Furthermore, members of treatment group were more likely to avoid exposure to smog episodes by spending more time inside with the windows closed than usual . Members of treatment group who take medication in the form of aerosol pumps also kept these devices on themselves more frequently than those of control group . The system however had no discernible effects on the awareness of air pollution risks, reduction of health symptoms related to smog and the use of health system services. The absence of health benefits could be related to the lower actual exposure to air pollution of such vulnerable groups during winter.Smog warning systems are components of adaptation strategies that are adopted by governments around the world to protect their citizens from extreme episodes of air pollution. As part of a growing research stream on the effectiveness of these systems, this article presents the results of a study on the impacts of an automated phone warning and advising system for individuals vulnerable to air pollution. A sample of 1328 individuals were recruited and randomly assigned to treatment and control groups. The treatment group received smog warning while the control group did not. Data were collected via three phone surveys, two before and one after issuing the smog warning. The comparison between treatment and control groups indicates that exposure to a smog warning improved information on the occurrence of smog episodes ( Smog episodes are severe air pollution periods characterized by a mixture of smoke and fog resulting from natural and/or anthropogenic factors . The freEfforts to curb greenhouse gas emissions are presently at the core of governments’ strategies to contain global warming as evidenced by the Paris Agreement on Climate Change . In addiIn parallel to the implementation of air quality warning systems, interest in the evaluation of their performance has increased in recent years. In accordance with the logic model of smog warning systems, special attention was paid to their impacts on the adoption of protective behaviors ,15,16, rThe objective of this study is to evaluate the impacts of an automated phone smog warning system (APWS) for individuals vulnerable to this hazard. Its contribution to this field of research is two-fold. First, it is so far among the rare if not the only study using an experimental design to assess the effects of smog warnings. As it is well known, randomization improves significantly the internal validity of impact evaluations. Second, while most studies analyzed the effects of alerts using mass media, the focus of this study is on the effects of automated phone warnings. APWSs have the advantage of enabling the delivery of personalized smog alerts and advice to targeted individuals while avoiding overcrowding public medias with these warnings .The structure of this article is as follows. In the next section, the study’s material and methods are presented. The The evaluation of the APWS is based on an experimental design in which a sample of study participants were voluntarily recruited and randomly assigned to treatment and control groups (see below). The APWS was programmed to issue automated phone smog warnings along protective advice to treatment group when Environment Canada predicts that the level of air pollution will reach levels considered prejudicial to population health. To assess its impacts, data on outcome variables were collected when the actual level of air pollution was equal or higher than the threshold triggering smog warnings . Comparisons between treatment and control on outcome variables was used to assess the effects of this system. A detailed description of the design of this experiment is provided in the following sections.Ethics Certification: The Protocol of This Study Was Approved by the Ethics Committee of the Institut National DE La Recherche Scientifique: Cer-15-370.2.1.The APWS was developed and tested between 2015 and 2017 by the Institut National de Santé Publique du Québec (INSPQ) and the Direction Régionale de la Santé Publique de la Montérégie (DSP Montérégie). It is part of Quebec government efforts to deal with climate change challenges facing this province. The APWS was designed to inform vulnerable individuals on the occurrence of excessive heat and smog episodes and provide them with advice on how to protect themselves from these hazards. Compliance with the recommended behaviors is presumed to mitigate the adverse effects of smog on health and reduce health services use. The results on the impacts of heat warnings were published in a previous issue of this journal . This ar3), particulate matter (PM2.5/PM10) and nitrogen dioxide (NO2). To facilitate the communication of health pollution risks to the population, the index was divided into four levels of health risks: low, moderate, high and very high risk. Environment Canada issues air quality warnings on its website when the predicted risk level is considered moderate, high or very high. These alerts are then disseminated by mass media in Canada such as TV and radio channels. The APWS was designed to issue warning alerts when the forecasted air pollution reaches levels representing a serious threat to the participants in this study. Regarding this, Environment Canada uses Air Health Quality Index (AHQI) to monitor air quality across different regions of Canada . AHQI isBe 65 years old or older;Present a heart or lung medical condition;Suffer from diabetes, kidney failure, mental health disorder or neurological disorderThe APWS was programmed to issue smog warnings along with protective tips when Environment Canada forecasts that the risk level of air pollution is moderate or higher on the AHQI’s scale. This level of triggering smog alerts was chosen because the objective of the APWS is to serve individuals that are particularly vulnerable to air pollution. Specifically, the APWS was tested on a sample of individuals having at least one of the following characteristics that, according to scientific literature and a committee of experts formed specifically to advise the authors of this study, are associated with vulnerability to heatwaves and air pollution : Be 65 yStay inside as much as possible and close windows.Outside, avoid physical efforts, even the short health walks.If you take medication in the form of aerosol pumps, always keep them with you.For any questions about your health, call Info-Santé at 811.In case of chest pain or difficulty breathing, call 911.The smog warning message was developed in consultation with experts in public health and after reviewing the relevant scientific publications . It wasThe APWS was programmed to issue automatically the prerecorded oral smog alert on the usual landline or mobile phone numbers of treatment group members before the onset of smog episodes. The automated phone message was not modulable according to the severity of smog episodes or vulnerability of study participants. The research team decided to test the performance of the APWS in the winter rather than in the summer season. The reason being that heat waves are frequently accompanied with smog episodes and therefore some advice provided to avoid smog exposure (like stay inside the windows closed), conflicts with tips intended to protect from heat . Phone surveys were used to collect data on participant characteristics such as gender, age, level of education and outcome. In addition, the questionnaire included questions measuring the main outcomes of the APWS.Two questions were used to determine whether respondents were informed or not about the smog episode. The first question asked respondents whether they were aware of the episode or not; the second, the date when they became aware of it, i.e., before, during or after smog episode. Data on participants’ knowledge of protective behaviors was collected via an open-ended question on the best ways of protecting themselves from the smog. The number of measures cited by respondents that matched the recommended behavior was then calculated for each respondent. The perception of the adverse effects of smog on health was measured with a five-level scale: 1: smog is not dangerous at all to 5: smog is extremely dangerous to my health. As mentioned earlier, recipients of the warning message were advised by the APWS to adopt the following behaviors during smog episodes: stay indoors with the windows shut, avoid intense outside physical effort and, for individuals using pump devices for respiratory medication, keep these devices always with them. A five-level scale was used to collect information on weather respondents stayed indoor the windows shut longer or less than usual during smog episode . Regarding physical efforts, respondents were first asked if they had made, or not, intense outdoor physical efforts during the smog episode. Those who responded affirmatively were then asked to rate on a five-level scale the length of this activity . Similarly, individuals who take respiratory medication were asked to report on a four-level scale the extent to which they kept the pump devices with them .Respondents had to report if they suffered from any of the following symptoms caused by air pollution during the smog episode or within the following two or three days: difficulty in breathing, chest pain, cough and eye irritation. A dichotomous variable was created that takes the value of 1 if an individual reported that he suffered at least one of these symptoms and 0 otherwise. Regarding the use of health services, respondents were asked to indicate whether, during the smog episode or the following two or three days, they had called a nurse, pharmacist or doctor; called 811 ; were hospitalized; visited an emergency room; consulted a doctor or nurse at a clinic; or consulted a pharmacist. A dichotomous variable was then created taking the value of 1 if the respondent used any of these services and 0 otherwise.The first draft of the questionnaire was reviewed by a panel of five public health experts and pretested on 22 individuals filling the admissibility criteria mentioned above. In addition, the questionnaire was tested by the survey firm for items clarity on a sample of study participants before the beginning of data collection.A sample of 1328 participants vulnerable to smog were recruited in this study from the city of Longueuil, Canada in 2015 and randomly assigned to treatment and control groups . t-test of means difference for independent samples was then used to estimate the effect of smog alert on this variable. A continuous variable was used to measure participants’ knowledge of coping strategies. A p = 0.46).Data on sample characteristics and baseline differences suggest that the randomization process ensured the equivalence between the treatment and control groups and, as a result, there was no need for further controls of preexisting differences.Data on the effects of the APWS were collected after the smog alert of 7 March and 8 March 2016. The main results are summarized in p = 0.01).We observed also a slight difference between treatment and control groups regarding the number of protection measures cited. Members of the experimental group cited an average of one out of three recommended behaviors against 0.91 for the control group (p = 0.40). This result could probably be explained by the fact that the warning message did not provide information on smog risks. p = 0.00). They also kept medication on them more often than usual comparatively to the control group . Concerning respondents’ behavior during the smog episode, 80% of the respondents reported that they stayed indoor the windows closed as much as usual . Experimp = 0.22). This advice seems to be less relevant for the participants in this study that, given their health conditions, only a very small proportion of them (5%) made intense physical efforts during winter smog episodes  and that of the second variable is equal to 1.03 (p = 0.92). Data on study participants indicate that approximatively 30% of respondents suffered smog related symptoms and 6.5% used health system services during the second smog episode . The comThis study suggests that the APWS allow recipients to be better informed on the occurrence of smog episodes and improve their knowledge about coping strategies. Smog alerts also seem to increase significantly compliance with two of the three recommended behaviors. The APWS however does not seem to raise awareness of smog risks, alleviate the symptoms related to this hazard and reduce the use of the health system. An APWS was used in this study to reach vulnerable groups and inform them about the occurrence of smog episodes and coping strategies. Recipients of smog warnings are supposed to be more informed on the occurrence and risks of smog episodes and have better knowledge on how to protect themselves comparatively to non-recipients. Furthermore, the improvement of information and knowledge is expected to encourage adherence to recommended behaviors which, in turn, results in the reduction of risks of suffering from smog symptoms and of using health system services.The results of this study indicate that automated phone warnings significantly improve information on the occurrence of smog episodes. As mentioned before, the APWS was programmed to send smog alerts to treatment group when Environment Canada issues a smog warning on its website. This substantial difference between the treatment and control groups can thus be considered as measuring the additional effect of the APWS to the effects of the smog warning system already in place in Canada, generally available through the media; at the time of the study it was not available through text or email messages, although it became available very recently. The baseline data of this study suggest that the current Canadian smog warning system has a low capacity of reaching vulnerable groups as only 30% of the respondents were informed of the first smog warning issued by Environment Canada. This is concordant with the results of a 2015 survey in Hamilton, Canada showing that 60% of the respondents were aware of the existence of the AHQI system and only 27% checked it . This loThis study also indicates that automated phone warning improves adherence to recommended measures of coping with smog episodes, thus confirming the findings of previous studies on this subject. For instance, Wen and Mokdad  found thThis study suggests that the APWS warnings failed to mitigate health symptoms associated with poor air quality as well as to reduce the use of health system services. This finding nurtures the uncertainty surrounding the health benefits of smog warnings in general. In a population-based cohort study, Chen and al.  show thaThe result of this study suggests that the APWS does not increase recipients’ awareness of the adverse effects of smog. Risk perception has been found to be an important determinant of the adoption of protective behaviors . The perThis study presents some limitations. While the warning system is intended to protect vulnerable groups from smog episodes in general, the scope of this study remains relative to the impacts of winter smog warnings. We should thus be cautious about the generalization of this study findings as the participants in this experiment, given their health conditions, are presumed to substantially reduce their outdoor activities in the winter. They therefore have low exposure to winter air pollution episodes comparatively to those of the summer season. The conclusions are also based on a single two-day smog episode which could limit its generalization potential. Finally, it should be reminded that participants in this study were not randomly selected from a defined population; this could also affect the generalization of findings. We used in this study an experimental design to measure the effects of an automated phone warning system on individuals vulnerable to smog. The comparison between treatment and control groups shows that exposure to smog warning improves information on the occurrence of smog episodes. Treatment group members also have more knowledge on how to protect themselves from this hazard. They are likewise more likely to adopt the recommended behaviors than members of the control group. The analysis however shows that the system has no discernible effect on the awareness of smog health risks, reduction of symptoms related to smog as well as on the use of health system. The low risk of target population exposure to smog may explain the absence of beneficial health effects of smog warning.


