R. wallichiana, R. yunanensis, WHOLE, Gclade2 (species from the second major clade in the Rhodiola phylogeny), GHM (species mainly distributed within the HM), Gplain (comprising species not found in the QTP and HM), Gmon (comprising monoecious species), and Gdio (comprising dioecious species).The most important bioclimatic variables associated with the distribution of The principal components analysis (PCA) of temperature variables generated two temperature axis (Temp1 and Temp2) that explained 55.6% and 32.1% (87.7% cumulatively) of the total variation in the temperature dataset. The PCA of precipitation variables produced two precipitation axis (Prec1 and Prec2) that explained 62.3% and 25.6% (87.8% cumulatively) of total variation in the precipitation dataset. BIO8 (Mean temperature of wettest quarter) and BIO9 (Mean temperature of the driest quarter) both had the largest loadings (contributions to the summed variance) for Temp 1 and were both positive. Mean Diurnal Range (BIO2) and Isothermality (BIO3) showed the highest loadings for Temp2  using Temp1, Temp2, Prec1 and Prec2 produced three discriminant functions, the first explained 76.9% among-group climatic variance, the second 20.1% Fig. .Figure 2plain and GQTP from other groups  and Isothermality (BIO3), whereas GQTP had the highest first discriminant function relating to highest Mean Diurnal Range (BIO2) and Isothermality (BIO3). For Gplain and GHM the second discriminant function distinctly separated these groups from others. GHM individuals tended to score highest on the second discriminant axis, while Gplain individuals tended to have intermediate scores, where these two groups had higher values for Mean Temperature of Wettest Quarter (BIO8) and Mean Temperature of Driest Quarter (BIO9).The Wilks’ Lambda value for each CDA axis was significant at α = 0.05, suggesting that at least some of the 14 focal species occupied geographic ranges that were climatically distinguishable. The first discriminant function best separated GRhodiola varies among species and groups under four climate scenarios: LIG, LGM, current and future (2050 RCP8.5)  Table –S4. MEAN5) Table , S8, whiRhodiola species expanded their ranges; an exception, R. henryi, was predicted to have undergone range contraction over this same period , whereas the results from Maxent showed that most species would have expanded their respective ranges Table , migratiiod Fig. –S4.TableRhodiola species are predicted to expand their ranges in MEAN ENSEMBLE, MMM ENSEMBLE, and Maxent, with the remaining species predicted to exhibit range contractions; this was especially evident for R. quadrifida for which all three modelling approaches predicted range contraction. In addition, we found that ranges were projected to shift more extensively (two or three orders of magnitude) between the current and future scenarios compared to between the LGM to current time points  m/year, 0.495 (±0.521) m/year and 7.07 (±5.42) m/year estimated using MEAN ENSEMBLE, MMM ENSEMBLE and Maxent, respectively. The three modelling approaches predicted that the mean northward rate of range movement should be ca. 0.288 (±0.288) km/year, 0.384 (±0.192) km/year and 1.24 (±1.19) km/year for . Maxent had relatively higher predictions than the other approaches. In contrast, some species displayed southward movement, including R. yunnanensis, R. alsia, R. himalensis , and Maxent  , and also applied each GCM to do ENM (MEAN ENSEMBLE), and then calculated the mean and standard deviations for all predictions. The predictions of MEAN ENSEMBLE for the LGM and 2050 scenarios overlapped those of MMM ENSEMBLE, implying that modelling with the MMM of the GCMs was reasonable28. We also found that Maxent30 models for Rhodiola outperformed other sub-model algorithms and predictions generally agreed with those from ensemble models, although Maxent consistently predicted more significant range shifts under future climate warming scenarios. Oppel et al.31 also reported that Maxent predicted larger areas than ensemble models, the cause of the difference between Maxent and ensemble models calls for further studies.Assertions of historical or future range shifts of species largely depend on the reliability of climatic data used and the ENM modelling process. In order to consider the range of plausible climates and of possible predictions by different modelsRhodiola species for which there are molecular phylogeographic information  were consistent with published studies, suggesting that the southeastern QTP and HM were the center from which Rhodiola lineages spread to occupy broader geographic ranges from the LGM to the present day  is similar to estimates of upward movement rate for the tree line in the Baima Mountains of the HM 37 and montane herbaceous plants in Europe . We have no estimates for rate of latitudinal movement in plants, although the northward rate of range shifting of Rhodiola (0.288~1.24 km/year) is comparable to that of animals (1.69 km/year)5.From the LIG to the LGM, concomitant with climate cooling, most LGM Fig. –S4. Alth4, most Rhodiola species broadened their geographic ranges rather than contracting