Introduction: Evidence has shown that utilization of antiosteoporotic medications does not correspond with risk, and studies on other therapies have shown that adequacy of pharmaceutical prescribing might vary between regions. Nevertheless, very few studies have addressed the variability in osteoporotic drug consumption. We aimed to describe variations in pharmaceutical utilization and spending on osteoporotic drugs between Health Areas (HA) in Spain.Methods: Population-based cross-sectional ecological study of expenditure and utilization of the five therapeutic groups marketed for osteoporosis treatment in Spain in 2009. Small area variation analysis (SAVA) methods were used. The units of analysis were the 168 HA of 13 Spanish regions, including 7.2 million women aged 50 years and older. The main outcomes were the defined daily dose (DDD) per 1000 inhabitants and day (DDD/1000/Day) dispensed according to the pharmaceutical claims reimbursed, and the expenditure on antiosteoporotics at retail price per woman ≥50 years old and per year.Results: The average osteoporosis drug consumption was 116.8 DDD/1000W/Day, ranging from 78.5 to 158.7 DDD/1000W/Day between the HAs in the 5th and 95th percentiles. Seventy-five percent of the antiosteoporotics consumed was bisphosphonates, followed by raloxifene, strontium ranelate, calcitonins, and parathyroid hormones including teriparatide. Regarding variability by therapeutic groups, biphosphonates showed the lowest variation, while calcitonins and parathyroid hormones showed the highest variation. The annual expenditure on antiosteoporotics was €426.5 million, translating into an expenditure of €59.2 for each woman ≥50 years old and varying between €38.1 and €83.3 between HAs in the 5th and 95th percentiles. Biphosphonates, despite accounting for 79% of utilization, only represented 63% of total expenditure, while parathyroid hormones with only 1.6% of utilization accounted for 15% of the pharmaceutical spending.Conclusion: This study highlights a marked geographical variation in the prescription of antiosteoporotics, being more pronounced in the case of costly drugs such as parathyroid hormones. The differences in rates of prescribing explained almost all of the variance in drug spending, suggesting that the difference in prescription volume between territories, and not the price of the drugs, is the main source of variation in this setting. Data on geographical variation of prescription can help guide policy proposals for targeting areas with inadequate antiosteoporotic drug use. Osteoporotic fractures, and among them hip fractures – the more severe clinical manifestation of osteoporosis – account for the majority of fracture-related health care expenditure and mortality in men and women aged 50 years and over . AlthougThis might be due to the existence of considerable uncertainty about how to quantify the risk, who should receive densitometry testing, who should be treated, what treatment should be used, and for how long . FurtherThe lack of strong, coherent, and widely accepted guidance for pharmacological treatment aimed at preventing fragility fractures may result in a wide variation in the patterns of drug use among neighboring populations with very similar demographic and epidemiologic characteristics. However, studies examining within-country geographical variations in pharmaceutical prescribing have been scarce  until reThe aims of this study are to describe variations in pharmaceutical utilization and spending on osteoporotic drugs between Health Areas (HA) in Spain in 2009 and to analyze the observed variation using small area variation analysis (SAVA) methods.Population-based cross-sectional ecological study of expenditure and utilization of the five therapeutic groups marketed for osteoporosis treatment in Spain.The units of analysis were the 168 HAs of the 13 Spanish Autonomous Communities (AC) participating in the study . These HAs, after excluding people not entitled to pharmaceutical benefits, covered a population of about 39.8 million inhabitants in 2009 (roughly 80% of the Spanish population at that time), of which 7.2 million were women aged 50 years and over.The sNHS – during the study period, previous to the 2012 healthcare reforms on entitlement and copayment – provided universal healthcare coverage through an extended network of public or public–private–partnership hospitals and primary healthcare centers with some features relevant to this study : health We selected all drugs (except zoledronate) marketed in 2009 in Spain for osteoporosis treatment categorized into five Anatomical Therapeutic Chemical (ATC) Classification groups: M05BA and M05BB , M05BX (strontium ranelate), G03XC , H05AA (parathyroid hormone and teriparatide), and H05BA . Zoledronate was not included because in Spain it is administered on an inpatient basis and there is no register of its use in the outpatient prescription data files.Utilization and expenditure data were supplied by the Regional Health Departments from their outpatient drug dispensation data files, containing information on the pharmacy claims reimbursed by the regional healthcare services in 2009. This database includes a description of the drug dispensed , the patient’s coverage (full or 40% copayment), and the HA in which the prescription was made. The databases transferred to the research team did not contain any information on individual patients. Figures about population entitlement in each HA were obtained from the Health Departments’ entitlement registries.W50/Day from here on.This was the defined daily dose (DDD) per 1000 inhabitants and day (DDD/1000/Day) dispensed in each HA according to the pharmaceutical claims reimbursed by the regional governments. The DDD is a technical unit of measurement used in drug utilization studies and assigned by the WHO Collaborating Center for Drug Statistics Methodology , equivalIndirect Standardized Drug Utilization Rate (ISR), obtained by comparing the observed DDD/1000W50/Day dispensed in each HA with an estimate of the expected DDD/1000W50/Day in the same HA if its actual population had the same stratified age and copayment drug utilization registered in a given reference population (the region of Madrid).(1) The pharmaceutical expenditure on osteoporotic drugs at retail price per women aged 50 years and over and per year; (2) the age–copayment The study, observational in design, uses retrospective anonymized non-identifiable and non-traceable data transferred to the research team meeting the requirements of the institutional providers. It is also in accordance with the CIOMS-WHO International Ethical Guidelines for Epidemiological Studies  and the Indirect Standardized Drug Utilization Rates in each HA were estimated by each therapeutic class as ith age–copayment group in a reference population in which these parameters were known. We used the available specific rates from the Madrid Region but recalibrated them to the observed total consumption of the 168 HAs. The indirect method used does not allow the comparison of one HA with another, but does admit a comparison with the reference pattern that by construction is equal to the mean utilization rate of the sNHS for the respective drug. Therefore, an ISR such as 1.50 for a given HA is interpreted as meaning that this HA has a 50% higher consumption of the respective drug than the sNHS average.First, we estimated the DDD/1000W50/Day by HA. Second, expenditure in each therapeutic class was aggregated by HA and divided by the number of women aged 50 years and over and by DDD dispensed to obtain the annual osteoporotic drug cost per woman aged 50 years and older. Subsequently, A descriptive analysis of drug use (crude and standardized) and spending per woman was carried out presenting the mean, median, and the values of the HAs in percentiles (P) 5, 25, 75, and 95. Variability among HAs was analyzed using the usual SAVA statistics  but, in Table 1). Seventy-nine percent of the antiosteoporotics consumed were bisphosphonates (91.9 DDD/1000W50/Day) followed by raloxifene (11.2 DDD/1000W50/Day), strontium ranelate (7.1 DDD/1000W50/Day), calcitonins (5.0 DDD/1000W50/Day), and parathyroid hormones including teriparatide (1.6 DDD/1000W50/Day). Regarding variability by therapeutic groups, biphosphonates showed the lowest variation (twice as high in the HA in the 95th percentile compared to that in the 5th percentile) while calcitonins and parathyroid hormones showed the highest variation . The standardization by age and copayment did not substantially modify the variability between HAs . Biphosphonates, despite accounting for 78.7% of utilization, only represented 62.9% of total expenditure on osteoporosis drugs, while parathyroid hormones with only 1.6% of utilization accounted for 14.9% of the osteoporosis pharmaceutical spending. Biphosphonates showed the lowest variation in expenditure (2.1 times higher between HAs in the 95 and 5 percentiles), followed by raloxifene (EQ5-95: 4.2) and strontium ranelate (EQ5-95: 5.4), while calcitonin (EQ5-95: 8.4) and parathyroid hormones (EQ5-95: 15.3) showed the highest variation. As with the utilization rates, the standardization by age and copayment did not substantially modify the variability in expenditure per woman between HAs (Supplementary Table Table 3) were, in general, moderate but positive and significant. Three pairs  showed coefficients above 0.40. The ACs explained a substantial part (14%) of the variance in osteoporosis drug utilization rates (Table 4), with differences between therapeutic classes, ranging between 15% in the case of biphosphonates and 54% in the case of parathyroid hormones. Supplementary Figure Spearman correlations between the consumption of the different therapeutic groups  and rates of prescribing explained almost all of the variance in drug spending. Furthermore, despite finding a moderate correlation between the use of the different therapeutic groups, our study also shows that antiosteoporotic drugs are prescribed as complementary treatments rather than as a substitute: HA, in general, uses all the groups of antiosteoporotics rather than choosing some groups over others.Regarding the variability between HA in the use of antiosteoporotic drugs, we found ratios at the 95th percentile more than doubling those at the 5th percentile for all antiosteoporotic drugs altogether, and ranging between a twofold difference for