Halostachys caspica C. A. Mey. (Chenopodiaceae) for the first time. By means of physicochemical and spectrometric analysis, they were identified as luteolin (1), chrysin (2), chrysin 7-O-β-D-glucopyranoside (3), quercetin (4), quercetin 3-O-β-D-glucopyranoside (5), isorhamentin-3-O-β-D-glucopyranoside (6), and isorhamentin-3-O-β-D-rutinoside (7). All flavonoids were evaluated to show a broad antimicrobial spectrum of activity on microorganisms including seven bacterial and one fungal species as well as pronounced antioxidant activity. Among them, the aglycones with relatively low polarity had stronger bioactivity than their glycosides. The results suggested that the isolated flavonoids could be used for future development of antimicrobial and antioxidant agents, and also provided additional data for supporting the use of H. caspica as forage.Seven flavonoids have been isolated from the aerial parts of  The structure was confirmed by comparison with literature data +, 253 [M-H]-; 1H-NMR  δ (ppm ), 6.96 , 12.81 , 6.21 , 10.91 , 6.52 , 8.06 , 7.59 ; 13C-NMR  δ (ppm), 163.1 (C-2), 105.2 (C-3), 181.8 (C-4), 161.4 (C-5), 99.0 (C-6), 164.4 (C-7), 94.1 (C-8), 157.4 (C-9), 104.0 (C-10), 130.7 (C-1'), 126.4 , 129.1 , 132.0 (C-4'). The structure was confirmed by comparison with literature data +; 1H-NMR  δ (ppm), 6.95 , 12.81 , 6.39 , 6.82 , 8.09 , 7.59 , 5.12 , 5.44 , 5.17 , 5.10 , 4.64 , 3.19-3.70 ; 13C-NMR  δ (ppm), 163.6 (C-2), 105.4 (C-3), 182.0 (C-4), 161.3 (C-5), 99.5 (C-6), 163.9 (C-7), 94.7 (C-8), 157.3 (C-9), 105.2 (C-10), 130.7 (C-1'), 126.5 (C-2'), 129.1 (C-3'), 132.1 (C-4'), 129.2 (C-5'), 126.3 (C-6'), 99.7 (glc-C-1), 72.9 (glc-C-2), 76.6 (glc-C-3), 69.2 (glc-C-4), 77.4 (glc-C-5), 60.7 (glc-C-6). The structure was confirmed by comparison with literature data +, 301 [M-H]-; 1H-NMR  δ (ppm), 9.56 , 12.48 , 6.17 , 10.76 , 6.39 , 7.66 , 9.33 , 9.33 , 6.87 , 7.53 ; 13C-NMR  δ (ppm), 147.9 (C-2), 135.9 (C-3), 176.0 (C-4), 160.9 (C-5), 98.4 (C-6), 164.1 (C-7), 93.5 (C-8), 156.3 (C-9), 103.2 (C-10), 122.1 (C-1'), 115.2 (C-2'), 145.2 (C-3'), 147.0 (C-4'), 115.8 (C-5'), 120.2 (C-6'). The structure was confirmed by comparison with literature data -; 1H-NMR  δ (ppm), 12.60 , 6.21 , 10.80 , 6.41 , 7.61 , 9.65 , 9.14 , 6.86 , 7.58 , 5.47 , 3.10-3.60 ; 13C-NMR  δ (ppm), 159.1 (C-2), 136.2 (C-3), 179.6 (C-4), 162.2 (C-5), 98.6 (C-6), 165.6 (C-7), 94.4 (C-8), 159.1 (C-9), 104.6 (C-10), 122.0 (C-1'), 116.0 (C-2'), 145.0 (C-3') , 150.2 (C-4'), 116.9 (C-5'), 122.7 (C-6'), 100.6 (glc-C-1), 74.9 (glc-C-2), 77.9 (glc-C-3), 70.2 (glc-C-4), 76.0 (glc-C-5), 61.7 (glc-C-6). The structure was confirmed by comparison with literature data +; 1H-NMR  δ (ppm), 6.20 , 6.39 , 7.57 , 3.90 , 7.05 , 7.69 , 5.40 , 3.07-3.83 ; 13C-NMR  δ (ppm), 156.2 (C-2), 133.0 (C-3), 177.5 (C-4), 161.1 (C-5), 98.8 (C-6), 164.0 (C-7), 93.8 (C-8), 156.4 (C-9), 104.2 (C-10), 121.7 (C-1'), 111.5 (C-2'), 149.9 (C-3'), 146.4 (C-4'), 115.6 (C-5'), 122.3 (C-6'), 55.7 (OCH3-3'), 101.0 (glc-C-1), 74.2 (glc-C-2), 77.2 (glc-C-3), 70.0 (glc-C-4), 76.6 (glc-C-5), 61.2 (glc-C-6). The structure was confirmed by comparison with literature data -, 647 [M+Na]+; 1H-NMR  δ (ppm), 12.50 , 6.19 , 10.89 , 6.41 , 7.86 , 3.83 , 9.83 , 6.92 , 7.53 , 5.44 , 4.41 , 0.98 , 3.07-3.83 ; 13C-NMR  δ (ppm), 156.7 (C-2), 133.2 (C-3), 177.4 (C-4), 161.4 (C-5), 99.1 (C-6), 165.0 (C-7), 94.1 (C-8), 156.5 (C-9), 103.9 (C-10), 121.2 (C-1'), 113.5 (C-2'), 149.6 (C-3'), 55.9 (OCH3-3'), 147.1 (C-4'), 115.5 (C-5'), 122.5 (C-6'), 101.5 (glc-C-1), 74.5 (glc-C-2), 76.6 (glc-C-3), 70.8 (glc-C-4), 76.1 (glc-C-5), 67.0 (glc-C-6), 101.1 (rha-C-1), 70.5 (rha-C-2), 70.8 (rha-C-3), 72.0 (rha-C-4), 68.5 (rha-C-5), 17.8 (rha-C-6). The structure was confirmed by comparison with literature data  × 100, where Ac was an average of six replicates of light absorption values at wavelength 510 nm of the negative controls, and At was an average of six replicates of light absorption values at wavelength 510 nm of the samples. The IC50 value was calculated using the linear relation between the inhibitory probability and concentration logarithm according to the method of Sakuma  × 100, where Gc is an average of three replicates of germinated spore numbers in the negative control, and Gt is an average of three replicates of germinated numbers in the treated sets. The IC50 value calculation for the spore germination inhibition was the same as that for antibacterial activity assay. The MIC value on the spore germination was defined as the lowest sample concentration that inhibited visible spore germination.Rice blast fungus,  reports . The samAcontrol-Asample)/Acontrol] × 100, where Acontrol is the absorbance of the control reaction containing all reagents except the test sample, and Asample is the absorbance of the test flavonoid. Tests were carried out in triplicate. BHT was used as the positive control. The IC50 value was calculated using linear relation between the flavonoid concentration and probability of the percentage of DPPH inhibition.Radical scavenging assay was determined by a microplate spectrophotometric method based on the reduction of DPPH according to our previous report . BrieflyAβ-carotene after 2 h assay/Ainitial β-carotene) × 100, Where Aβ-carotene after 2 h assay is the absorbance of the sample with β-carotene-linoleic acid mixture after 2 h period of incubation, and Ainitial β-carotene is the absorbance of the initial mixture. All tests were carried out in triplicate. The IC50 value calculation for β-carotene bleaching inhibition was the same as that for antibacterial activity assay.The antioxidant activity of the flavonoids was evaluated by the β-carotene-linoleic acid bleaching method according to our previous report . BrieflyH. caspica showed a broad spectrum of antimicrobial activity on microorganisms including bacterial (Gram-positive and negative) and fungal species, as well as pronounced antioxidant activity. The bioactivities of flavonoid aglycones with relatively low polarity were better than those of the glycosides. The isolated flavonoids may be contributed to the antimicrobial and antioxidant activity of the extract and fraction, though some synergistic or antagonistic effects should be occurred in the either extract or fraction which need to be clarified. The present study suggested that the crude extract and fraction as well as the flavonoids from H. caspica could be used for future development of naturally-occurring antimicrobials and antioxidants. It also provided additional data for supporting the utilization of H. caspica as forage. A greater research effort should be devoted to confirm antimicrobial and antioxidant activities of the flavonoids from H. caspica which may be applied in food, agriculture and medicinal industry as a source of antimicrobial and antioxidative agents. The underlying antimicrobial and antioxidant mechanisms of the flavonoids, their contents in plant materials as well as their preparation in large scale also need to be further studied.In this study, we reported for the first time that seven flavonoids from


Panax notoginseng, which has been widely used in China for the treatment of strokes. In this study, an ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) combined with preparative high performance liquid chromatography (PHPLC) method was developed to rapidly identify both major and minor saponins in ZSYXST. Some high content components were removed through PHPLC in order to increase the sensitivity of the trace saponins. Then, specific characteristic fragment ions in both positive and negative mode were utilized to determine the types of aglycone, saccharide, as well as the saccharide chain linkages. As a result, 94 saponins, including 20 pairs of isomers and ten new compounds, which could represent higher than 98% components in ZSYXST, were identified or tentatively identified in commercial ZSYXST samples.“Zhu She Yong Xue Shuan Tong” lyophilized powder (ZSYXST), consists of a series of saponins extracted from  Panax notoginseng, is commonly used for treating strokes in the clinic. It can dilate blood vessels, promote blood circulation − (m/z 1171.5509) was about 4, which indicated that there was more than one sugar linked at C-20. In the MS/MS spectrum, characteristic fragment ions at m/z 963.5411 [M − H − 162]−, 801.4900 [M − H − 162 − 162]−, 635.4389 [M − H − 162 − 162 − 162]−, 477.3889 [M − H − 162 − 162 − 162 − 162]−, Glc-Glc diagnostic fragment ions at m/z 221.0657, 323.0960, and PPD-B type characteristic fragment ions at m/z 477.3889 were all observed. According to the MS fragment rules, 60 was tentatively identified as PPD-B-S1(Glc-Glc)-S2(Glc-Glc).In the positive MS spectrum, compound 5, 38, 39, 79, 81, 82, 85, 86, 87 were identified or tentatively identified as ginsenoside C-Y1, quinquenoside IV, notoginsenoside G, chikusetsusaponin VI, ginsenoside Ra0/quinquenoside V, notoginsenoside Fa, ginsenoside Ra3, ginsenoside Rb1, isomer of ginsenoside Rb1, respectively  and [M + COOH]− (m/z 1007.5460) peaks of compound 25 were observed in the negative MS spectrum, and the peak ratio of [M − H]− to [M + COOH]− was 1.5, which indicated more than one sugar was located at the C-20 position. Diagnostic fragment ions at m/z 799.4883 [M − H − 162]−, 637.4389 [M − H − 162 − 162]−, 475.3826 [M − H − 162 − 162 − 162]−, 391.2872 as well as m/z 405.3973, 423.4204, 441.4223 were observed in negative and positive mode, respectively. As a result, compound 25 was identified as notoginsenoside R3, and compounds 37 and 71 were identified as isomers of 25 because similar fragment ions were observed in their spectra.