with future climate warming  in the northern and western parts of the QTP remain uncolonized. Second, warmer temperatures and higher precipitation under future warming conditions could transform unsuitable regions, such as those currently with permafrost, into suitable habitats for Rhodiola at higher latitudes and elevations. Moreover, increased vegetation coverage could also play a role in expansion of Rhodiola species ranges. Cannone et al.41 reported that vegetation coverage, particularly that of shrubs, increased dramatically in the alpine belt of Italian Central Alps from 1952 to 2003, coincident with higher precipitation and diminution of permafrost. The extent of shrub coverage has also increased in the Arctic over the past 50 years42. Rhodiola species typically co-occur with shrubs15, and thus we predict that areas suitable for colonization by Rhodiola will be increasingly available as shrub vegetation expands in the QTP and environs. Overall then, our results provide evidence that vascular plant species richness in montane communities will increase with climate warming9.Contrary to the expectations under the “nowhere to go” hypothesising Fig. –S4. DeChR. quadrifida would have a more restricted geographic range under future climate warming . Sister species and closely related species existing in sympatry should have distinct niches for them to coexist, and thus are expected to respond differently to climate change46. In our study we did detect significantly different patterns of range shifting between sister species and related species within particular clades  and biotic factors (e.g. evolutionary history and interspecific competition)ies Fig. .Rhodiola species distributions, implying that cold-adapted plants exhibit sensitivity to climate warming. Results from our PCA and CAD analyses suggested that divergence in ecological niches accounted for the different distribution patterns among Rhodiola species  increased from the LIG to the LGM, and then decreased from the LGM to the current and future scenarios. This was in contrast to higher elevation taxa (e.g. GQTP) which were predicted to continue to increase from the LIG using Maxent (Supplementary Table 46.Model performance was higher for groups than for the genus in its entirety (WHOLE)  phylogeographic data availability, (2) variation in breeding system (dioecious or monoecious), (3) distinction in evolutionary history (sister group vs. different clades in phylogeny tree), (4) dissimilarity in spatial distribution patterns (dispersed or clumped over the QTP), and (5) availability of at least 39 occurrence records. Based on these criteria, a total of 14 species were selected for ENM analysis . Among these 14 targeted species, we included three pairs of sister species  that, despite overlapping ranges, show some allopatry15. The distributions of the 14 species can be classified into four groups: 1) GQTP = species for which the bulk of their distributions occur on the QTP , 2) GHM = species that are mainly distributed within the HM , 3) Gwide = species widely distributed from the QTP to northern China (R. dumulosa and R. kirilowii), and 4) Gplain, = not distributed on the QTP or other high elevation (<3000 m) regions (R. henryi)  and monoecy  (Table We selected riteria:  phylogeois Table . These sis Table 16 (Gclads) Table .www.gbif.org; Chinese Virtual Herbarium Data Portal, CVH: www.cvh.org.cn) and published research . We removed duplicate records and records with obvious errors in their geographic coordinates and thinned records that occurred within the same 1 km pixel so that only one occurrence remained. After pruning and verifying our data, we were left with 1,444 records for ENM spanning 39 to 200 unique locations  , current conditions (average from years 1950 to 2000) and 2050 RCP8.5 (average from 2041 to 2060) from WorldClim . Future climate data were accessed from Phase 5 of the Coupled Model Intercomparison Project (CIMP5)51, where we selected scenario 2050 RCP8.5, the highest emission scenario, so that we could forecast the warmest future environments for alpine species2. For LGM and 2050 which include multi-GCMs, the 19 bioclimatic variables were averaged for 4 GCMs for the LGM  and 5 precipitation variables )  statistics using Biodiversity R package in R version 3.1.2clade1, Gclade2, GQTP, GHM, Gwide, Gplain, Gdio, Gmon) and finally for all 14 species combined (WHOLE).We used three methods in our modelling process: (1) MEAN ENSEMBLE ; (2) MMM ENSEMBLE (using the multi-model mean (MMM) of all GCMs to do ensemble ENM); (3) Maxent . For each method, we did ENM for individual species, clade, distribution, and breeding system groups defined above  to calculate the predicted suitable area, mean elevation, and mean center of the predicted distribution in ArcGIS for each model based on 25% habitat suitability. The predicted suitable area was calculated using rectangular projections. We calculated the mean elevation of each suitable area in meters, and found the mean center of the suitable area by calculating the mean longitude and latitude .t represents the suitable area at time ‘t’; tn represents time for