biphosphonates and an 18-fold difference for some costly drugs such as parathyroid hormones. These figures are similar to those reported in previous studies in Portugal  and NorwWe also found that variation in drug utilization is transferred almost entirely to variation in expenditure per capita  and rates of prescribing explained almost all of the variance in drug spending. This finding suggests that the different amounts of medications prescribed in each territory, rather than their price, are the main source of variation in osteoporosis drug spending in the sNHS setting, in contrast to a large US study using Medicare Part D data, concluding that regional variation in pharmaceutical spending results largely from differences in the costs of drugs rather than prescription volume . The disLastly, and although the correlation between the use of the different therapeutic groups is moderate, our study also shows that antiosteoporotic medications were used as complementary treatments rather than as a substitute: HAs, in general, use all the groups of antiosteoporotics rather than choosing some groups over others. This finding does not have a clear clinical rationale in osteoporosis, where, in contrast to other conditions such as hypertension, it is exceptional that two different drugs are used simultaneously in the same patient. This tends to amplify variations in global spending and has important implications for the design of practical policies aiming to reduce variability and pharmaceutical expenditure .The evidence accumulated shows wide geographical variation in healthcare utilization and spending, which is independent of patient characteristics and unrelated to the quality of care or patient outcomes . The resOur findings are subject to several limitations. First, the nature of the design  limits the generalization of the results from the geographically aggregated population to the individual basis. Second, the use of data on dispensed drugs may provide an imprecise estimate of a physician’s prescriptions (as some prescriptions issued might not be filled). Third, indirect adjustment for age and copayment may be insufficient to control for differences in the risk of being treated with antiosteoporotic drugs. Fourth, we assigned all DDDs consumed to women aged 50 years and over. Although variations between HAs in utilization in men or young women could affect our estimates of variability, we expect this strategy to have a minor impact on our figures, because the group of women aged 50 years and over is the population group that consumes the vast majority of anti-osteoporotic drugs. Fifth, the results presented correspond to 2009 and might not be extrapolated to the current situation of antiosteoporotic drug use and spending in Spain . A very recent study  shows trAlthough the level and patterns of use and expenditure might have changed, to our knowledge no other study has assessed the variability in anti-osteoporotic drug prescribing and spending in a comprehensive manner to date, across the Spanish territory, nor in other settings. Therefore, we are presenting novel results, with the most recent information available at the Health Area level regarding this relevant topic.In summary, this study highlights a marked geographical variation in the prescription of antiosteoporotic treatments in a setting with universal healthcare coverage, the variation being more pronounced in the case of costly (but low-prescription) drugs such as parathyroid hormones. The differences in rates of prescribing explained almost all of the variance in drug spending, suggesting that the difference in prescription volume between territories, and not the price of the drugs, is the main source of variation in this setting. Our study also shows that antiosteoporotic drugs are prescribed as complementary treatments rather than as a substitute, overall: HAs prescribe all the groups of antiosteoporotics rather than choosing some groups over others. Our findings are useful to identify inconsistencies in the clinical management of a major health problem for older people  as is osteoporosis, and the prevention of its more severe clinical manifestation: hip fracture. Data on geographical variation of prescription can help guide policy proposals for targeting areas with inadequate antiosteoporotic drug use.Aragon: Ma Concepción Celaya, Mercedes Aza, Félix Pradas, Enrique Bernal-Delgado, Anselmo López Cabañas, and María Elfau Maizal; Andalucia: Carmen Beltrán Calvo, Teresa Molina López, Carmen Suárez Alemán, Carmen Vela, Lola Pérez Pacheco, Ma Ángeles García Lirola, and Francisco Rivas Ruiz; Asturias: Gracia Modroño Riaño; Balearic Islands: María Vega Martín Martín and Eusebi Castaño; Canary Islands: Ma Dolores Fiuza Pérez, Jose Luis Alonso Bilbao, Jose Antonio Díaz Berenguer, Alberto Espiñeira Francés, Antonio Cabeza, and Conrado Dominguez Trujillo; Castilla y León: Nieves Martín and Judit Ceruelo; Catalonia: David Magem; Extremadura: Galo Agustín Sánchez Robles, Lola Sainz, Félix Fernández, and Gregorio Montes; Madrid: Encarna Cruz Martos and Ángel Mataix; Murcia: Rafael Herrero Delicado; Navarra: Maite Artázcoz Sanz, Lourdes Muruzábal Sitges, Ana Azparrén Andía, Francisco Javier Garjón Parra, Javier GorrichoMendivil, and Juan Erviti López; Basque Country: Felipe Aizpuru Barandiarán, Amanda López Picado, Elena Ruiz de Velasco Artaza, JosuneIribar, and María Armendáriz Cuñado; Valencia Region: Gabriel Sanfélix-Gimeno, Salvador Peiró Julián Librero, and María García-Gil.GS-G had full access to all of the data in the study and took responsibility for the integrity of the data and the accuracy of the data analysis. GS-G and SP were responsible for the study concept and design. GS-G and JL-L carried out the data preparation and the statistical analysis. SP, GS-G, and CR-B drafted the manuscript. GS-G, SP, GM-R, JL-L, and CR-B participated in the analysis and interpretation of data, critical revision of the manuscript for important intellectual content, and all approved the final version submitted for publication.Most of the members of the Drug Utilization in the Spanish National Health System Research Group, work in institutions which depend from the Health Departments of the different Autonomous Communities. These departments or the institutions participating in this research, including the Instituto de Salud Carlos III (which provided the grant for this study), did not play any role in the design of the study; the collection, analysis, or interpretation of data; the writing of the manuscript; or the decision to submit it for publication, and may not necessarily share the contents of this paper.SP has received research grants from various pharmaceutical companies, and fees for participation in scientific meetings sponsored by pharmaceutical companies. GS-G participated in 2014 in an advisory meeting of Boehringer Ingelheim. The other 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.


Using a diseased nursery field and artificially inoculated open field experiments, we detected the canopy spectra of wheat at different developmental stages and under different degrees of disease severity. At the same time, destructive sampling was carried out for physical tests to investigate the change of physiological parameters under the condition of disease. Selected vegetation indices (VIs) were mostly comprised of green bands, and correlation coefficients between these common VIs and plant water content (PWC) were generally 0.784–0.902 (p < 0.001), indicating the green waveband may have great potential in the evaluation of water content of winter wheat under powdery mildew stress. The Photochemical Reflectance Index (PRI) was sensitive to physiological response influenced by powdery mildew, and the relationships of PRI with chlorophyll content, the maximum quantum efficiency of PSII photochemistry (Fv/Fm), and the potential activity of PSII photochemistry (Fv/Fo) were good with R2 = 0.639, 0.833, 0.808, respectively. Linear regressions showed PRI demonstrated a steady relationship with PWC across different growth conditions, with R2 = 0.817 and RMSE = 2.17. The acquired PRI model of wheat under the powdery mildew stress has a good compatibility to different experimental fields from booting stage to filling stage compared with the traditional water signal vegetation indices, WBI, FWBI1, and FWBI2. The verification results with independent data showed that PRI still performed better with R2 = 0.819 between measured and predicted, and corresponding RE = 8.26%. Thus, PRI is recommended as a potentially reliable indicator of PWC in winter wheat with powdery mildew stress. The results will help to understand the physical state of the plant, and provide technical support for disease control using remote sensing during wheat production.Plant disease and pests influence the physiological state and restricts the healthy growth of crops. Physiological measurements are considered the most accurate way of assessing plant health status. In this paper, we researched the use of an  Climate change is affecting crop disease prevalence, significantly influencing crop yield and quality worldwide  positively correlates with water content in beans  at canopy scale in winter wheat under powdery mildew stress. The objectives of this study were to (1) analyze the traditional water vegetation indices (VIs) to determine canopy PWC of winter wheat in a diseased field and artificially inoculated open field; (2) assess the response of leaf chlorophyll content (Chl content) and PWC under powdery mildew stress; (3) quantify the relationship between PRI and disease index (DI), leaf Chl content, and fluorescence parameters and (4) evaluate the method using PRI to measure water conditions under powdery mildew stress based on the ground-based canopy hyperspectral technology. The results will provide a basis for disease grading and classification, and further provide technical support for early recognition and precise control during crop production.