[M − H]90 produced [M − H]− ions at m/z 943.5121 and [M + COOH]− ions at m/z 989.5183 in negative mode, while the peak ratio of [M − H]− to [M + COOH]− was 8, which indicated there are two or more sugars at the C-20 position. Diagnostic ions of PPT type were observed at m/z 405.3433, 423.3545, 441.3794, and diagnostic ions of PPT-A type were observed at m/z 457.3656. In the MS/MS spectrum, characteristic fragment ions at m/z 781.4300 [M − H − 162]−, 619.3881 [M − H − 162 − 162]−, 457.3656 [M − H − 162 − 162]− and Glc-Glc ions at m/z 221.0662 were obtained. Based on the MS cleavage rules (90 was tentatively identified as PPT-B-S1(Glc-Glc-Glc). Compound ge rules , literat83 could be supposed to 1106 through negative ions at m/z 1105.5357 [M − H]— and 1151.5401 [M + COOH]− (− to [M + COOH]−(0.1). Furthermore, diagnostic ions of PPT type were observed at m/z 405.3459, 423.3565, 441.3661. In the MS/MS spectrum, fragment ions at m/z 943.5543 [M − H − 162]−, 781.4996 [M − H − 162 − 162]−, 619.3441 [M − H − 162 − 162 − 162]−, 457.3654 [M − H − 162 − 162 − 162 − 162]− could be deduced to represent neutral losses of four glucose moieties successively from the precursor ions. Glc-Glc diagnostic ions at m/z 221.0664 could be detected. Characteristic ions at m/z 457 indicated the aglycone type of 83 should be PPT-B (83 could be tentatively identified as PPT-B-S1(Glc-Glc-Glc-Glc). Compound 84 with a similar fragmentation behavior could be tentatively identified as an isomer of 83 -S2(H). Compound 27, 29, 31, 33, 40, 41, 47, 55, 66, 73, 78, 93 and 94 were unambiguously identified as notoginsenoside N, notoginsenoside R6, 20-O-glucoginsenoside Rf, notoginsenoside R1, ginsenoside Rg1,ginsenoside Re, pseudoginsenoside Rt3, sanchirhinoside A3, sanchirhinoside B, notoginsenoside U, ginsenoside Rg2, ginsenoside Rk3, and ginsenoside Rh4, respectively , yesanchinoside E (28), an isomer of yesanchinoside E (30), an isomer of 20-O-glucoginsenoside Rf (32), notoginsenoside M or N (34), an isomer of notoginsenoside M or N (35), gypenoside LXIV (36), an isomer of notoginsenoside R3 or notoginsenoside R6 (37), PPT-A-S1(Glc-Glc-Xyl/Ara)-S2(Glc-Glc) (42), quinquenoside L16 (43), PPT-C-S1(Glc)-S2(Glc-Glc) (44), PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (45), chikusetsusaponin L5 (46), an isomer of chikusetsusaponin L5 (48), an isomer of notoginsenoside R2 (49), an isomer of notoginsenoside R2 (52), an isomer of 20-O-glucoginsenoside Rf (53), an isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (54), PPT-A-S1(Rha-Xyl)-S2(Glc) (56), an isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (57), an isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (58), an isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (61), an isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (63), an isomer of 20-O-glucoginsenoside Rf (67), an isomer of notoginsenoside R3 or an isomer of notoginsenoside R6 (68), ginsenoside Rg1 (69), an isomer of 20-O-glucoginsenoside Rf (70), an isomer of notoginsenoside R3 or an isomer of notoginsenoside R6 (71), an isomer of notoginsenoside R2(72) and ginsenoside F1 (78), respectively . Furthermore, diagnostic ions of OCO type was observed at m/z 421, 439, 457  and [M + COOH]− (m/z 993.4633) of compound 64 were observed in the negative MS spectrum, and the peak ratio of [M − H]− to [M + COOH]− was 0.25. Diagnostic fragment ions at m/z 815.3567 [M − H − 132]−, 653.3778 [M − H − 132 − 162]−, 491.3608 [M − H − 132 − 162 − 162]−, 391.8554, as well as m/z 421.3447, 439.3612, 457.3701 were observed in negative and positive mode, respectively. As a result, 64 was identified as OCO-B-S1(Glc-Glc-Xyl/Ara)-S2(OH).[M − H]1 could be deduced as 966 according to quasi−molecular ions at m/z 965.5198 [M − H]− and 1011.5278 [M + COOH]−. The peak ratio of [M − H]− to [M + COOH]− was about 0.1, which indicated that there was one or less sugar at C-20. The aglycone type could be identified as OCO type through diagnostic ions at m/z 439, 457. In the MS/MS spectrum of [M − H]−, fragment ions at m/z 785.9465 [M − H − 180]−, 653.3969 [M − H − 180 − 132]−, 491.3843 [M − H − 180 − 132 − 162]−, 415.3350 and fragment ions at m/z 191 indicated there was a Glc-Xyl/Ara chain in compound 1. Characteristic ions at m/z 415 and 491 indicated the aglycone would be OCO-A type. According to the peak ratio rule of [M − H]− to [M + COOH]− and the structural features of OCO-A, we could deduce that the Glc-Xyl chain was linked at the C-6 position of the aglycone. As a result, compound 1 was tentatively identified as OCO-A-S1(Glc)-S2(Glc-Xyl/Ara), compound 2 was identified as an isomer of 1 because a similar MS cleavage pathway was observed. The molecular weight of compound 18 could be supposed to have a molecular weight of 814 through negative ions at m/z 813.4677 [M − H]− and 859.4739 [M + COOH]− . Compound 21 with similar fragmentation behaviors at m/z 651.4113 [M − H − 162]−, 489.3635 [M − H − 162 − 162]−, was tentatively identified as an isomer of OCO-E-S1(Glc-Glc).Compound + COOH]− . There m439, 457 . In the olecules . Charact20, [M − H]− ions at m/z 959.5194 and [M + COOH]− at 1005.5220 as well as their peak ratio (0.25) were observed. In the MS/MS spectrum, characteristic fragment ions at m/z 797.4207 [M − H − 162]−, 635.3567 [M − H − 162 − 162]−, 473.3460 [M − H − 162 − 162 − 162]− and 221.0431 [Glc-Glc]− were also observed. In the positive spectrum it produced ions at m/z 421, 439, 457 which could be used to identify an OCO type compound. In MS/MS mode, OCO-D type fragment ions at m/z 473 could be obtained. According to the MS fragment rules, 20 was tentatively identified as OCO-D-S1(Glc-Glc-Glc). Similar diagnostic fragment ions were observed in 22 -S2(CH3), an isomer of OCO-B-S1(Glc-Rha-Glc)-S2(CH3), an isomer of OCO-B-S1(Glc-Rha-Glc)-S2(CH3) and OCO-B-S1(Xyl-Glc-Glc-Glc)-S2(OH) -S2(OH) .6 (23), notoginsenoside R3 (25), notoginsenoside N (27), notoginsenoside R6 (29), 20(S)-20-O-glucoginsenoside Rf (31), notoginsenoside R1 (33), notoginsenoside G (39), ginsenoside Rg1 (40), ginsenoside Re (41), pseudoginsenoside Rt3 (47), sanchirhinoside A3 (55), vina-ginsenoside R4 (65), sanchirhinoside B (66), notoginsenoside R2 (72), notoginsenoside U (73), ginsenoside Rg2 (78), notoginsenoside Fa (82), ginsenoside Ra3 (85), ginsenoside Rb1 (86), ginsenoside Rd (91), ginsenoside Rk3 (93), and ginsenoside Rh4 (94) were isolated from ZSYXST by the authors. Their structures were elucidated by 1D and 2D NMR spectra [HPLC grade acetonitrile (ACN) and formic acid were purchased from Merck Technologies Inc. , and Tedia Company Inc. . Deionized water was obtained from a Millipore Milli-Q water system . All other reagents were of analytical purity. ZSYXST sample was obtained from Guangxi Wuzhou Pharmaceutical (Group) Co., Ltd. . Twenty-one reference compounds: sanchirhinoside A spectra ,44.18 column , . The mobile phase consisted of water (A) and ACN (B), using 19%–20% B at 0–25 min, 20%–30% B at 25–35 min, 30%–35% B at 35–45 min, 35%–45% B at 45–70 min, 45%–90% B at 70–75 min, 90% B at 75–76 min, 90%–19% B at 76–80 min, the flow rate was 8 mL/min.ZSYXST (10 mg)was dissolved in 19% acetonitrile (1 mL)to obtain sample 1, which was then centrifuged for 10 min at 14,000 rpm, and the supernatant of sample 1 was applied to a Shimadzu LC-8A PHPLC system , equipped with a binary pump, an UV detector and a fraction collector(FRC-10A). Chromatographic separation was achieved on a Cosmosil-5CUtilizing an online collector simulation system, the major peaks 0–2 of ZSYXST were easily removed through the control of the collection parameters, set at level of 500 µV, peak slope of 1000 µV/s and the delay volume of 200 µL. Finally, sample 2 (1.3 mg) without major components was obtained, and the peaks of lower content ingredients became more obvious finally .3 column  under 40 °C. The mobile phase consisted of 0.1% formic acid water (A) and ACN (B), using 10% B at 0–10 min, 10−40% B at 10–90 min, 40%–100% B at 90–91 min, 100% B at 91–100 min, 100%–10% B at 100–100.1 min, 10% B at 100.1–105 min. The flow rate was 0.3 mL/min, and the sample volume injected was 2 µL.Sample 2 (1 mg/mL) and ZSYXST sample (1 mg/mL) were analyzed on an Agilent 1290 Ultra high performance liquid chromatography system , equipped with a T2) flow rate, 8.0 L/min; drying gas temperature, 350 °C; nebulizer, 30 psig; capillary voltage (Vcap), 3500V. Cone voltage was 120 V and 175 V in the positive and negative mode, respectively. Collision energy (CE) was dynamically adjusted from 45 to 70 V according to the m/z of precursor ions in the negative MS/MS mode. The mass analyzer scanned over m/z 100–2000. All the data were recorded and processed by Agilent Mass Hunter Workstation software (Version B.02.00).The Q-TOF/MS analysis was performed on an Agilent 6520 Accurate-Mass Q-TOF/MS system. The conditions of the ESI source were: drying gas (NP. notoginseng and P. ginseng.In this work, a novel UHPLC-Q-TOF-MS combined with PHPLC method was established. The sensitivity of some minor components in ZSYXST could be enhanced significantly by this method. Combining the characteristic ions in positive and negative mode, the types of aglycone, saccharide, as well as the linkage positions of the saccharide chains of saponins were quickly determined. As a result, based on the exact mass, fragmentation behaviors, retention times and literature, 94 saponins, including 20 pairs of isomers, which could represent over 98% of the components in ZSYXST were identified or tentatively identified and ten of these saponins were identified as new compounds. This method could provide a powerful platform for profiling the compounds in ZSYXST and also could be useful for identification of saponins of