different periods ; ΔT equals the years from the periods tn to tn+1, ΔT1 ≈ 98,000 (from LIG to LGM), ΔT2 ≈ 22,000 (LGM to current), ΔT3 ≈ 75 (current to future) and for  or longitude and latitude of distribution center (ulon and ulat) and Vt equals the mean elevation or mean center at time ‘t’.The following equations were used to estimate the range shifts between different time periods:entclass1pt{minima49.Wilcoxon rank sum tests were used to evaluate whether models differed in AUC values for models with all clades, breeding and distribution groups, individual species, and genus29, with higher gain indicating a variable that contributes highly to the species’ spatial distribution. For a better understanding of the contributions of temperature and precipitation variables to the distribution of Rhodiola species, we first performed separate Principal Components Analysis (PCA) on the four temperature variables and five precipitation variables using records for all species (WHOLE)49. These ordinations produced two major temperature axes (Temp1 and Temp2) and two major precipitation axes (Prec1 and Prec2). To further reduce the dimensionality of the PCA data, we then used the scores of PC axes to do canonical discriminants analysis (CDA) (considering both temperature variables and precipitation variables) for GQTP, GHM, Gplain and Gwide . Additionally, χ2 tests were used to compare Gmon and Gdio, as well as between groups pairs from GQTP, GHM, Gplain and Gwide to examine the effects of breeding systems and ecological niche differentiation on the response to climate change. These analyses were performed using SPSS Statistics ver. 22.To test the influence of evolutionary history on range shifts induced by climate warming, we did χSupplementary Information


Drosophila melanogaster, high-altitude melanism has evolved in separate mountain ranges in sub-Saharan Africa, potentially as an adaptation to UV intensity. We investigated the genetic basis of this melanism in three populations using a new bulk segregant analysis mapping method. We identified 19 distinct QTL regions from nine mapping crosses, with several QTL peaks overlapping between two or all populations, and yet different crosses involving the same melanic population commonly yielded distinct QTL. The strongest QTL often overlapped well-known pigmentation genes, but we typically did not find wide signals of genetic differentiation (STF) between lightly and darkly pigmented populations at these genes. Instead, we found small numbers of highly differentiated SNPs at the probable causative genes. A simulation analysis showed that these patterns of polymorphism were consistent with selection on standing genetic variation. Overall, our results suggest that, even for potentially simpler traits like pigmentation, the complexity of adaptive trait evolution poses important challenges for QTL mapping and population genetic analysis.Unraveling the genetic architecture of adaptive phenotypic divergence is a fundamental quest in evolutionary biology. In  And yet, genome-wide association studies (GWAS) of female abdominal pigmentation variation  within four temperate populations all revealed major effects of the two melanin synthesis genes tan and ebony and the transcription factor bric-a-brac 1 (bab1), although other minor effect genes were also detected  and showed evidence of a strong partial selective sweep (Pigmentation displays strong geographic trends in Drosophila (ebony in Ethiopia) was found to be consistent with natural selection acting on standing genetic variation.Here, we investigate the genetic basis of melanic flies in three populations from Ethiopia, Cameroon, and Uganda, presenting the first application of a bulk segregant analysis (BSA) approach designed for osophila . Many ofThe populations in the present study were all studied by 1 flies produced from a cross between a darkly pigmented EF, CO, or UG line and a lightly pigmented ZI line, respectively . All parental lines had been inbred for eight generations. Each mapping cross between a melanic population and Zambia involved distinct inbred lines . To choose the parental lines, we tested lines from each population for the presence of eight common chromosomal inversions by PCR using primers and amplification conditions from In(2L)t, In(2R)NS, In(3L)P, In(3R)P, In(3R)K, and In(3R)Mo), and two sub-Saharan inversions on the X chromosome (In(1)A and In(1)Be). We also used the pigmentation score data of the fourth abdominal segment of females that we generated in We set four, three, and two experimental crosses each seeded with F1 males and 125 random F1 females were mixed together (N = 500). After combining the F1 flies from both reciprocal crosses, offspring were allowed to interbreed for 20 nonoverlapping generations at a population size of ∼1500 individuals in 28 × 14 × 15-cm plastic cages, each provided with 14 vials with standard Drosophila medium  and kept at ∼20°. Each generation, adults were allowed to lay eggs on the food for 1 week and then discarded, and food vials were replaced when the first new adult flies in the cage were 7–10 days old. After 20 generations , we allowed