−1, available phosphorus 28.44–34.52 mg kg−1, available K 114.68–120.12 mg kg−1, and organic matter 10.2–15.7 g kg−1 pre-planting. Nitrogen fertilizer application (135 kg hm−2 nitrogen) in the study plots was higher (12.5%) than the local field practice, plus 120 kg hm−2 P2O5, and 90 kg hm−2 K2O as basic fertilizer. A second dose of nitrogen fertilizer (135 kg hm−2 nitrogen) was combined with the irrigation at the jointing stage. The study plots were watered 3 times at the jointing stage, booting stage, and flowering stage, and the corresponding local fields were watered 2 times at jointing stage and flowering stage. Other aspects of field management followed standard local practices for wheat production.These experiments were conducted in the wheat growing seasons of 2014–2016, located in Zhengzhou, China. The powdery mildew susceptible varieties, Yanzhan 4110, was selected as the test material. The field site consisted of loam soil. The 0–30 cm soil basic indicators were total N 0.97–1.20 g kg2 (2.1 × 4 m). Experiment 2 was carried out at an open field located in the experimental farm of Henan Agricultural University . Wheat was sown on 15 October 2014, and each plot area was 20.3 m2 (7 × 2.9 m) during the 2014–2015 growing season. There were four disease treatments with three replications. The healthy treatment was not inoculated, the low level treatment was inoculated every 4 days, the medium level was inoculated by every 2 days, and the severe treatment were inoculated every day. Experiments 3 and 4 were repeated trials of Experiments 1 and 2 at the same location in the following year. Wheat was sown on 22 October 2015 and 12 October 2015, respectively, during the 2015–2016 growing season. To avoid powdery mildew cross-contamination between plots, powdery mildew resistant triticale was sown between experimental plots. Each plot was divided by a 0.6 m spacing which contained two rows of triticale. Triticale has a plant height of 2 m. The data from Experiments 1–3 were used to build the model and the independent Experiment 4 was used to test the model.Experiment 1 was carried out at a diseased nursery field located in the experimental station of National Engineering Research Center for Wheat, . Wheat was sown on 22 October 2014, in the north-south direction with 20 cm row spacing, and the area of the plot was 8.2 mInfected plants with soil were excavated and placed into prepared pots  for inoculating the field experiments from the jointing stage. After 16:00, infected plants with powdery mildew fungi were brought to the inoculated field. The infected plants were shaken above the experimental plot to infect the wheat and then the infected plants were placed in the center of the experimental plots. The inoculated plot was covered with a transparent shed which remained open in the day and closed at night; the inside of shed was kept moist and the temperature at night was 10°C. After 10 days, symptoms began to appear, and once the wheat infection became obvious during the late jointing stage, the shed was removed. The meteorological data during powdery mildew infection are shown in Figure 2 of wheat canopy was selected to measure canopy reflectance spectra, five sequential readings were averaged to obtain one spectrum, and to analyze biochemical and biophysical canopy features. Instrument optimization was performed prior to sample acquisition, with the average of 50 dark current and white reference, respectively. A white BaSO4 calibration panel was taken before canopy measurements to calculate baseline reflectance. DC measurement can be updated at any time, but should be updated more frequently in the beginning of a given session, as the instrument warms up. Finally, reflectance spectra were obtained by determining the ratios of data acquired for a sample to data acquired for the white reflectance standard.Wheat canopy reflectance spectra were measured by FieldSpec HandHeld spectrometer  over 350–2,500 nm spectral region, at 0.5 m above the wheat canopy with a field of view of 25° , which ensured that almost all the PSII reaction centers were in the open state. The maximum fluorescence (Fm) was determined by saturating pulse light of 8,000 μmol photons m−2 s−1 over a 0.8 s period (reaction centers fully closed). Both the values of Fo and Fm were measured on a leaf under full dark-adaptation for 30 min. The difference between Fm and Fo generated the variable fluorescence . The maximum quantum efficiency of PSII / Fm) has been widely used as a sensitive indicator of plant photosynthetic performance . Fluorescence parameters were measured at 10:00–11:30 local time. The minimum fluorescence (Fo) was induced by a weak pulse of modulating light applied  to calculate the severity of powdery mildew. Powdery mildew severity was divided into nine grades, as follows: 0, 1, 10, 20, 30, 45, 60, 80, and 100%. The DI was computed using the following formula  of powdery mildew incidence, and f is the value of total leaves in each level of disease severity.Here, x is the value of each incidence level, n is the value of the highest level (F). The sample was then denatured at 105°C for 30 min and at 80°C until a constant weight . PWC was calculated using the method of weight ratio  of leaf chlorophyll enthaler .R2 and a self-developed computer program based on the software of MATLAB 7.0. Data from Experiments 1–3 were used to establish the relationship between the spectral indices and PWC. Data from Experiment 4 were used to validate the PWC monitoring models; the estimated values were compared with the measured values to assess reliability and accuracy of the equation output under actual conditions. Root mean square error (RMSE), relative error (RE), and precision  were applied to check the applicability between the estimated and measured data. RMSE and RE obeyed the following Equations (1) and (2), respectively:Several spectral indices previously reported in the literature were selected to check the correlation with PWC; optimized VIs are listed in Table i and Oi are the estimated and measured values, respectively, and n is the sampling number. Estimation was superior if RE < 10%, good if RE = 10–20%, and acceptable if RE = 20–30%  across each developmental stage used for reflectance data acquisition . These water band indices were linearly correlated with PWC at the booting stage (R2 = 0.60–0.64), but less so at flowering and filling stages . Typical water band indices were unsuitable across different stages for detection of water status under powdery mildew stress, and this may be due to significant superimposed effects from physiological and biological changes under biotic stress, and which requires us to seek out efficient indicator of physiological parameters under disease stress.To understand whether the reported water band indices effectively monitored the water dynamic change under disease stress in winter wheat, we performed correlation between representative water indicator and PWC at booting, anthesis, and filling stage using data from Experiments 1–3 Table . The linp < 0.001). The four VIs of SPRI, RGRcn, RIDA and RVI  were followed to give better correlation (r > 0.8).Previous VIs were developed mostly under abiotic stress. Nine out of the 12 published VIs in Table r < 0.413), VARIgreen and Lo with PWC were generated the weakest significance correlation at the 0.05 probability level (r < 0.521), and other VIs with PWC gave higher significance correlation at the 0.01 probability level (r < 0.815). As shown in Figure R2 values of 0.817 and RMSE of 0.217, minimizing the possible heterogeneity from different growth stages.Since the collected data was from two different types of test systems and three growth stages, correlation between VIs and PWC varied according to different growth conditions. By comparison, correlation in Experiment 1 was better than the other two experiments, and correlation at booting stage was poorer than the other two stages. For example, at booting stage, both RIDA and AI with PWC show no significant correlation at the 0.05 probability level . The 1:1 relationship between the observed and estimated values confirmed the accuracy of the derived model . Although, 900–970 nm is the most sensitive band range of water absorption in near-infrared regions . This may be due to a mixture phenomenon of physiological signal coverage and then confuse water absorption signal on canopy spectra under powdery mildew stress. Therefore, assessing PWC under powdery mildew stress using the traditional water indices , is difficult at canopy level.The sensitivities of the visible and near-infrared wavebands to the disease were different depending on disease type , and a significant relationship with PWC (R2 = 0.817) and fluorescence parameter . When white powdery mycelium covers the leaf surface, it causes a loss of pigment, fading leaves, and deteriorated leaf structure. These factors are closely linked with the green waveband. Reduced photosynthesis is closely linked to the green waveband in the visible range at canopy level  under powdery mildew stress , and leaf angle distribution (LAD), etc. Previous studies have shown that the main factors affecting spectrum are LAI and LAD at the canopy level, and major changes are in the near-infrared region 760–900 nm . Physiological indicators  significantly responded to increasing disease severity levels. The ordinal regressions between the PRI and plant physiological parameters under powdery mildew stress applied in the field, showed that PRI was sensitive to physiological change, such as Chl content (R2 = 0.639), Fv/Fm (R2 = 0.833), and Fv/Fo (R2 = 0.808). The green band range (510–570 nm) was valuable for detection of physiological parameters under the powdery mildew stress, and the unified relationship between PRI and PWC generated higher precision (R2 = 0.817), and lower error (RE = 8.26%). This showed that the PRI model has better compatibility between different test conditions and sampling periods, and PRI could be used as a plant water indicator of infected wheat at canopy level. This result will provide a theoretical basis for monitoring plant moisture dynamic variation under biotic stress in winter wheat using remote sensing technology.The physiological analysis of plant disease is extremely important for understanding relationships between VIs and growth parameters under biotic stress. Ground based remote sensing data acquired with the FieldSpec HandHeld spectrometer  were used to detect the variation of PWC of winter wheat infected with powdery mildew. The common water band indices, WBI, FWBIWF and XL conceivedand designed the research; WF and SQ analyzed the data and wrote the manuscript; YH, YW, WL, and YZ provided data and data acquisition capacity; and LH assisted manuscript writing and editing.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.