Although numerous studies of diversity have been conducted in cassava, there is no comprehensive assessment of global genetic diversity. Here we draw on previous studies and breeders’ knowledge to select diversity sets from the International Institute of Tropical Agriculture (IITA) and the International Center for Tropical Agriculture (CIAT) genebanks and breeders’ germplasm, as well as elite germplasm and landraces from eastern, southern and central (ESC) Africa to make a global assessment of diversity in cassava, using a SNP based GoldenGate (Illumina Inc.) assay. A synthesis of results from genetic distance and ADMIXTURE analysis essentially revealed four populations (i) South American germplasm characterised by relatively higher genetic diversity with hypothetical ancestral founder genotypes from Brazil, (ii) a smaller group of African introduction germplasm which is more distantly related to all other germplasm, (iii) West Africa germplasm dominated by IITA breeding lines, containing sources of cassava mosaic disease resistance, and IITA genebank accessions from West Africa, both characterised by slightly lower diversity, and (iv) a less cohesive group of African germplasm, termed ‘Other’, with moderate levels of diversity and a majority of germplasm from ESC Africa. This study highlights opportunities for heterosis breeding, purging of duplicates in genebanks and the need for conservation of ESC Africa landraces. Manihot esculenta Crantz.) are a major food staple in Africa and provide calories for nearly half a billion people worldwide. Africa contributes 61% of global cassava production, followed by Asia (29.5%) and Americas (9.5%). Nearly 178 Million tonnes (Mt) of cassava are produced in Africa, which is twice as much as maize (84Mt) [Starch-rich storage roots of cassava (Manihot esculenta ssp. flabellifolia (Pohl), a perennial woody shrub [th and 18th centuries [Cassava is clonally propagated through stem cuttings with a typical 12-month cropping cycle. Botanical seed is produced with separate male and female flowers that are adapted for outcrossing. The center of origin for cassava is in Central and South America although the specific geographic origins are still debated. Evidence based on molecular markers suggests that it was domesticated as much as 10,000 years ago, within the south-western rim of the Amazon basin (in modern day Brazil), and is derived from its closest wild relative, dy shrub . During dy shrub ,5. Subsedy shrub . InformaM. esculenta genepool. Strict quarantine regulations that are in place are essential to help restrict the spread of a number of diseases, which are currently geographically limited. Frog Skin Disease occurs exclusively in South America. Cassava Mosaic Geminiviruses which cause cassava mosaic disease (CMD) leading to on average 24% yield losses in Africa [A lack of understanding of the global structure of diversity of cassava and a previous lack of adequate diagnostics and certification procedures to meet quarantine regulations for movement of germplasm have been main factors that have constrained the use of diversity within the n Africa , exist in Africa , 9 are cn Africa . One wayn Africa . The purM. esculenta predominantly of South American and Asian origin and 883 associated wild relatives. The International Institute of Tropical Agriculture (IITA) hosts a collection of 2,712 M. esculenta accessions predominantly of West African origin. The conservation of cassava germplasm from ESC Africa is under-represented in international collections [The International Center for Tropical Agriculture (CIAT) holds a collection of 5,760 accessions This study utilises data generated under the Generation Challenge Program (GCP) to select subsets of germplasm from the IITA and CIAT genebanks to maximise diversity for further SNP genotyping in this study. The GCP study genotyped at CIAT 3,000 cassava accessions from CIAT, EMBRAPA and IITA for 22 simple sequence repeat (SSR) markers. IITA, CIAT and EMBRAPA contributed 1,000, 1,500 and 500 clones respectively to that study . As partBreeders from IITA and CIAT selected germplasm representing diversity with specific attributes important for breeding. Approximately 100 breeding lines were requested along with documentation on the traits of interest. The IITA breeding lines were primarily from West Africa.Germplasm from ESC Africa was selected from  in whichHere, to ensure there were no genotypes with an exceptionally high similarity, a modified Rogers Genetic Distance (MRD) was calculated from SSR data  among thTo assess the diversity of the ESC Africa subset relative to that of 1,401 varieties analysed by , and theDNA from the selected 121 CIAT breeding lines, 100 IITA breeding lines, 144 CIAT and 4 EMBRAPA genotypes maintained at the CIAT genebank, 102 IITA genebank accessions, and 117 varieties from ESC Africa were sent for EST-derived SNP genotyping using a previously established 1,536 Illumina GoldenGate array  at the SInitially loci followed by individuals with more than 6% missing data were removed from the dataset. Relationships among the six pre-defined groups were estimated using Nei’s genetic distance and illustrated using the unweighted neighbor-joining method of clustering in DARwin v6.0.21 . This waOf the 3,000 germplasm lines genotyped under GCP using 22 SSR markers from CIAT, data was obtained from 2,494 accessions . PrincipComparison of the number of alleles represented in genotyping of the 2,494 accessions (GCP set) compared to the genebank subset is given in Approximately ten varieties selected by breeders from each of seven countries according to the presence of interesting traits and usefulness in the breeding program are provided in Genotyping data for 555 accessions for 1,536 SNPs were obtained. SNPs and accessions with more than 6% missing data were deleted leaving a final matrix of 1,124 polymorphic SNPs and 522 genotypes. Prior knowledge that many breeding lines in ESC Africa originated from IITA, Nigeria, through the International Seedling Nurseries, was supported by an initial analysis of data based on the five populations , which showed the relatively close relationship between ESC Africa germplasm and IITA genebank (0.99) and IITA breeders (0.98). To avoid any bias that this mix of germplasm may cause in the analysis, ESC Africa germplasm was further divided into ESC Africa elite (42 genotypes) and ESC Africa landrace (75 genotypes). The final dataset of 522 genotypes consisted of 121 CIAT breeding varieties, 96 IITA breeding varieties, 87 CIAT genebank accessions, 101 IITA genebank accessions, 75 ESC Africa landraces and 42 ESC Africa elite genotypes. In addition, closely linked SNPs (derived from same EST fragment) were filtered leaving a set of 901 SNPs. These data are available as AMOVA analysis with 999 permutations indicated that 91% of variation was found within populations with only 9% among populations, and zero among individuals. Average F statistics across loci and six populations were Fst = 0.040, Fis = -0.077 and Fit = -0.034, indicating limited differentiation among populations. Pairwise population differentiation (Fst) calculated via the frequency option of AMOVA using GenAlEx 6.5 , 18 is gFrom Interestingly, another cluster  is dominated by landraces from Mozambique, Madagascar and Tanzania and also contains two clones from the CIAT genebank, MAL60  and TAI1 (from Thailand). These are the only accessions from the respective countries included in this study. Included in this cluster are also five accessions from the IITA genebank, namely TMe-3181 from Malawi, TMe-3690 and TMe-3638 from Ghana, TMe-3554 from DRC, and TMe-2998 from Benin.Another cluster (Cluster 3 DRC) is dominated by landraces from DRC and breeding lines from Madagascar together with a clutch of IITA genebank accessions largely from West Africa  as well as one from Angola and one from Chad. Some of these genebank accessions are essentially identical based on the SNP data generated in this study.A large cluster is dominated by IITA breeding lines interspersed with IITA genebank accessions and many breeding lines from ESC Africa (Cluster 4 West Africa). Only three genotypes classified as ESC Africa landraces cluster with this group; namely Nambiyo from DRC, Tukumbo from Rwanda and Rantsan’akoho from Madagascar. Interestingly three IITA genebank accessions are essentially identical to TMEB1  and close to