adult flies to lay eggs on fresh food for 2 days before replacing the vials and waiting for F20 flies to emerge. We then visually phenotyped 3–5-day-old females (N = 600) for pigmentation on the fourth abdominal segment under CO2 anesthesia, focusing on either pigmentation intensity near the anterior margin  or the width of the posterior black stripe , as in N = 60) and the 10% lightest (N = 60) females.We followed the experimental design described in 20 pigmentation subgroups for each cross. For each library, genomic DNA was extracted from a pool of 30 females by chloroform extraction and ethanol precipitation. DNA was then fragmented using the Bioruptor sonicator (Diagenode), and paired-end libraries with approximately 300-bp inserts were prepared using the NEBNext DNA Library Prep Reagent Set for Illumina (New England Biolabs no. E6000L). Library concentration and quality were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies) and were sequenced at the UW–Madison Biotechnology Center on the Illumina HiSequation 2000 platform with 100-bp paired read lengths.Genome libraries were independently prepared for the parental lines and the four FD. melanogaster reference genome (release 5.57) using default parameters in BWA v0.6.2-r126 (http://picard.sourceforge.net). Alignment around indels was then improved using GATK v3.2  summarized the difference in parental strain ancestry between the high and low phenotypic pools. With respect to the melanic parental line allele, da was estimated asHp is the frequency of the major allele in the melanic parental line, Lp the frequency of the melanic allele in the nonmelanic parental line, Hf the frequency of the melanic allele in the F20 dark subgroup, and Lf is the frequency of the melanic allele in the F20 light subgroup. Only sites with parental strain frequency difference H – pLp ≥ 0.25 were considered. The five chromosomal arms  were subdivided into 2728, 3131, 2357, 2956, and 2935 windows, each of roughly 8.4-kb on average, whose boundaries were determined according SNP density in ZI genomes  and secondary QTL peaks that may flank them  for its effect size (the proportion of phenotypic variance explained) and genomic location. Empirically-observed secondary peaks are also compared against these single QTL simulations, asking how often the observed “secondary deviation”  is generated when no secondary QTL is actually present. That analysis yields a P-value for each secondary peak (the null probability that the associated primary QTL would generate the observed secondary deviation by chance). If significant secondary peaks are present, simulations of multiple linked QTL are conducted. Rejection sampling conditioned on each peak’s height is used to refine the strength and location estimates of the primary and secondary peaks while accounting for these linked peaks’ effects on each other’s da values  are conducted to assess the significance of primary peaks. Peaks with at least a 95% true positive probability are carried forward in the analysis. For each of these significant primary peaks, simulations with a single QTL are conducted, with rejection sampling conditioned on the peak’s d values .STF for these windows between Zambia and the melanic population in question. Genomes from ZI (n = 197), CO (n = 10), EF (n = 68), and Uganda (n = 40) were obtained from the Drosophila Genome Nexus  of the window STF relative to the empirical distribution of STF of all windows per chromosomal arm, where Q denotes the proportion of windows with an equal or greater STF value. Strong selective sweeps from new mutations are expected to produce high window-based STF values due to hitchhiking effect, but such outliers may not include soft sweeps targeting alleles that already occur on multiple haplotypes; STF in each window and the quantile of this value according to the empirical distribution of this estimate for all windows per chromosomal arm.To test whether significant QTL contained highly differentiated windows between the two populations from which the parental lines were drawn, we estimated D. melanogaster populations  implies an instantaneous growth of the ancestral population (ZI) prior to an initial split, with a bottleneck taking place in the ancestor of the RG and EF populations. A second split between the two derived populations takes place followed by a bottleneck and a growth phase in each descendant population, with ongoing migration occurring among all populations. Parameters were optimized using δαδι and several runs were conducted to confirm that convergent parameter estimates were obtained. We then used nonparametric bootstrapping to infer parameter uncertainties. For this, we generated 100 bootstrapped AFS from the empirical data, and parameter SD were estimated using the Godambe Information Matrix (GIM) approach as implemented in δaδi version 1.7. Ancestral effective population size (eN) was inferred by dividing δaδi-estimated Watterson’s θ over four times the mutation rate, using an estimate from D. melanogaster of 3.27 × 10−9  and the frequency of the favored allele at the start of the sweep. In these simulations, we explored the