To facilitate precise local ablation of hepatocellular carcinoma (HCC) in a setting of combined ablation and transarterial chemoembolization (TACE), we evaluated accuracy and efficiency of a novel technique for navigated positioning of ablation probes using intrahepatic tumor referencing and electromagnetic (EM) guidance, in a porcine model.An angiographic wire with integrated EM reference sensor at its tip was inserted via a transarterial femoral access and positioned in the vicinity of artificial liver tumors. The resulting offset distance between the tumor center and the intrahepatic endovascular EM reference was calculated. Subsequently, EM tracked ablation probes were inserted percutaneously and navigated toward the tumor center, relying on continuous EM guidance via the intrahepatic reference. Targeting accuracy was assessed as the Euclidean distance between the tip of the ablation probe and the tumor center . Procedural efficiency was assessed as time efforts for tumor referencing and tumor targeting.In 6 animals, 124 targeting measurements were performed with an offset distance < 30 mm , resulting in a mean TPE of 2.9 ± 1.6 mm. No significant correlation between the TPE and different intrahepatic offset distances  was shown as long as the EM reference was placed within the liver. However, the mean TPE increased when placing the EM reference externally on the animal skin (p < 0.01). TPE was similar when targeting under continuous ventilation or in apnea (p = 0.50). Mean time for tumor referencing and navigated targeting was 6.5 ± 3.8 minutes and 14 ± 8 seconds, respectively.The proposed technique allows precise and efficient navigated positioning of ablation probes into liver tumors in the animal model. We introduce a simple approach suitable for combined ablation and TACE of HCC in a single treatment session. Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide . HoweverImage-guided thermal ablation using radiofrequency or microwaves are increasingly performed for patients with early stage HCC and as part of downstaging or bridging strategies in patients awaiting transplantation ,3. WhileFurther important aspects of a successful ablative therapy are accurate tumor visualization and precise placement of ablation probes. In a combined treatment approach in particular, tumor visibility can be altered due to prior chemoembolization . To enhaTo address the challenge of precise thermal ablation in an angiographic setting and thus facilitate a combined therapeutic approach for TACE and ablation in a single treatment session, we propose a technique termed Multi-Operational Selective Computer-Assisted Targeting (MOSCAT) of HCC. This approach combines i) internal tumor tracking using an endovascular intrahepatic reference, taking advantage of the almost exclusive arterial vascularization of HCC and the selective arterial catheterization already performed during TACE, and ii) consecutive percutaneous positioning of tracked ablation probes using dynamic EM-based navigation technology.In a series of ex vivo experiments, we have previously described stability of EM tracking regarding environmental interferences in the angiography suite, feasibility of the applied image evaluation approach as well as ex vivo targeting accuracy of the proposed technique . In the German Large White) ranging in weight from 60 to 63 kg were used in this study. Animals were bred at the breeding farm Peter Reber, Oberdettigenstrasse 57, CH-3043 Uettligen, a facility supervised by the Swiss Pig Health Service (SUISAG). SUISAG is the authority responsible for the administration of the herd-book, the carrying-out of performance testing and the estimation of breeding values on behalf of Suisseporcs, the official breeding association of the Swiss pig breeders. Health assessment prior to conduction of the experiments included the evaluation of age-specific size, nutritional status, behavior/activity, body posture, breathing, bristles, color of skin/ears/snout, signs of external injuries, scars, abscesses, swollen joints, secretions from eyes/snout, diarrhea, and interest in food and water. Animals were fed commercial feed (Melior #3411 and whey) three times daily up to 12 hours prior to the experiments, with free access to water up to the time of transport to the University Hospital of Bern on the day of the experiment.Experiments were conducted in accordance with the international and local guidelines for care and handling of experimental animals. The study was approved by the Bern veterinary office, the responsible committee on the ethics of animal experiments (license # BE 140/14). Six swine  midazolam and 1 mg IV atropine. Following endotracheal intubation, general anesthesia was maintained with propofol 200–500 mg/h and fentanyl 200–300 μg/h IV as required. Vital parameters were continuously recorded and confined to narrow margins. Mean arterial blood pressure was maintained between 50 and 70 mmHg, heart rate between 60 and 90 beats per minute and end-expiratory partial carbon dioxide pressure between 30 and 40 mmHg. Lactated Ringer's solution was administered for fluid homeostasis, with additional crystalloid fluid and hydroxy-ethyl starch boluses as required.After preparation of the groin, the external iliac vessels were exposed and an arterial  and a venous sheath were inserted. Artificial tumors were created using a mixture of 1% agarose, 10% contrast agent (Iopamidol 300 mg/ml) and distilled water, and shaped to form a diameter of approximately 2 cm using rubber forms. A right subcostal laparotomy was performed to insert the pre-formed artificial tumors, which were fixed between the liver lobes using organic glue. The abdominal incision was closed immediately after insertion of the artificial tumors using a continuous suture.The animals were euthanized immediately after completion of data acquisition, using 40 mmol/L potassium chloride IV for induction of cardiac arrest, after deepening anesthesia with an additional 200 mg bolus of propofol.Experiments were performed in the angiography suite equipped with a high-resolution angiography system , allowing direct measurement of distances on fluoroscopic images. Basic angiographic material and contrast media were identical to those used in humans. A 5 French catheter  was used for catheterization of the hepatic artery after celiac and superior mesenteric arteriography. A 2.7 French microcatheter  was inserted into a segmental or subsegmental hepatic artery and advanced toward an intrahepatic artificial tumor under fluoroscopic guidance.A commercially available navigation system  was equipped with EM tracking . The navigation system displays the relative 3D displacement of the targeting device with respect to the chosen target in a cross-hair viewer, with the 3D information separated into orientation and distance. To generate the EM workspace, a window field generator  was attached underneath the angiography table and aligned using a water level. The animal was positioned on the table such that its upper body and liver were covered by the EM field .To position an EM reference in the vicinity of an intrahepatic tumor in a clinically feasible environment, a prototype of an angiographic wire with integrated EM reference sensor was developed, with corresponding dimensions in order to fit through a 2.7 French microcatheter (inner diameter: 0.65 mm). It consisted of the smallest available EM sensor , enforced by a steel core wire and covered by a polyamide tube  .For percutaneous navigated targeting of intrahepatic tumors, microwave ablation probes  were equipped with an externally bonded EM sensor .Both the EM tracked wire and the EM tracked ablation probe were calibrated to their tip by pivoting, a calibration method which estimates the position of the tip as the center of a rotation .t = r + o], where r is the EM reference and o the offset, measured as illustrated in The EM tracked wire was inserted into the groin access through the 2.7 French microcatheter and positioned in direct vicinity of an intrahepatic artificial tumor. The resulting offset distance between the tumor and the EM reference mimicked the expected distance when the latter would be placed into tumor supplying arteries of HCC lesions. To assess the position of the tumor center (target point t) relative to the EM reference (r), the distances between the two points were measured in the x, y, and z axes in two perpendicular fluoroscopic images  before each targeting series. The target point (t) was calculated in 3D as [Consecutively, EM tracked ablation probes were inserted percutaneously and navigated toward the intrahepatic target, relying completely on the navigation system for instrument guidance. Separate skin incisions were used for probe insertion to create different angles and lengths of targeting trajectories. During navigated targeting, the C-arm was moved to a maximum distance from the working space to eliminate its influence on the EM field and thus on targeting accuracy . On compThe workflow of the MOSCAT of HCC technique is illustrated in a Video Video in .Targeting measurements were carried out in two different scenarios , left. TCatheterization of the hepatic artery and positioning of the microcatheter and EM tracked wire under fluoroscopic guidance was performed by an experienced interventional angiologist. Percutaneous navigated positioning of ablation probes was performed by a surgeon experienced with stereotactic ablation of liver tumors.Targeting accuracy was assessed as the Target Positioning Error (TPE) that resulted after navigated positioning of the ablation probes. TPE was calculated as the Euclidean distance from the tip of the ablation probe to the defined target . Distances were measured in 3D in two perpendicular fluoroscopic images, using the same approach as described for the offset distance measurement . Matlab Experiments were carried out in six individual animals. As primary endpoint, TPE was assessed when the EM reference was placed in direct vicinity (≤ 30 mm Euclidean distance) relative to the intrahepatic tumor, corresponding to the clinically most relevant position when the EM reference is placed into tumor supplying arteries of HCC lesions .As secondary endpoints, factors potentially influencing targeting accuracy were assessed. To this end, TPE was measured i) with the EM reference at five different distances relative to the artificial tumor, to evaluate the influence of the offset distance magnitude on targeting accuracy , ii) with the EM reference placed on the animal skin, to evaluate the effect when positioning the EM reference at an external location , and iii) when targeting in complete apnea versus under continuous positive pressure ventilation, to evaluate the influence of the applied breathing modality on targeting accuracy .Procedural efficiency of the technique was assessed as the time required for i) tumor referencing , ii) offset measurement and calculation (measurement of the 3D offset distance in fluoroscopic images before each offset targeting series), and iii) tumor targeting .t test was used for between-group analyses of intra- versus extrahepatic position of the EM reference and for different breathing modalities with respect to targeting accuracy. P values < 0.05 were considered statistically significant. GraphPad Prism V 7.0  was used for statistical analyses and generation of graphs.For assessment of the primary endpoint, a sample size of 100 targeting measurements was calculated based on accuracy measurements conducted in preliminary in vivo experiments . Linear regression analysis was used to estimate correlation between targeting accuracy and offset distance. The unpaired student’s All experiments were conducted as planned, with preliminary termination of targeting measurements in one animal due to increased blood loss from the hepatic puncture sites. Mean diameter of pre-formed artificial tumors was 23.1 ± 8.7 mm, as assessed in fluoroscopic images.After selective catheterization of a segmental or subsegmental hepatic artery and positioning of the microcatheter under fluoroscopy guidance, the EM tracked wire was successfully positioned in the vicinity of an intrahepatic artificial tumor in all six animals . No lossA total of 505 targeting measurements were performed in six animals, consisting