TMe-3070. Another series of essentially identical genotypes are TMEB3,7,11,12,14, TMe-3084 and TMe-62. TMEB419, a popular variety in East Africa, is also essentially identical to TMe-3599. Interestingly one CIAT breeding line, CG1320-10, clusters with the African breeding germplasm. It is a cross between Mex1 and Pan51 with excellent cooking qualities.Another cluster of germplasm is dominated by Kenyan, Rwandan and Ugandan germplasm with a few genotypes from DRC, Tanzania and Mozambique. This is largely an East African group . A few IITA genebank accessions from several countries in West Africa  also cluster with this group. Strangely the only two accessions from Venezuela from the CIAT genebank also cluster with this group (VEN164 and VEN173).A final, much more diverse cluster is dominated by IITA genebank accessions and landraces from Tanzania, Uganda, Madagascar and Mozambique. It contains a cluster of nine essentially identical West African genotypes from IITA genebank and Longo Asara, a landrace from Madagascar, TMe-3195 from Angola and TMe-3206 from Chad. These genotypes are in turn almost identical to TMEB693, with a further five very closely related accessions and the landrace Fernando Po from Mozambique. Other genotypes in this cluster are much more diverse (Cluster 6 Others).The ADMIXTURE program assigns individuals proportionally to hypothetical founder populations. The number of sub-populations (K) was varied from 2 to 30. The most appropriate value of K which produced the lowest 10-fold cross-validation error compared to other K values was quite difficult to discern , so one A second ancestral founder population (Group 2) from ADMIXTURE analysis is characterised by Nase 3  and three landraces from Ghana (TMe3698), Guinea (TMe3459) and Niger (TMe3219). The old IITA breeding lines of TMEB 3,7,11,12 and 14 have the vast majority of this ancestry, with a small proportion of ancestry from the early introductions of cassava to West Africa. The third ancestral founder group is from Brazil, the presumed center of origin and domestication of cultivated cassava (Group 3). The group consists of four landraces from Brazil (Saracura Lii (BRA234), Paraiba (BRA296), Vermelhina (BRA445) and Pirassununga (BRA775)). ESC Africa germplasm largely comprised a mixture of ancestry from all three groups.There were only four private alleles (occurring exclusively in one population) all within the CIAT breeders’ population; namely locus Me.MEF.c.0738 , locus Me.MEF.c.0259  and locus Me.MEF.c.0368  which interestingly are all chlorophyll A-B binding proteins, and locus Me.MEF.c.0285  which is a translation initiation factor 5A (eIF-5A). Of these private alleles three are synonymous and Me.MEF.c.0285 is a non-synonymous mutation. These were present in just seven samples. This is the first study that evaluates diversity of cassava germplasm on a global basis, with a substantial number of genotypes from several geographical regions. The goal of this study was to understand present patterns of global cassava diversity from a historical perspective.Based on SSR data of nearly 3000 accessions, 82.5% of alleles were included in the genebank subset of 250 accessions thus the subset can be considered fairly representative of genebank diversity. The entire ESC Africa germplasm set (117 accessions) could also be considered a good representation of both landraces and elite clones from the region as it consisted of germplasm from seven countries and contained 85.4% of SSR alleles present in the set of 1,401 genotypes analysed by . ESC Afrvice versa for Frog Skin Disease in the Americas. It is interesting that there does not appear to be evidence for a strong genetic bottleneck  during the introduction of cassava to Africa. This may be due to frequent introductions of a broad diversity of germplasm and the clonal nature of propagation, which restricts recombination events and thus genetic drift. It is possible that if germplasm could be moved safely, that heterosis between these two genepools could be exploited in breeding. Germplasm from the CIAT breeding program and the genebank are closely related with little differentiation . Interestingly, in cassava, unlike some other crops such as maize, landraces are often indistinguishable from improved clones and are often considered for release. Breeding has not significantly separated improved clones from landraces.In terms of genetic similarity, the greatest distance between populations appears to be between CIAT and IITA breeding germplasm and IITA breeding germplasm and CIAT genebank accessions. This is supported by both Nei’s genetic distance and Fst values. These Fst values are not particularly high indicating no substantial differentiation, thus reflect a relatively recent introduction of cassava into Africa from the Americas and limited opportunity for divergence due to a restricted frequency of recombination events due to the predominant clonal nature of propagation and a relatively long reproductive cycle (12 months). The limited differentiation that has occurred, within the last ~400 years since the introduction of cassava to Africa, will be the result of selection due to biotic and abiotic factors and farmer and consumer preferences. It is likely that the differentiation has been accentuated in recent years by the difficulties of germplasm movement between the two continents. Quarantine regulations restrict the movement of African cassava germplasm to the Americas due to the presence of CMD and CBSD in Africa but not Asia, and The most closely related groups were the ESC Africa elite germplasm and IITA breeders’ germplasm (Nei’s genetic distance = 0.012) reflecting the movement of germplasm from IITA to ESC Africa, most likely through the International Nurseries Program. This justifies the separation of ESC Africa elite germplasm from ESC Africa landraces. Interestingly ESC Africa elite germplasm is also closely related to IITA genebank accessions (Nei’s genetic distance = 0.13), whereas germplasm from IITA breeders and IITA genebank are less closely related (Nei’s genetic distance = 0.17). It is likely that breeding programs in ESC Africa are incorporating germplasm from the IITA genebank as well as ESC Africa landraces.Levels of diversity according to Ne, percentage of polymorphic loci, I, He and uHe were greatest in CIAT breeders’ germplasm, followed by the CIAT genebank . This isM. glaziovii made at the Amani breeding program in Tanzania [Interestingly the ESC Africa landraces had similar levels of diversity to IITA genebank . The relTanzania –29. LeveEquatorial Guinea, neighbouring the islands of São Tomé and Príncipe, centres of activity for the Portuguese in the 16th and 17th centuries [The analyses of genetic variation and identification of hypothetical founder groups complement each other and reveal four albeit rather loose groups . The firenturies . It was enturies . The Mozenturies , 5, 6. Tenturies  (Supplemhttps://www.genesys-pgr.org/), these three IITA genebank accessions were collected from Nigeria in 1971. Of all the germplasm shipped to Africa from South America in those early years, much would have succumbed to CMD, however it is possible that some may have survived in their entirety and become incorporated and thought of as African germplasm. Interestingly the IITA genebank accession TMe-3 did not cluster with the IITA breeding clones TMEB 3,7,11,12,14 which contain the CMD2 gene. This “forensic introgression” needs to be further investigated.The second group is that from South America, with hypothetical founder genotypes from Brazil. This group includes Cluster 1 from genetic distance analysis , and theM. glaziovii inter-specific breeding program in Tanzania [M. glaziovii, however this could only be discerned by further whole genome sequencing or fingerprinting. It is represented by Cluster 4 in the dendrogram , - 3 (DRC) and—5  in the dendrogram. Cluster 2 is dominated by Mozambican, Madagascar and Tanzanian landraces. The occurrence of an IITA genebank accession from Malawi (TMe-3181), which neighbours Mozambique, fits well with this group. The inclusion of germplasm from Malaysia and Thailand in this group is likely to reflect Indian ocean trade routes. TAI1 was a popular landrace grown from the 1960s to 1980s in Thailand and was the first officially released variety in that country under the name Rayong 1. It was thus likely a prime candidate for introduction into East Africa. The four accessions in this group from Benin, Ghana and DRC do not fit the pattern and are less easy to explain.vice versa. A number of IITA genebank accessions from West Africa  as well as one from Angola and one from Chad are associated with this cluster, however some of these are essentially identical. Within this group is some differentiation defined by these clusters. The disaggregation of Mozambican, Madagascar and Tanzanian landraces  from DRC, Rwanda, Kenyan and Ugandan germplasm  was also observed to some extent by [Cluster 3 (DRC) is dominated by germplasm from DRC and Madagascan breeding germplasm. This is an interesting association and possibly reflects introduction of DRC germplasm into Madagascar, or xtent by .It is hoped that the insights provided through this study will enhance both the conservation and use of cassava genetic diversity. Efforts are underway to broaden the genetic base of IITA breeding germplasm, and exploit heterosis, by introgressing CMD resistance into South American and SE Asian germplasm through the international nurseries established in Hawaii. Similarly, introgression of the putative immunity to CBSD found in South American cassava germplasm into African breeding populations is ongoing. It is clear that there are a number of duplications within the genebank at IITA, and efforts are underway to purge these duplicates. Landraces from ESC Africa clearly have diverse ancestral backgrounds and occupy an intermediary position between West African germplasm and that from South America. This germplasm is under-represented in the IITA collection, and efforts are underway to conserve germplasm from the region and increase its representation in IITA’s genebank. Although this study has revealed some interesting relationships and raised some hypotheses, further studies of ‘forensic introgression’ are clearly needed to more fully understand and substantiate the findings.