hypothesis that the most differentiated SNP observed at ebony was a beneficial mutation (in Ethiopia only), and we set the target of selection to be the middle of the simulated 5001-bp locus. We simulated 10 different selection strengths and 6 starting beneficial allele frequencies for a total of 60 scenarios of selection. The 10 selection coefficients we studied were 0.00005, 0.000075, 0.0001, 0.0025, 0.005, 0.0075, 0.001, 0.0025, 0.005, and 0.0075. The six starting beneficial allele frequencies were 1/2Ne, 0.005, 0.01, 0.025, 0.05, and 0.1.We first ran 10,000 neutral simulations to test whether the values observed at STF as low as that observed at ebony. To emulate the observed SNP frequencies in simulations with selection, we employed a subsampling strategy. We initially simulated larger samples of chromosomes for the Zambia (n = 250) and Ethiopia (n = 50) populations, then pared them down to 189 and 33 chromosomes, respectively. Subsampling was then pursued to achieve a nontrivial probability of a simulation matching our observed allele counts in both populations. Simulations lacking sufficient numbers of both alleles in either population to achieve these subsamples were rejected. Although we used the –SFC flag of msms to condition on the beneficial allele’s persistence in the Ethiopian population at the time of sampling, simulations could still be rejected if this allele was unsampled or sampled at low frequency. For each selection scenario, we ran a maximum of 2,500,000 simulations in an attempt to get 1000 accepted simulations.In these simulations, our primary interest was to ask which models could replicate the observed maximal SNP frequency difference while generating a window Ne (corresponding to new mutations), only hard sweeps could be generated. For all other initial frequencies, we required at least two unique copies of the beneficial allele to be present at sampling, to study soft sweeps specifically.We also filtered simulations to clearly distinguish between the predictions of hard sweeps and soft sweeps. For the case of an initial beneficial allele frequency of 1/2Table S1.Raw sequence data are available from the NIH Short Read Archive with accession numbers given in i.e., SIBSAM. We performed nine mapping crosses, each pairing an inbred line from one of our melanic populations  with an inbred line from a lightly pigmented ancestral range population (Zambia). Mapping focused on two female pigmentation traits, A4 abdominal background color and A4 stripe width. For each cross, offspring interbred for 20 generations at a population size of ∼1500. In the final generation, the 10% darkest and lightest flies among 600 scored females were collected and sequenced as bulk pools for each cross.We used a newly-developed BSA method  to map pMaterials and Methods; A key goal of SIBSAM is to distinguish two or more linked QTL with overlapping mapping signals, which may occur when complex traits are mapped on this experimental scale. SIBSAM uses detailed experimental simulations in a three phase approach to make QTL inferences , each with a false positive probability below 5% (Table S2). Effect sizes ranged from 6.29 to 37.11% for primary peaks and from 5.66 to 25.92% for secondary peaks. Based on overlapping genomic confidence intervals, these can be summarized into 19 major QTL regions, ranging from 4-kb to 14-Mb long, of which 10 were unique to different single crosses  was shared among five crosses. This region consists of the 2.4-Mb telomeric end of chromosome X containing the important melanin synthesis gene volution , althougjing, which has been shown to affect abdominal pigmentation (bab1 and was shared by one cross from each of the three melanic populations. The other region (Q18) was shared by two crosses from Ethiopia and one cross from Uganda, and includes the melanin synthesis gene ebony. In Uganda, a strong effect of ebony on pigmentation has previously been illustrated  centered on cramped, which can influence abdominal pigmentation in a temperature-sensitive manner . While outliers for window STF could indicate loci subject to population-specific natural selection, some of these may be unrelated to pigmentation evolution. In light of the strong trend toward major QTL occurring near well-known pigmentation genes, we discuss the genetic differentiation of these genes in particular, in addition to the full QTL regions that contain them.To investigate whether pigmentation-associated peaks harbor genes that have been strongly differentiated between the lowland ZI population and each of the melanic populations, we estimated their differences in allele frequencies using ebony in Uganda ; however, genetic differentiation tends to be broadly elevated across this low recombination telomeric region, so genetic variation at yellow should be interpreted with caution. Elevated STF was also detected for a window upstream of the two transcription factor paralogs bric-a-brac , which may affect pigmentation , Q10 . Three peaks of differentiation (Q < 0.05) were found at Q8 surrounding the melanin synthesis gene black but no peak at the gene itself . None of these peaks contain