of 90 baseline measurements and 415 offset measurements. Of the offset measurements, 124 measurements yielded an offset distance of ≤ 30 mm between the center of the artificial tumor and the EM reference (i.e. primary endpoint).The relevant results for targeting accuracy and procedural outcomes are summarized in Mean TPE for baseline measurements was 2.6 ± 1.6 mm (n = 90). Mean TPE for offset measurements with an offset distance ≤ 30 mm (primary endpoint) was 2.9 ± 1.6 mm (n = 124), with a mean offset distance between the center of the tumor and the EM reference of 24.5 ± 2.1 mm.Exemplary results of baseline and offset targeting measurements are shown in When analyzing all offset measurements performed with the EM reference positioned within the liver at different offset distances relative to the artificial tumor (range: 20.6 mm to 61.0 mm), mean TPE was 3.2 ± 1.6 mm (n = 365). No significant correlation was observed between targeting accuracy and the magnitude of the offset distance, as long as the EM reference remained within the liver . However, mean TPE increased to 5.6 ± 2.3 mm when the EM reference was placed externally on the animal skin, with a mean offset distance of 102.1 ± 22.5 mm . Accordingly, the rate of targeting measurements that resulted in a TPE of ≤ 3 mm was 52% when the EM reference was positioned within the liver versus 16% when the EM reference was located externally on the skin .In the first five animals, navigated targeting was performed in apnea or with intermittent ventilations between the targeting procedure and obtaining fluoroscopic images for image evaluation. In the sixth experimental animal, the influence of different breathing modalities on targeting accuracy was explicitly assessed. To this end, both baseline and offset targeting measurements were performed either in apnea or under continuous ventilation, with 20 measurements in each group. Mean TPE when targeting in apnea versus under continuous ventilation was 2.0 ± 1.4 mm and 2.0 ± 1.1 mm for baseline measurements (p = 0.91), and 2.9 ± 1.3 mm and 2.5 ± 1.2 mm for offset measurements (p = 0.50), respectively .A displacement of the artificial tumor relative to the surrounding liver tissue and thus to the EM sensor was noticed during the insertion of the ablation probe. This "tumor shift" was 2.2 ± 1.5 mm on average for targeting measurements with an offset distance ≤ 30 mm, and 2.7 ± 2.4 mm for measurements with an offset distance > 30 mm with the EM reference inside the liver.Mean time for positioning of the EM tracked wire with integrated EM reference was 6.5 ± 3.8 minutes (n = 6). Mean time for assessment and calculation of the 3D offset from two 2D fluoroscopic images was 5.2 ± 1.3 minutes. Mean time for navigated positioning of ablation probes  was 13.4 ± 8.3 seconds (n = 505).This study confirms accuracy and efficiency of a novel approach for minimally invasive navigated targeting of intrahepatic tumors, in a porcine model. To our knowledge this is the first work describing the combination of an angiographic approach for intrahepatic tumor referencing with percutaneous navigated targeting and positioning of ablation probes.The potential benefits of using stereotactic navigation systems for targeting and ablation of liver tumors as compared to traditional ultrasound (US) or computed tomography (CT)-guidance include a more accurate and efficient targeting, with a reduction of radiation doses in some settings ,19,22. TA large body of research quantifies the technical performance of electromagnetic tracking systems in the medical field . In the The calculated average displacement of the tumor target relative to the internal reference sensor ("tumor shift") of 2.2 ± 1.5 mm might be due to local tissue compression during the insertion of the rigid ablation probe. In our experimental model this might also be influenced by a tumor displacement due to a less solid fixation of artificial tumors in the surrounding liver parenchyma than in real liver tumors. When excluding targeting measurements with a tumor shift > 3.7 mm (mean tumor shift + 1 SD), mean TPE was reduced to 2.7 ± 1.4 mm (n = 107).The MOSCAT of HCC technique utilizes a continuous internal tracking of the tumor close to its true location, introducing several hypothetical advantages which we verified and confirmed in this work. Firstly, a synchronous movement of the tumor target relative to the intrahepatic tracking reference might be assumed during motion of the whole organ (e.g. due to breathing). In our series, no significant loss of targeting accuracy was shown when performing navigated targeting under continuous ventilation as compared to targeting in apnea (p = 0.50). The importance of this finding is emphasized when considering the substantial efforts performed to overcome the issue of organ motion in soft tissue navigation are considered. In most reports this involves measuring and adapting for organ motion using complex algorithms ,27,34. SFrequent barriers to introducing novel navigation techniques into clinical practice are the associated technical complexity, additional procedural efforts and cost investments ,35. We dMost studies reporting a combined treatment approach of TACE and ablation use US or CT imaging with or without fluoroscopic guidance for placement of ablation probes ,36,37. HThe MOSCAT of HCC technique is potentially an efficient method to combine TACE and ablation in a single treatment session. As we applied the same angiographic material as clinically used for TACE, a relatively simple combination of both treatment approaches is imaginable. Hypothetically, the sequence would include the positioning of the microcatheter into a suitable position for TACE under fluoroscopic guidance, followed by introduction of the EM tracked wire and percutaneous navigated positioning of the ablation probe. Before applying thermal energy for ablative therapy, the EM tracked wire would be removed such that TACE could be performed according to the standard techniques, with the microcatheter already in place. Then, thermal ablation over the precisely positioned ablation probe could be initiated. Both local destructive treatments would thus be integrated and combined to a single and efficient minimally invasive treatment approach for HCC.With regard to an evaluation of the proposed technique in a clinical setting, several issues must be considered. Firstly, the MOSCAT of HCC technique focuses on a tumor-directed navigation without registration of surface structures. Hence, additional US guidance is required to control the trajectory of the ablation probe between skin insertion and the intrahepatic tumor ,38. In oIn conclusion, the MOSCAT of HCC technique allows precise and efficient positioning of ablation probes into intrahepatic tumors in a porcine model. Operating in the available angiographic imaging space, the technique allows dynamic instrument guidance for tumor targeting due to continuous intrahepatic endovascular tumor referencing. We introduce a simple approach for combined TACE and ablation in one treatment session, for selected patients with HCC. Clinical applicability and the impact of the technique on treatment outcome for HCC will be included in a future clinical trial.S1 Video(MOV)Click here for additional data file.


Monoclonal antibodies (mAb) are promising therapeutics in multiple sclerosis and multiple new candidates have been developed, hence increasing the need for some agreement for preclinical mAb studies. We systematically analyzed publications of experimental autoimmune encephalomyelitis (EAE) studies showing effects of monoclonal antibodies. A PubMed search retrieved 570 records, out of which 122 studies with 253 experiments were eligible based on experimental design, number of animals and presentation of time courses of EAE scores. Analysis of EAE models, treatment schedules, single and total doses, routes of administration, and onset of treatment from pre-immunization up to 35 days after immunization revealed high heterogeneity. Total doses ranged from 0.1 to 360 mg/kg for observation times of up to 35 days after immunization. About half of experiments (142/253) used total doses of 10–70 mg/kg. Employing this range, we tested anti-Itga4 as a reference mAb at varying schedules and got no, mild or substantial EAE-score reductions, depending on the mouse strain and onset of the treatment. The result agrees with the range of outcomes achieved in 10 reported anti-Itga4 experiments. Studies comparing low and high doses of various mAbs or early vs. late onset of treatment did not reveal dose-effect or timing-effect associations, with a tendency towards better outcomes with preventive treatments starting within the first week after immunization. The systematic comparison allows for extraction of some “common” design characteristics, which may be helpful to further assess the efficacy of mAbs and role of specific targets in preclinical models of multiple sclerosis. Monoclonal antibodies are increasingly used and developed as therapeutics in multiple sclerosis (MS). Prominent candidates are natalizumab targeting α-4 integrin and two “old” antibodies against lymphocyte surface markers, alemtuzumab CD52) and rituximab (CD20), which have been repurposed for multiple sclerosis, and the recently from the U.S. Food and Drug Administration (FDA)-approved ocrelizumab, also targeting the B-cell antigen, CD20 and is the first for relapsing remitting (RRMS) and primary progressive forms of MS (PPMS) ,2. The s and rituTo assess the efficacy of novel MS-specific mAbs, and to further repurpose monoclonal antibodies, they have to be tested in terms of efficacy and safety in pre-clinical models, raising the need for some agreement about experimental settings and study designs to increase comparability and predictability for the efficacy in humans. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS, but not beyond dispute because of the differences of human MS and rodent EAE ,16,17. TBecause of the shortcomings of EAE models, there is some agreement among EAE researches to test novel candidates in at least two different models, because the predictive value increases with the number of models in which efficacy can be demonstrated. Mostly drugs are continuously or once daily administered during the course of the EAE disease and a crucial decision is when to start the therapy, for example at onset or peak of clinical symptoms or shortly after immunization or during intervals. Above these considerations, which also apply for mAbs, the latter are very versatile in terms of the potential dosing regimens or schedules. Even in humans, monoclonal antibodies may be administered every 4 weeks or at intervals of 6 months, and mostly no longer than 2 years because longer treatments increase the risk of viral infections. These human schedules cannot be re-translated directly to the mouse so that there is presently no obvious rationale for choosing a specific regimen in a rodent EAE study.To address the current uncertainty in terms of preclinical study designs for evaluation of mAbs in EAE models, the present systematic review summarizes results of various mAb-EAE studies in mice, rats and marmosets ranging from 1990–2017 and provides a searchable Excel spreadsheet detailing designs and schedules. The review extracts some agreeable design strategies, supported by experimental data for anti-Itga4.Overall, 122 studies were eligible based on the selection criteria . They ast-tests for comparison of the clinical scores between treatment groups, either for each time point individually, or by using the cumulative scores or without further information. Further 23 studies used ANOVAs to compare treatment effects either using one-way ANOVA for area under the curves (AUCs), or two-way ANOVAs for “time” by “group”. In 47 studies, the non-parametric Mann Whitney U, Kruskal Wallis or Wilcoxon tests were employed. Contingency tables for EAE incidence were presented in 11 studies using either χ2 statistics of Fisher’s exact test and 27 studies did not employ statistical methods or did not report on the type of statistics used. Although EAE scores are ordinal-scaled data, several studies used parametric statistical tests. Nineteen studies employed unpaired Student’s Several studies presented the mean or median