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Elaphurus davidianus) were once widely distributed in the swamps  of East Asia. The dramatic recovery of the Milu population is now deemed a classic example of how highly endangered animal species can be rescued. However, the molecular mechanisms that underpinned this population recovery remain largely unknown. Here, different approaches  were utilized to elucidate the aforementioned molecular mechanisms. The comparative genomic analyses revealed that the largest recovered Milu population carries extensive genetic diversity despite an extreme population bottleneck. And the protracted inbreeding history might have facilitated the purging of deleterious recessive alleles. Seventeen genes that are putatively related to reproduction, embryonic  development, and immune response were under high selective pressure. Besides, SCNN1A, a gene involved in controlling reabsorption of sodium in the body, was positively selected. An additional 29 genes were also observed to be positively selected, which are involved in blood pressure regulation, cardiovascular development, cholesterol regulation, glycemic control, and thyroid hormone synthesis. It is possible that these genetic adaptations were required to buffer the negative effects commonly associated with a high‐salt diet. The associated genetic adaptions are likely to have enabled increased breeding success and fetal survival. The future success of Milu population management might depend on the successful reintroduction of the animal to historically important distribution regions.The Milu (Père David's deer,  This spOdocoileus virginia) (21%–22%) . This region is historically synonymous with the Milu Cao, . AlthougPrevious genetic studies using mitochondrial and microsatellite loci have helped to investigate the population genetics of the Milu. These analyses have revealed that the Milu exhibit extremely low genetic diversity caused by reduced foundation population sizes  in Dafeng Milu Natural Reserve in 2013. The blood samples were deposited in vacuum blood collection tube (Heparin anticoagulation) and shipped to the laboratory by dry ices.2.2An adult female Milu in Dafeng Milu Natural Reserve was used for de novo sequencing. Samples from an additional five animals were utilized for resequencing. Genomic DNA was extracted using Puregene Tissue Core Kit A (Qiagen). Libraries with different insert sizes were constructed at Majorbio (Shanghai), and the insert sizes of the libraries were 180, 500, 800 bp, 3, 5, 8, and 10 kb. For short‐insert libraries (180–800 bp), 6 μg of DNA was fragmented to the desired insert size, end‐repaired, and ligated to Illumina paired‐end adaptors. Ligated fragments were size selected at 180, 500, and 800 bp on agarose gels and were purified by PCR amplification to yield the corresponding libraries. For long insert sizes  mate‐pair library construction, 60 μg of genomic DNA was used; we circularized DNA, digested linear DNA, fragmented circularized DNA and purified biotinylated DNA, and then performed adaptor ligation. The libraries were sequenced using a HiSeq2000 instrument. The other five resequencing samples were sequenced with read and insert lengths of 101 and 500 bp, respectively.denovo . Finally, variants from each individual were generated by filtering positions with low depth . The SNP positions in 18 polar bear genomes  was set to 6 years. Before ROH analysis, we used VCFtools (v0.1.12b)  model , and the genome and annotation data for P. hodgsonii, C. hircus, L. vexillifer, B. grunien, and C. ferus were downloaded from the NCBI database. The longest predicted translation product was chosen to represent each gene, and gene models with an open reading frame <150 bp in the genomes were removed. Next, these protein sets were pooled, and self‐to‐self BLASTP , and Ka (nonsynonymous substitutions per nonsynonymous site) values for each remaining gene family using the free‐ratios model and an F3x4 codon frequency model implemented using CODEML in PAML4.7  as Milu or cow lineage‐specific fast evolving GO categories, which were summarized and visualized by REViGO  were extracted from orthologous gene clusters identified by OrthoMCL. PRANK  ratios of filtered reliable codons in 3,549 single‐copy gene families were calculated using the branch‐site model of CODEML in PAML4.7 Yang, . Milu wa2.9X‐axis. The saline standard concentration values were used as the Y‐axis. A total of 0.5 g of plant materials was weighed in a beaker and mixed with 100 ml of distilled water. After the mixture was heated using an electric stove for 30 min, the resultant solution was strained into a new volumetric flask with 25 ml of distilled water. The solution was stored in a 50‐ml centrifuge tube and was subsequently used to determine EC values.A total of 5, 10, 15, 20, 25, 40, 60, 80, 100, 120, 140, 160, 180, and 200 mg of NaCl were weighed, respectively, in separate beakers. A total of 50 ml of distilled water was subsequently mixed with each quantity of NaCl to prepare saline standards. The electric conductivity (EC) value of standard saline was determined using a conductivity meter, and the resultant values were used to generate the 33.1Odocoileus virginianus    had a scaffold N50 value of 2.85 Mb  but slightly higher than that of the crested ibis , and the baiji . Also, patterns of SNP density distributions were explored by fitting a two‐component mixture model to the observed SNP densities using the expectation‐maximization algorithm  in vertebrates  was significantly overrepresented  that are known to be involved in vessel, platelet and cardiovascular development, thyroid hormone synthesis, cholesterol regulation, insulin regulation, and glycemic control , has been identified as a new endogenous mammalian hormone implicated in glycemic control. The trace elements iodine is essential for thyroid gland functioning and thyroid hormone biosynthesis and metabolism  and inland regions (freshwater). However, long‐term exposure to high‐salt food can lead to higher risks of high blood pressure and cardiovascular disease, and damage to blood vessels, kidneys, and bones in humans  is proportionally expanded in the Milu genome  and antigen processing and presentation of peptide antigens via MHC class I (p = 3.54 × 103). The proteins encoded by these families are essential in eliciting immune system responses against non‐self‐antigens  values in the Milu following Milu‐cow‐human, Milu‐TA‐human, and Milu‐baiji‐human comparisons, respectively. These accelerated evolving GO categories were predominantly found to be involved in responses to immunity, development, DNA repair, and excretion .However, as revealed by our analyses, the species themselves  also inherently contributes to its survival and recovery. Juvenile mortalities are very high in many captive mammals (including ungulate species and inbred cheetah populations), which are most likely caused by inbreeding depression and other factors often associated with inbreeding , will be useful and necessary for these purpose. Using these large‐scale population data, we can evaluate the genetic diversity, population demography, and recent adaptions after genetic bottlenecks (such as selective sweep) of Milu subpopulation, therefore pave the way to the future success of Milu population management.The authors declare no competing commercial interests.L.Z. conceived the study, L.Z. headed and H.H. managed the sequencing project, L.Z. and J.D. prepared sequencing data, L.Z., C.D. X.Z., and Z.W. coordinated the bioinformatics activities, L.Z., C.D., X.Z., S.Z., Z.W., G. L, and S.Q. designed experiments and analyzed the data, S.H. and Y.D. participated in project design, L.Z., C.D., Z.Y., and G. L. wrote and edited the manuscript with input from all other authors. All authors have read and approved the manuscript.1zhulf@ioz.ac.cn).This whole‐genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JRFZ00000000. The version described in this paper is version JRFZ01000000. Correspondence and requests for materials should be addressed to L.Z. ( Click here for additional data file.