any known pigmentation-related genes.In Cameroon, significant differentiation was found within five QTL detected in this population, including windows similar to that found in Uganda within Q1  in the QTL of this population did not show strong signals of window genetic differentiation, including ebony in spite of the major effect of this QTL in the cross with the darkest phenotype.In Ethiopia, strong differentiation was found in all but 2 (Q10 and Q16) of its 12 QTL, but in general these peaks did not fall near pigmentation candidate genes. The four melanin synthesis genes  for Ethiopia vs. Zambia. In spite of the lack of strong window STF signal cited above, all four of these genes had one or more individual SNPs with strong differentiation, yet without any strong pattern of differentiation at linked SNPs  fell in the first intron. Although this region is not known to affect abdominal pigmentation in cosmopolitan D. melanogaster, in D. pseudoobscura and D. virilis, two species that are completely dark and whose phenotype resembles the dark Ethiopian phenotype, intronic enhancers affect body pigmentation  which had often been detected in within-species studies focusing on the posterior abdominal segments in Drosophila , with an initial bottleneck to ∼1% of the ancestral population size followed by exponential growth. Although we could not investigate all possible historical models, and the effects of natural selection on genomic variation could impact the precision of these estimates, the demographic estimates obtained here serve our primary objective of providing a neutral model capable of generating population genetic data resembling that of our empirical populations.To identify a plausible null model, we used δaδi , from ouSTF and maximum SNP STF under this null model. Compared with neutral simulations based on this demographic model, we found that our window STF observed at ebony was only marginally elevated (Q = 0.07), but maximum SNP STF deviated more strongly from neutral predictions . Thus, simulation replicates in which the beneficial allele was fixed or else failed to rise much in frequency were rejected. For each simulation scenario, we tested how often the SNP STF entailed by the above counts was paired with a window STF at least as low as we observed for ebony.Next, we conducted simulations based on the hypothesis that the most differentiated SNP at STF criteria were soft sweeps with higher initial frequencies (Table S6). Weak hard sweeps could also match the STF disparity, but these scenarios almost never allowed the beneficial allele to rise to high frequency on the time-scale of the Ethiopian population (Table S6). Hence, selection from standing genetic variation appears to be the strongest hypothesis for the patterns of genetic variation observed at ebony in the Ethiopian population.The scenarios that best matched the above frequency and ebony is indeed related to pigmentation evolution, and whether other ebony variants may contribute as well. Contributions of multiple SNPs or haplotypes at a locus have previously been described for ebony in Uganda , or contributions to Ethiopia melanism from other SNPs not highlighted by our population genetic analysis. Different mutations at the same genes drive parallel pigmentation changes in humans . These QTL have estimated effect sizes above 0.33, a strength that resulted in 100% detection power in SIBSAM test simulations , none of these loci showed a clear window signal of high genetic differentiation. Instead, we found a recurring pattern of strong frequency differences limited to just one or a few SNPs. For ebony in Ethiopia, simulations confirmed that this pattern seems best explained by selection on standing genetic variation. Although the functional significance of our high STF variants remains to be confirmed, these narrow intervals of high genetic differentiation support the examination of individual SNPs in scans for local adaptation (e.g., While QTL often occurred near well-known pigmentation genes (D. melanogaster. These results provide one partial set of answers to the basic questions posed at the beginning of this article concerning the genetics of adaptation. However, it will be important to conduct similar studies of different adaptive traits in different organisms to assess the generality of our findings.Our work begins to provide one case study of the genetic architecture of adaptive trait evolution, while offering a foundation for detailed molecular studies to confirm the relevant genes and mutations responsible for parallel melanic evolution in Overall, our work highlights significant challenges to mapping the genetic basis of adaptive population differences. Even when relatively large effect loci are present, their detection may depend upon the specific genetic backgrounds used for QTL mapping and genetic targets of local adaptation may not be detected in typical population genetic scans. Nevertheless, continued advances in the analysis of genomic data and molecular methods for confirming adaptive variants have the potential to expand our understanding of the nuanced process of adaptive trait evolution.