time course of EAE scores without indices of inter-variability and the majority of the studies did not present sufficient data to calculate the effect size according to Cohen’s D. Therefore, we used an estimate based on the graphical presentation of the EAE time courses. Because variances were not available, the number of animals per treatment group was used for weighting of the effect sizes. Funnel plots did not reveal a bias towards publication of positive effects B and theIn humans, single doses of monoclonal antibodies in MS patients range from 1–2 mg/kg for daclizumab, 3–6 mg/kg for natalizumab, 1, 3 or 10 mg/kg for alemtuzumab and 10–15 mg/kg for rituximab. Hence, translated to mice, one may consider a single dose of 1–15 mg/kg as a reasonable start, and most studies used doses in this range (92 studies with 181 experiments). There was much less consensus in terms of the dosing frequency, intervals and start of treatment. Particularly the latter ranges from 7 days before immunization ,37,38 upMost studies used dosing intervals of 1–3 days , but theTo address the impact of treatment schedules relative to disease onset we assessed the effects of anti-Itga4 in four independent experiments , testingMonoclonal antibodies were directed against 78 different targets  includinMonoclonal antibody treatments aggravated EAE symptoms in 30 experiments of 26 studies A. For soTargeting of natural killer cell surface molecules caused dual effects. Blocking Klrb1, which likely confers inhibitory functions on natural killer T (NKT) cells aggravated EAE symptoms , whereasUnlike other MS drugs, efficacy of monoclonal antibodies did not substantially differ between C57BL6 and SJL/J mice in studies, in which both strains were directly compared. Seventeen studies compared two or more different strains or active immunization vs. adoptive transfer. The outcomes were similar, except for anti-CD70 and anti-CD137, which failed or aggravated symptoms on adoptive transfer (effect size 0 or −2) but completely prevented or strongly reduced EAE on active immunization (effect size 6 or 4–5) ,72. InveThe present compilation of EAE studies that tested mAbs illustrates the heterogeneity of treatment schedules, dosages, models and outcomes and reflects the uncertainty in terms of study design, which may be most qualified to reveal the impact of the target rather than that of timing and dosing. The heterogeneity of the studies reflects the EAE dilemma, which is the best model for human MS but cannot be translated 1:1 to human MS, neither in terms of mechanisms nor therapeutic success ,16,17. OCorrect timing of treatment and monitoring are crucial. Particularly, for mAbs targeting autoimmune B cells, opposing effects were observed with early and later treatments, because different B cell subtypes promote or inhibit EAE pathophysiology ,77. PartFor quantitative comparisons and regression analyses, we used an estimate of the effect size based on the graphical presentation of the time courses of the EAE scores, body weights where available, and the change of the AUC under mAb treatment. Although the effect size scores were independently extracted by two researchers with high agreement (±1 of the effect size score), and without knowledge of the interpretation of the result by the respective authors, the estimate is an approximation of the outcome and might miss details, which may be relevant for the efficacy of a certain mAbs. In particular, although listed in We systematically searched PubMed for all publications describing controlled studies that investigated effects of monoclonal antibodies in EAE models in laboratory rodents or primates up to August 2017, irrespective of dose, dosing schedule, route of administration and observation time , minor (1), mild (2), moderate (3), substantial (4), strong (5) or very strong reduction of the scores . For studies, in which the monoclonal antibody increased the clinical scores compared with the control group, that is, where treatment worsened the disease, the effect size was scored as “−1” or “−2”. More severe worsening did not occur. If the effect consisted exclusively in a delay of clinical symptoms without change of disease intensity the effect was considered to be minor, mild or moderate  depending on the time of the disease free period. The latter mainly applied to marmoset studies. The effect size score was weighted based on the number of animals per treatment group (n), by multiplication of the effect size score with n/10. A group size of 10 was used as “standard”, because it is the recommended number of animals per group for EAE studies and was the mean and most frequently used number of animals per group. To assess the associations of effect sizes vs. total doses, vs. single doses or vs. treatment onset, weighted effect size scores were plotted vs. the respective parameters including all experiments irrespective of the target of the mAb, or only those studies which addressed dose responses or onset-dependent responses. Linear regression analyses were used to assess the dependent or independent nature of each two parameters, and were performed for unweighted and weighted effect size scores.The effect sizes were calculated according to Cohen’s d (where possible) using reported results of t-tests or Mann Whitney C57BL6/J mice were immunized according to a standard protocol using the Hooke Kit™ MOG35-55/CFA emulsion PTX , which contains 200 µg myelin oligodendrocyte glycoprotein (MOG) 35–55 emulsified in 200 µL Complete Freund’s Adjuvant (CFA). The emulsion was injected subcutaneously at two sites followed by two intraperitoneal (i.p.) injections of 200 ng pertussis toxin (PTX) in phosphate buffered saline (PBS), the first 1–2 h after MOG35-55, and the second 24 h thereafter.SJL mice were immunized according to a standard protocol using Hooke Kit™ PLP139-151/CFA emulsion PTX (EK-0123), which contains 200 µg myelin proteolipid protein (PLP) 139-151 in 200 µL CFA . The emulsion was injected subcutaneously at two sites followed by two i.p. injections of 200 ng PTX in PBS, the first 1–2 h after PLP135-151, and the second 24 h after PLP135–151.EAE scores and body weights were assessed daily to evaluate the severity and stage of the disease by an observer who was not aware of the treatments. Score 0 means no obvious changes in motor functions. Score 0.5 is a distal paralysis of the tail; score 1 complete tail paralysis; score 1.5 mild paresis of one or both hind legs; score 2 severe paresis of hind legs; score 2.5 complete paralysis of one hind leg; score 3 complete paralysis of both hind legs and score 3.5 complete paralysis of hind legs and paresis of one front leg. Mice reaching scores ≥ 3.5 were euthanized.Anti-Itga4 monoclonal antibody or placebo was injected i.p. or IV using different schedules and doses in 4 independent experiments in SJL/J or C57BL6 mice each consisting in 10/10 mice for mAb and placebo groups. Experiments were approved by the local Ethic committee for animal research , adhered to the European and Germany regulations and to the guidelines of GV-SOLAS and were in agreement with ARRIVE.


Phylloscopus collybita abietinus) and the Siberian chiffchaff (P. tristis)—two recently diverged species which differ in morphology, plumage, song, habitat, and migration—to quantify the regional variation in genome-wide genetic diversity and differentiation, and to identify candidate regions for trait variation. We find that the levels of diversity, differentiation, and divergence are highly heterogeneous, with significantly reduced global differentiation, and more pronounced differentiation peaks in sympatry than in allopatry. This pattern is consistent with regional differences in effective population size and recurrent background selection or selective sweeps reducing the genetic diversity in specific regions prior to lineage divergence, but the data also suggest that postdivergence selection has resulted in increased differentiation and fixed differences in specific regions. We find that hybridization and backcrossing is common in sympatry, and that phenotype is a poor predictor of the genomic composition of sympatric birds. The combination of a differentiation scan approach with identification of fixed differences pinpoint a handful of candidate regions that might be important for trait variation between the two species.Identification of candidate genes for trait variation in diverging lineages and characterization of mechanistic underpinnings of genome differentiation are key steps toward understanding the processes underlying the formation of new species. Hybrid zones provide a valuable resource for such investigations, since they allow us to study how genomes evolve as species exchange genetic material and to associate particular genetic regions with phenotypic traits of interest. Here, we use whole-genome resequencing of both allopatric and hybridizing populations of the European ( Until re.g., e.g., Of late, there has been gradually increasing interest in understanding how adaptation to the local environment can drive the formation of reproductive barriers between diverging populations . This haRecent genome-scan approaches using population samples from a range of taxa at different levels of divergence have shown that a common pattern encountered is a mosaic of highly and lowly differentiated genomic regions , elevatePhylloscopus collybita abietinus) and Siberian chiffchaff (P. tristis) as a study system to quantify patterns of genetic diversity and differentiation across the genome. The chiffchaff superspecies complex is part of the Eurasian Old World leaf warbler group, and, historically, the complex has been divided in four distinct species: Canarian chiffchaff, Iberian chiffchaff, mountain chiffchaff, and common chiffchaff (P. collybita) has been divided into five different subspecies, including subspecies abietinus (eastern European common chiffchaff) and tristis (Siberian chiffchaff). P. tristis has recently been given full species status  only, and to accommodate consistency with recent previous analyses of this particular pair of taxa  is distributed from the Ural mountains to eastern Siberia during breeding season, and migrates to south-central and south-eastern Asia , is smaller in size, sings a faster and more diverse song, and preferably inhabits boreal forests  use population whole genome resequencing data to get global (genome-wide) and regional (locus specific) estimates of diversity and differentiation in allopatry and sympatry, ii) to use a genome-scan approach to characterize the landscape of differentiation between typical abietinus and tristis morphotypes in allopatry and in sympatry to detect regions that appear impermeable to gene-flow, and iii) to identify candidate genes for local adaptation and reproductive isolation between the species.Here, we present a population genomic survey using both allopatric and sympatric samples of the common chiffchaff . A single allopatric abietinus male (Pcol09) was selected for deep sequencing, and two different paired-end libraries (180 and 380 bp) were generated for this individual and sequenced on four lanes—this generated, in total, 100.2 Gb of raw sequence data (91.1 Gb after filtering out bases with Q-scores <30) for this individual (Table S1 in File S1). Libraries were prepared by the SNPandSEQ Technology Platform in Uppsala, and sequencing was performed in two separate batches, one batch using Illumina HiSeq2000 technology (100 bp read length) at the SNPandSEQ Technology Platform in Uppsala  under accession number: PRJEB21643 (Table S1 in File S1). The sample names Pcol01-Pcol704 follow the nomenclature established during the sampling stage, when abietinus and tristis were classified as subspecies of the common chiffchaff , and we decided to keep the sample names for easier comparison of results from studies of the same system  assembly as a reference  were aligned to the F. albicollis assembly using BWA version 0.7.8 (https://github.com/lh3/seqtk) was used for the conversion of the FASTQ-file format to a FASTA formatted final assembly. Ambiguous bases in the reference-assisted assembly were replaced by N if they represented more than two alleles, and diallelic ambiguities were assigned to either allele randomly using an inhouse developed python script (https://github.com/venta380/chiffchaff_project/). The reference assisted chiffchaff genome assembly covered 89% of the entire F. albicollis reference genome, and spanned, in total, 1.04 Gb. It should be noted that a reference assisted assembly is blind to structural changes and differences in synteny can affect the interpretation of genome-wide patterns of genetic variation. Although the divergence time of Ficedula and Phylloscopus is ∼40 MY  genome assembly, to obtain comparative data for high-quality avian genome assemblies. In order to assure sufficient mapping efficiency of the population resequencing data, one random sample (Pcol41) from the resequenced sample set was mapped to the chiffchaff assembly using BWA version 0.7.8  are presented in Table S2 and Table S3 in File S1.A reference-assisted nuclear genome assembly of the on 0.7.8 . The cons ∼40 MY , and theon 0.7.8 , and mapon 0.7.8 . All assF. albicollis mitochondrial genome as template to identify mtDNA reads. Mapped reads were subsequently used in de novo assemblies of chiffchaff mtDNA genome assemblies using the iterative mapping and assembly tool MITObim  revealed that gene order and gene sizes were extremely conserved between the two lineages —note that the mtDNA genome is circular and the presentation in linear format generates a random cut point so that start and stop positions are different in the two assemblies.Complete chiffchaff mtDNA genome assemblies were generated for each of the 40 samples using mapping with BWA , with th MITObim . The proF. albicollis reference genome was created using Picard Tools version 1.127 (available at: https://github.com/broadinstitute/picard). All 40 resequenced samples were mapped individually to the chiffchaff reference assembly using BWA version 0.7.8 (https://github.com/broadinstitute/picard), and the sequences flanking indels were realigned using IndelRealigner in GATK (Pcol09) were mapped back to the assembly using BWA  was found to have considerably lower mapping quality than all other samples, and was removed from the downstream analysis in order to reduce biases in the results as a consequence of differences in mapping efficiency, coverage, and variant calling. Sequence polymorphisms were called for each sample individually using HaplotypeCaller, subsequently joint genotyped using GenotypeGVCFs, and filtered using standard settings for the VariantRecalibrator in GATK  SNP calling algorithms using BWA version 0.7.8  and SAMton 0.7.8 . The ressing BWA , and the2r < 0.2 (https://github.com/venta380/chiffchaff_project/). A population structure analysis was conducted using the R Bioconductor package LEA   was conducted on genotypes that were covered with a minimum of seven reads per individual in all samples in each of the four populations. The PCA was performed with 220,097 SNPs using the R Bioconductor package SNPRelate , using aStudies)  on the sStudies)  to clustStudies) , 2015.The mitochondrial genome was assembled for each individual independently (see above), and all mtDNA genome sequences were aligned with MAFFT version 7 , generatF. albicollis repeat annotation libraries  as reference.Before any additional analyses, interspersed and tandem repeats in the chiffchaff assembly were hard masked using RepeatMasker version 4.0.6 (abietinus (n = 10), allopatric tristis (n = 9), sympatric abietinus (n = 10), and sympatric tristis (n = 10)], with a minimum sequence coverage of five reads , were retained for subsequent analysis. Genotype calls were translated to specific polymorphism classes for both the allopatric and sympatric species comparison; fixed differences between the two species, private polymorphisms in abietinus, private polymorphisms in tristis, and shared SNPs (polymorphic in both species).The sequence coverage depth at each genotype was calculated using the geno-depth option in Vcf-tools version 0.1.14 (πƟ), Watterson’s theta (WƟ) and Tajima’s D] and genetic differentiation (STF) between allopatric and sympatric species pairs. As the ancestral state for segregating sites was not known, the folded allele frequency likelihoods and allele frequency spectra were computed using GATK’s genotype likelihood model as implemented in ANGSD (https://github.com/venta380/chiffchaff_project/) to calculate absolute divergence (XYD) between allopatric and sympatric species pairs, and independent estimates of πƟ for each respective population based on the allele frequencies estimated using ANGSD. The rationale behind this was that the inhouse calculated XYD estimates, and the πƟ estimates from ANGSD were not calculated using identical settings, and to compare intraspecific polymorphism levels with absolute divergence we needed to apply the same settings for these summary statistics. All summary statistics were averaged over nonoverlapping windows of 10 kb across the genome. The levels of linkage disequilibrium  for each respective population were calculated for SNP pairs <100 kb apart using Plink version 1.90 (https://github.com/venta380/chiffchaff_project/), with the filtered LD output from each population specific analysis from Plink  package version 0.902  for each comparison by standardizing with the genomic average and variance across windows according to the following formula:eN) for the Z-chromosome was reduced as compared to the autosomes, and, hence, the expected baseline levels differ between these chromosome classes. Autosomal windows with STZF values higher than the 99th percentile in the distributions were considered outliers  for the allopatric and the sympatric comparison, respectively. In order to evaluate if the overall sequence coverage affected the STF distribution , per-site genome coverage was calculated using the coverageBed utility in the BEDTools version 2.25.0 (STF was checked. We found only weak and inconsistent correlations between sequence coverage and STF (range of Pearson’s r = −0.007 to 0.025 for the four different populations). To visually inspect the landscape of diversity and differentiation in the allopatric and the sympatric species comparisons, all population genetic summary statistics were plotted against physical positions along chromosomes using the ggplot2 library in R (https://cran.r-project.org/). The most extreme differentiation regions in each comparison  were further investigated by extracting the coordinates for clusters (≥10 outlier windows present in a 0.5-Mb genomic region) of the windows with the highest level of relative differentiation, and intersect these with annotation data from the F. albicollis genome . The rationale behind this strategy was to identify coding genes to investigate if any particular functional category of genes was over-represented in the most highly differentiated regions. We used a binomial distribution to estimate the probability of this level of clustering or higher, since the sample size (50 windows per block) was much smaller than the total population . The probability of 10 outlier windows clustering in a 500 kb (50 windows) region was estimated at 7.133 × 10−11. Importantly, relaxing the significance level did not affect the results quantitatively, i.e., very few additional blocks were identified if the limits were set to 5  or even three outliers  within a 50-window block (data not shown). Enrichment analysis of specific gene ontology (GO) terms for genes located in chromosome regions with higher than expected density of relative differentiation outliers was performed using GOstat with a custom reference database based on GO terms from the F. albicollis gene set as available in Ensembl . The identities and potential functions of the identified genes in highly differentiated regions were inferred using orthology information as available in the Biomart tool in Ensembl (http://www.ensembl.org/biomart/martview/c02baa1e6506b4840e7e34ceac0a49da: 2017-06-30).To assess the genomic distribution of the most highly differentiated regions between n 2.25.0 , and thehttps://github.com/venta380/chiffchaff_project/) to sort previously called SNPs into four categories for the allopatric species comparison : fixed differences between the two species, private polymorphisms in abietinus, private polymorphisms in tristis and shared SNPs (polymorphic in both species). Only SNPs covered by at least five reads in each individual and where information was present for at least nine individuals in each of the populations were retained for this analysis  to minimize the bias toward fixed differences when representation in individuals is low. We identified the functional relevance of each SNP using the annotation software snpEFF version 4.3 , which is the closest relative to chiffchaff with extensive genome annotation information. The annotations were grouped into classes “downstream region,” “upstream region,” “exon,” “intron,” “intergenic region,” and “other” . The relative frequencies of different polymorphisms were then compared across annotation classes for autosomes and the Z-chromosome separately to get information about the relative effects of selection and genetic drift in driving the patterns of differentiation, heterozygosity, allele sharing, and accumulation of fixed differences between abietinus and tristis.To further assess the potential functional relevance of different types of polymorphisms, we used custom PyVCF 0.6.8 scripts . All statistics of the chiffchaff assembly are presented in Table S2 in File S1. The assessment of the chiffchaff assembly completeness based on conserved gene sets showed that 80.9% of vertebrate and 50.3% of eukaryotic genes were completely covered. The fractions of fragmented genes in the vertebrate and eukaryotic gene sets were 6.5 and 4.9%, respectively, and missing genes constituted 12.6 and 44.8% of the gene sets (Table S3 in File S1). For comparison, the high-quality F. albicollis and C. corone assemblies cover only slightly higher fractions of complete vertebrate  and eukaryotic (50.3% in both species) genes, and have a slightly lower proportion of completely missing genes; 2.5 and 10.1% in the vertebrate gene set (Table S3 in File S1).The total length of the chiffchaff assembly was 1.04 Gb, similar to previously available avian genome assemblies , but there were no fixed differences on the Z-chromosome in the sympatric comparison , and all sympatric tristis had >75% of the assigned proportions shared with allopatric tristis while sympatric abietinus varied from being assigned with 100% to abietinus to >90% with tristis . Allopatric tristis clustered in two separate groups, birds from Southern Siberia being in a derived clade while birds from Central and Eastern Siberia and the Ural Mountains were scattered in basal lineages in the “tristis”-type mtDNA group, and birds from the Southern sympatric zone showed higher diversity in mtDNA haplotypes than birds from the Northern sympatric region . The analyses of mtDNA genetic variation within and between populations showed that diversity was higher in abietinus than in tristis in allopatry , and the pattern was even more pronounced in sympatry . MtDNA genetic divergence was also higher between allopatric than between sympatric abietinus and tristis . Overall, this picture a scenario where abietinus and tristis diverged in allopatry roughly 1–2 MYA  as compared to the autosomes combined (πƟ = 0.0054 ± 0.0030 and 0.0048 ± 0.0030) – the same pattern was observed in sympatry (D) and linkage information. In summary, the global estimates of Tajima’s D were generally negative, in line with a bottleneck + expansion model for all populations, but less pronounced in allopatric tristis  than in the allopatric regions (0.220 ± 0.064) , and the level of genetic differentiation was significantly higher on the Z-chromosome than on the autosomes , with reduced divergence on the Z-chromosome as compared to autosomes , although the quantitative difference between allopatry and sympatry was less pronounced , except for the Z-chromosome where XYD was considerably reduced in sympatry as compared to allopatry , and also a negative correlation between differentiation and absolute divergence, both in allopatry  and in sympatry . Tajima’s D was, in general, more negative in regions of high differentiation in comparison to the remaining parts of the genome , with the exception of sympatric abietinus that showed a