Calanus finmarchicus (haploid genome size of 6.34 Gbp). We optimized a capture enrichment-based protocol based on 2656 single-copy genes, yielding a total of 154 087 high-quality SNPs in C. finmarchicus including 62 372 in common among the three locations tested. The set of capture probes was also successfully applied to the congeneric C. glacialis. Preliminary analyses of these markers revealed similar levels of genetic diversity between the two Calanus species, while populations of C. glacialis showed stronger genetic structure compared to C. finmarchicus. Using this powerful set of markers, we did not detect any evidence of hybridization between C. finmarchicus and C. glacialis. Finally, we propose a shortened version of our protocol, offering a promising solution for population genomics studies in non-model species with large genomes.Advances in next-generation sequencing technologies and the development of genome-reduced representation protocols have opened the way to genome-wide population studies in non-model species. However, species with large genomes remain challenging, hampering the development of genomic resources for a number of taxa including marine arthropods. Here, we developed a genome-reduced representation method for the ecologically important marine copepod  Its pohaploid; ,36 to mihaploid;  and a fehaploid; . All stuhaploid;  to a larhaploid; . The queCalanus individuals from different locations. This protocol requires a high amount and high quality of DNA to start with, but as the amount of DNA extracted from one individual of Calanus finmarchicus is rather low, due to the body-size of the organism , pooling several individuals together was the only option. The enzyme pair to be used for the digestion was selected based on the results from an in silico digestion of the very small fraction of the genome sequenced so far (less than 0.5%). Although we would normally expect a small portion of genome to be sufficient for in silico digestion, it seems obvious that the large (and probably duplicated) genome of C. finmarchicus may have altered the success of this approach in selecting an optimal restriction enzyme pair. Indeed, in the SimRAD-based method )et al. ) and limmpromise . Moreoveromising .Calanus  appears genetically well differentiated from the two other locations on the first axis of the PCA (PC1: approx. 12%). Individuals from the Skjerstadfjord are clustered closely together, while the individuals from the Lurefjord are more distanced from one another. Both FST and PCAs suggest a more recent and stronger genetic structure for C. glacialis compared to C. finmarchicus populations. It is important to keep in mind though, that we have been sampling C. glacialis genome using C. finmarchicus originated probes. Consequently, we may have missed some naturally more variable regions in C. glacialis by capturing mostly regions conserved enough between species to be recognized by the capture probes; another possibility is that due to the lower number of individuals we would be missing more variants  in C. glacialis  [Calanus species.The vast majority (greater than 99.99%) of the SNPs identified with the capture-based protocol did not show any sign of recent and/or strong positive selection. Assessment of genetic diversity and heterozygosity levels revealed very similar results between the two species of  figures  and 3. Aequenced . Samplesig. 2 in ). These ig. 2 in ,72. Only [et al.  reportedte loci) . HoweverCentropages typicus, a 2b-RAD-seq approach yielding 675 SNPs revealed genetic structure between the northwest and the northeast Atlantic Ocean [Euphausia superba [ca 24 Gbp haploid) with no primary reference available, particularly due to the fact that most of the markers they discovered were from multicopy genomic regions and had to be removed from downstream analyses [Population genomics studies of marine zooplankton have been very scarce . In the ic Ocean , which wic Ocean . AnotherC. finmarchicus and C. glacialis represents a very powerful tool to investigate the potential for hybridization and introgression between the two species. Indeed, using microsatellites, the presence of hybrids between C. finmarchicus and C. glacialis has been suggested at the Canadian east coast [Finally, the obtained SNP set that is shared by st coast . Howeverst coast ,77. The


Real-time functional magnetic resonance imaging (fMRI) is a promising non-invasive method for brain-computer interfaces (BCIs). BCIs translate brain activity into signals that allow communication with the outside world. Visual and motor imagery are often used as information-encoding strategies, but can be challenging if not grounded in recent experience in these modalities, e.g., in patients with locked-in-syndrome (LIS). In contrast, somatosensory imagery might constitute a more suitable information-encoding strategy as the somatosensory function is often very robust. Somatosensory imagery has been shown to activate the somatotopic cortex, but it has been unclear so far whether it can be reliably detected on a single-trial level and successfully classified according to specific somatosensory imagery content. Using ultra-high field 7-T fMRI, we show reliable and high-accuracy single-trial decoding of left-foot (LF) vs. right-hand (RH) somatosensory imagery. Correspondingly, higher decoding accuracies were associated with greater spatial separation of hand and foot decoding-weight patterns in the primary somatosensory cortex (S1). Exploiting these novel neuroscientific insights, we developed—and provide a proof of concept for—basic BCI communication by showing that binary (yes/no) answers encoded by somatosensory imagery can be decoded with high accuracy in simulated real-time (in 7 subjects) as well as in real-time (1 subject). This study demonstrates that body part-specific somatosensory imagery differentially activates somatosensory cortex in a topographically specific manner; evidence which was surprisingly still lacking in the literature. It also offers proof of concept for a novel somatosensory imagery-based fMRI-BCI control strategy, with particularly high potential for visually and motor-impaired patients. The strategy could also be transferred to lower MRI field strengths and to mobile functional near-infrared spectroscopy. Finally, given that communication BCIs provide the BCI user with a form of feedback based on their brain signals and can thus be considered as a specific form of neurofeedback, and that repeated use of a BCI has been shown to enhance underlying representations, we expect that the current BCI could also offer an interesting new approach for somatosensory rehabilitation training in the context of stroke and phantom limb pain. Brain-computer interfacing refers to the use of a signal originating from neural  activity to control an external device , as long as they are technically available and compatible with the user’s faculties. Presently, motor and visual imagery are among the most frequently used mental-imagery strategies in the context of fMRI-BCIs. Both have been shown to generate robust brain activation patterns that can be used as a control signal in the context of fMRI-BCIs for communication  investigate whether somatosensory imagery is a suitable strategy for operating a hemodynamic BCI, and provide a proof-of-concept that it is suitable for online BCI-based communication; and (2) explore to what extent its classification is driven by somatotopic information. For this proof-of-concept study, we exploited the high signal to noise ratio of ultra-high-field 7T fMRI.Somatosensory imagery recruits the core imagery-generating network  were recruited among students and staff members of the Faculty of Psychology and Neuroscience at Maastricht University . Participants were screened for MRI counter-indications and signed an informed consent form prior to their participation. Students received monetary compensation or credit points after the experiment. The study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the Faculty of Psychology and Neuroscience.Ten neurologically healthy participants he had found a subjectively effective imagery strategy.During the scanning session, the participants’ heads were stabilized with foam padding to minimize head movement. They were instructed not to move and to keep their eyes closed during the functional runs. In a pilot experiment, participants reported that somatosensory imagery was easier to perform with eyes closed. As the eyes were closed throughout the entire run this did not constitute a confounding factor. Participants received auditory instructions indicating when they had to start imagining touch to the right hand (“hand”) and the left foot (“foot”), and when they could stop (“rest”). Each participant completed five (participant 7) to six somatosensory-imagery classifier-training runs. Every classifier-training run contained a pseudorandom sequence of nine left-foot (LF) and nine right-hand (RH) somatosensory mental imagery trials of 18 s duration, each followed by a 16, 18 or 20 s rest period . Seven pAfter scanning, participants were asked to fill out a questionnaire in which they had to rate the experienced quality of their somatosensory imagery  and the extent to which it evoked a tactile sensation  on a ten-point scale . They also rated the percentage of time that they were successful at maintaining the imagery and described the particular imagery strategy they used.SD = 0.65) and of their foot imagery 6.6 (SD = 0.49). The average score in response to the question to what extent they experienced a tactile sensation during imagery was 5.3 (SD = 2.3) for the hand and 4.9 (SD = 1.9) for the foot. The reported average percentage of time that somatosensory imagery worked during the hand imagery epochs was 75%  and 73%  for the foot imagery epochs. Paired t-tests did not reveal any significant differences between foot- and hand-related ratings at p < 0.05.All subjects reported that they were able to evoke somatosensory imagery. The type of strategies used ranged from imagining a warm feeling (four subjects), imagining vibratory stimulation to the finger or toe (two subjects), to imagining touch  on a 7T scanner  with a 32 receiver channel (Nova) head coil.The scanning session included the acquisition of an anatomical scan (8 min) and five to six classifier training runs (12 min/run). In seven subjects, there was sufficient time to end the session with one to seven additional answer-encoding runs  in which the participant answered one or more binary questions using somatosensory imagery (4 min/run).For the anatomical scan a Magnetization Prepared 2 Rapid Acquisition Gradient Echoes sequence . In each session, five volumes were acquired in opposite phase encoding direction to be used for (offline) correction of EPI distortions.3. The first two volumes of each functional run were discarded. The remaining volumes were adjusted based on slice-scan time, realigned to the first functional image of the session using rigid body transformations, temporally filtered to remove linear trends and nonlinear periodic signals with a frequency below 0.01 Hz, corrected for EPI distortions, coregistered to the anatomical scan interpolating to 2 mm3 isotropic, and normalized to Talairach space. This procedure includes the definition of subject-specific anatomical landmarks  that are used to rotate each brain in the AC–PC plane followed by piecewise, linear transformations to fit each brain in the common Talairach “proportional grid” system . The anatomical scan was down-sampled to 1 mmThe multivariate analyses were limited to the voxels included in anatomical masks. Masks were obtained by creating anatomical regions of interest (ROIs) in Talairach space based on the Jülich probability maps for the primary  were extracted by linearly fitting each voxel within the mask with a design matrix consisting of a