positive correlation between Tajima’s D and the level of genetic differentiation . The analysis of the levels of genetic diversity and absolute divergence shows that the correlation is very strong, both in allopatry and in sympatry, with only little deviation from the linear relationship between πƟ and XYD . We observed a higher variance in absolute divergence in allopatry than in sympatry, and a more pronounced clustering of high differentiation windows at the lower left end in the graph (low genetic diversity and low absolute divergence) in autosomes than for the Z-chromosome .Global estimates of nuclear genetic diversity showed highly heterogeneous patterns across the genome of both species, both in allopatric and sympatric populations . In the allopatric comparison, 19 blocks representing 13 chromosomal regions , were identified as significantly enriched for STZF outliers, and the analogous analysis in sympatry revealed 13 windows, representing 10 different regions located on chromosomes 1, 1A, 5, 10, 12, 13, 17, 20, and 21 (File S1). The identified regions were highly overlapping between the two different comparisons, but, again, regions were narrower and more pronounced in sympatry . The GO enrichment analysis revealed no over-representation of specific terms for genes in differentiation outliers in allopatric comparison , and only one term associated with regulation of cellular component organization in the sympatric comparison for two partly overlapping gene sets (Table S6 in File S1)Windows with mparison . We testsympatry . The chrabietinus and tristis (ARF4), sensory perception of sound (OTOG), and pigmentation (HPS5)  .abietinus and tristis, and we applied more stringent filtering  to minimize the risk of including erroneously called fixed differences in this analysis. Among all high-quality SNPs selected for this analysis , we found that almost half of them (43.6%) were shared polymorphisms, while only 7609 SNPs (0.27%) represented fixed differences. We generated annotation information for all SNPs from all of the eight SNP sets  using the annotation available for the F. albicollis genome assembly . For autosomes, there was an excess in the proportion of fixed differences in genes, or chromosome regions in the vicinity of genes, while there was an underrepresentation of fixed differences in intergenic sequences, but this pattern was not present on the Z-chromosome (File S1), this indicates that patterns of elevated differentiation cannot entirely be explained by restricted recombination and less efficient selection, but also that diversifying selection has driven certain alleles to fixation between abietinus and tristis. We therefore took the analysis one step further by classifying all the fixed differences between the species in allopatry. The results showed that of the 7609 fixed differences identified, the majority were as expected located in introns, intergenic regions or noncoding regions adjacent to genes (n = 7458), but we also identified 134 exonic fixed differences corresponding to 116 synonymous and 18 nonsynonymous positions, and 17 fixed differences potentially affecting splice signals. The genes associated with the fixed differences at nonsynonymous positions or splice sites are all potential candidates for underlying species-specific characteristics of abietinus and tristis, respectively, and are presented in Table S7 in File S1. Two of the genes had more than one nonsynonymous substitution, and, based on functional studies in other organisms, these may be involved in the innate immune response (FYB) and lipid metabolism (PRUNE2). Another two genes had both nonsynonymous and splice altering differences, and these genes have functions related to gene expression regulation via histone methylation (KDM4C), microtubule activity, and intracellular transport (KIF24).To obtain further information about the underlying forces generating a heterogeneous distribution of genetic diversity and differentiation, we assessed the functional relevance of different categories of SNPs. This analysis was focused on the allopatric comparison, only since we did not identify any fixed differences between sympatric romosome . Since fCYTB) in the same set of chiffchaff samples. They investigated population structure in abietinus and tristis in relation to phenotypic variation in allopatry and sympatry, and verified that the intermediate phenotypes and mixed singers observed in the area around the Ural mountains represented admixed individuals rather than a third taxon previously suggested to be present in the area , and had sufficient quality to use as a backbone for mapping reads from resequencing efforts involving allopatric and sympatric populations of abietinus and tristis. The level of complete and partial coverage of conserved gene sets was, for example, similar to high-quality avian genome assemblies .abietinus and tristis to have a thorough background for more detailed analyses of genomic regions that showed patterns of differentiation consistent with diversifying selection and/or genetic incompatibilities. Our estimates of genetic diversity, genetic differentiation, allele frequency distributions, LD, and population structure for both mtDNA and nuclear DNA supported the scenario suggested by abietinus showing slightly higher proportion of private polymorphisms and a higher genetic diversity, but a higher proportion of low frequency variants (more negative Tajima’s D) than allopatric tristis, despite the smaller sampling range of abietinus than tristis  and the collared flycatcher , where average nucleotide diversity ranges from 3.2 × 10−3 to 4.0 × 10−3  on the Z-chromosome as compared to the autosomes in both abietinus and tristis and the level of genetic differentiation (STF) was on average higher on the Z-chromosome in both allopatry and sympatry. Interestingly, the level of absolute divergence (as estimated by XYD) was considerably reduced on the Z-chromosome as compared to the autosomes, similar to previous observations in the greenish warbler species complex (abietinus and tristis (eN and increasing the effect of drift), and should also increase the effect of linked selection on the Z-chromosome. Second, it is plausible that the Z-chromosome harbors a gene set that is nonrepresentative for the genome as a whole, e.g., enriched for genes involved in local adaptation or reproductive isolation, as has been suggested for the X-chromosome in Drosophila  of the Z-chromosome is reduced as compared to autosomes. With an equal sex-ratio, eN for the Z-chromosome is 3/4 (75%) of the eN for autosomes, since females only have a single Z-chromosome . In birds, males also generally have higher variance in reproductive output than females, further reducing the effective population size of the Z-chromosome and increasing the effect of genetic drift. Fourth, since the Z-chromosome is in hemizygous state in female birds, recessive mutations are expressed in females, and this can contribute to both faster evolution  determining the difference in relative importance of genetic drift and selection in different parts of the genome, and that a major proportion of the fixed differences we observe are the result of stochastic fixation of alternative alleles in the two species in allopatry. However, the observation that a larger proportion of autosomal fixed differences are present in exonic sequence or adjacent to genes indicates that at least a proportion of the fixed differences on the autosomes result from selection driving alternative alleles to fixation in abietinus and tristis, respectively. In addition, it is possible that stochastic allele frequency changes and random accumulation of fixed differences may lead to diverging phenotypes between species in allopatry, and it is therefore of interest to investigate the functional relevance of observed differences between species, even if it is apparent that the major fraction of these were not initially driven to fixation by adaptation to different environments  or by different preferences for secondary sexual traits . In the following paragraph, we discuss the detailed analyses of functions of genes in highly differentiated regions, and genes with alternative alleles fixed between the two species.Similar to the observations when comparing the Z-chromosome to the autosomes, the highly differentiated regions across chiffchaff species in general showed reduced diversity and absolute divergence. There was also a strong correlation between absolute divergence and genetic diversity. Again, similar to the situation for the Z-chromosome, this may be translated to a scenario where linked selection and regional lowered effective population size has resulted in loss of genetic variation prior to the divergence of the two lineages . The levabietinus and tristis that may reflect species specific adaptations. First, we selected all genes that were located in genomic regions enriched for relative differentiation outliers, and analyzed if any functional categories were overrepresented in this class of genes. This analysis showed no significant enrichment in the allopatric comparison and a single term (regulation of cellular organization) in sympatry. Such an analysis is characteristically rough, and, although it may give some preliminary insights into gene categories associated with high differentiation, it is difficult to draw detailed conclusions regrading functional relevance (STF) within each of the regions enriched for relative differentiation (STZF) outliers, and found that these top windows overlapped with 26 protein coding genes. The genes were mainly involved in functions related to ion transport, expression regulation , and muscle activity/development, but also associated with processes directly relevant to phenotypic traits of interest in our study system; for example, sensory perception of sound (OTOG), and pigmentation (HPS5) (OTOG and HPS5 are located in the peak of the differentiation island on chromosome 5. OTOG has been associated with sound perception and locomotion in mouse (Mus musculus domesticus) (Danio rerio)  . Both OTo rerio) , and recbra fish , and, agabietinus and tristis and selected the positions where the substitutions resulted in amino acid and splice signaling changes. Again, we found that several genes were related to metabolism and gene expression , but there was a diverse array of other functions in this class of genes. Of particular interest were two genes that had multiple (n = 2) fixed differences resulting in nonsynonymous changes; FYB and PRUNE2. FYB has been shown to be involved in the innate immune response by stimulating interleukin-2 expression in T-cells in the mouse (PRUNE2 has been associated with many different functions (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRUNE2); the gene is, for example, associated with pyrophosphatase activity that could indicate involvement in lipid metabolism. Two additional genes contained both a nonsynonymous and a splice-altering fixed difference; KDM4C and KIF24. KDM4C regulates gene expression by targeting trimethylated histones in humans . The structural changes do not affect the qualitative interpretation of the results in this study since they constitute but a minor part of the genome, and do not overlap with any of the major differentiation islands observed.It should be noted that a reference-assisted assembly cannot detect structural changes between the target organism and the reference. A consequence might be that visual inspection of patterns of genetic variation might be affected. Since birds in general have conserved karyotypes and comparatively few large structural rearrangements , this isTo conclude, this study contributes to the increasing body of knowledge about genome divergence processes under natural settings, and highlights the chiffchaff species complex as an important model system to study the genomics of speciation and adaptation in songbirds. In a general perspective, this work will help in the overall understanding of the speciation process, and, in particular, how secondary contact affects patterns of genome differentiation between diverging lineages.www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300152/-/DC1.Supplemental material is available online at Click here for additional data file.