constant term (intercept) and a modeled hemodynamic response, within a temporal window of 30 s, spanning from one volume (2 s) before the auditory cue to 14 volumes (28 s) after the auditory cue. The modeled hemodynamic response was obtained by convolving a canonical hemodynamic response function (hrf) with a box-car predictor whose value was one in the volumes corresponding to the imagery blocks, and zero otherwise. The fit was done separately for each trial, and the t-estimates of the hemodynamic model were used as features per voxel. This procedure resulted in 45 (participant 7)/54 feature examples per class (nine per run).Features . Subsequently, proportions correct classifications were computed with edge correction [/number of trials] and logit transformed before statistically testing the hypotheses of interest using SPSS .Data were imported in Matlab  using the NeuroElf  for feature extraction and Spider toolboxes for multivariate classification. A linear support vector machine  and the number of training runs (5) as within-subject factors. To test whether accuracy increased with the number of training runs, within-subject planned polynomial contrasts were used.To test the suitability of the current approach for (online) communication, a simulated real-time classification was performed using Turbo-BrainVoyager  to emulate the procedure that would be performed during the scanning session. In the last participant, a proof-of-concept online communication experiment was performed in which the answer runs were also decoded online during the scanning session using an SVM based on data from all available training runs.3), which were 3D-motion corrected using the standard TBV incremental procedure spatially realigning each volume to the first recorded volume by rigid body transformation. The lowest probability (largest) S1 and S2 mask was back-transformed from Talairach space into native space. After spatially smoothing the functional data with a 4 mm full-width-at-half-maximum (FWHM) kernel, t-values were computed by fitting a general linear model with the same predictors as used in the offline procedure, within the same temporal window of 30 s, spanning from one volume (2 s) before the auditory cue to 14 volumes (28 s) after the auditory cue. T-values from the S1 and S2 mask were used as features for SVM training and testing using LIBSVM (2000–2009 Chih-Chung Chang and Chih-Jen Lin).The online and simulated real-time analyses were methodologically identical. Both analyses were based on slice data in native resolution . The resulting edge-corrected and logit transformed accuracies were entered into a repeated-measures ANOVA with the number of training runs (1–6) as within factor was used to statistically assess the effect of the amount of training data.The effect on decoding accuracy of the anatomical region chosen for feature extraction was evaluated by repeating the cross-validation procedure with five runs (four for subject 7) for training and one run for testing with features extracted from the anatomical masks of the primary  and secondary  somatosensory cortex at different probability levels  and probability level  as within-subject factors, to test whether there was a difference between S1 and S2 in terms of decoding accuracy, and whether this was possibly affected by the probability level, which was further assessed using within-subject planned polynomial contrasts. For the ANOVA, only probability levels of 80% or lower were taken into account which corresponded to masks with more than 500 voxels, to ensure stable classification results.The extent to which the classification was based on somatotopically different weight patterns in the S1 was assessed using the average discriminative weights across the different training splits. Discriminative weights were extracted from single-subject maps  that represented the average weights across the different training splits, using SVM classifiers trained on features from the most inclusive anatomical mask, with all possible combinations of 4/5 runs for training. Because the representations of hand and foot are most clearly separated on the x-axis (left-to-right), we computed the subject-specific median x-coordinates for the 200 largest positive (foot imagery) and 200 largest negative weights (hand imagery) and compared them using a Wilcoxon signed-rank test. Finally, the Spearman rank correlation was computed between the subject-specific differences in median x-coordinates for foot and hand and the leave-one-run-out classification accuracy.Ten healthy participants were instructed by auditory cues to perform alternating somatosensory imagery of the hand or foot  while ulAfter establishing that all participants subjectively experienced somatosensory imagery , we went on to test the suitability of this strategy to induce activation that can be reliably decoded for the purpose for BCI communication. We hypothesized that using a machine-learning algorithm, the contents of somatosensory hand or foot imagery can be accurately decoded on the single-trial level with similar accuracy for each imagery class, with accuracy increasing when increasing numbers of runs are used to train the classifier (hypothesis 1a). Our second hypothesis (1b) was that somatosensory imagery would yield accurate decoding of answer runs in simulated real-time as well as during a proof-of-concept online brain-based communication session.p < 0.05, based on permutation testing) was achieved in eight out of 10 subjects with one training run, and for all subjects for two or more trainings runs (SD = 0.12) when using five runs (four in one subject) for training and one run for testing. The effect of imagery class and the number of training runs were tested in an imagery class  by training runs (5) repeated measures ANOVA on the edge corrected and logit transformed accuracy data. This revealed a significant effect of the number of training runs on the decoding accuracy  = 8.823, p < 0.001, Greenhouse-Geisser (GG) corrected for non-sphericity with ε = 0.276). Planned within-subjects contrasts showed significant effects for the linear  = 40.869, p < 0.001) and quadratic contrasts  = 7.808, p < 0.023). There was no effect of the imagery class  = 1.728, p = 0.225), nor an interaction between imagery class and number of training runs  = 0.423, p = 0.558, GG corrected for non-sphericity with ε = 0.285).Testing the first hypothesis (1a), we found that significant above-chance classification accuracy  = 1.470, p = 0.229; SD = 0.17), mounting to 0.92 (SD = 0.08) when disregarding one subject who, after the session, indicated he misunderstood the instructions for the answer runs. In the proof-of-concept, online communication session with the last subject all answers to the questions were accurately decoded. Note that methodologically the simulated real-time and real-time analyses were identical, except for the fact that the simulated real-time analysis was performed after the scanning session.To test the second hypothesis (1b) and provide a proof-of-concept for brain-based communication, we used the answer runs that were available from seven subjects  that classification accuracy would be higher in the S1, where somatotopy is most pronounced, compared to secondary somatosensory cortex and (2b) that the discriminative weights obtained from the SVM would show a somatotopic distribution.F = 10.581, p = 0.012; F = 20.174, p < 0.001, GG corrected with ε = 0.340). Decoding accuracy increased with decreasing probability level  = 48.685, p < 0.001) and quadratic  = 10.024, p = 0.013) within-subjects contrasts were significant. The region by level interaction was not significant  = 0.593, p = 0.611, GG corrected with ε = 0.391). Significant above-chance classification accuracy (p < 0.05) was achieved in all ten subjects at the lowest probability level (largest mask) in S1, vs. seven out of ten subjects at the lowest probability level in S2.To test the first hypothesis (2a), we compared results obtained from anatomical probability masks of S1 vs. S2  between the median x-coordinates of the voxels associated with the 200 largest positive (foot) weights and the median x-coordinate of the voxels associated with the 200 largest negative (hand) weights. The median x-coordinate across subjects was 8.50 (SD = 14.5) for the 200 largest positive weights and −21.5 (SD = 18.4) for the 200 largest negative weights (p < 0.001).Next, we tested somatotopy of the discriminative SVM weights (hypothesis 2b). Visual inspection of discriminative SVM weight maps revealed a pattern that roughly corresponded to the expected somatotopy in 4 out of 10 subjects  investigated whether somatosensory imagery is a suitable encoding strategy for a hemodynamic (fMRI-based) BCI, aiming to provide a proof-of-concept of its suitability for online BCI-based communication; and (2) tested to what extent its decoding is based on somatotopic information. In accordance with our first set of hypotheses, we found that the contents of somatosensory imagery could be decoded above chance in each of our participants with similar accuracy for hand or foot, and that decoding accuracy increased with an increasing number of training runs. We also found that answers to binary questions encoded by seven healthy participants using the somatosensory imagery BCI could be successfully decoded in simulated real-time as well as during a proof-of-concept online communication session tested in one subject. In accordance with our second set of hypotheses, we found that decoding accuracy was significantly higher in the S1 compared to the secondary somatosensory cortex and that discriminative weight patterns generated by healthy participants through somatosensory mental imagery showed a somatotopic distribution.3 offline vs. smoothing with 4 mm FWHM Gaussian kernel in native space in simulated-real-time). On the other hand, the increased accuracy for the answer run classification might also be related to enhanced imagery quality during the answer run, due to extended practice, focus on a single imagery strategy  and possibly increased motivation because of the social interaction component of answering a genuine question.The average classification success was 82% for the offline leave-one-run out cross-validation procedure on the training runs, and 92% for the classification of the answer runs in simulated real-time (86% when including S03 who indicated afterward that he misunderstood the instructions). The fact that the classification success was higher for simulated real-time classification of the answer runs could be explained by the additional training run that was used for training the classifier in simulated real-time, whereas in the offline leave-one-run out procedure one run necessarily had to be left out for testing. However, the evaluation of the effect of additional training runs in the offline condition suggested that the beneficial effect of adding training runs leveled off after four runs. Another possible explanation might lie in the slight differences in the analysis pipelines for the simulated real-time and offline analyses regarding slice-scan-time-correction and data transformation , others used imagery of more naturalistic somatosensory stimulation involving a combination of somatosensory submodalities. Such imagery, e.g., combining warmth and touch might increase vividness and evoke stronger activation. Work by Kosslyn and Thompson in the visual domain suggests that the level of vividness and detail is related to primary visual cortex activation during imagery  population. This would require further research. However, based on the high accuracies during training and during answer runs, we deem the prospects for such a future study will be positive.differential activation profiles. Moreover, we found higher classification rates during answer runs than during training runs, possibly due to increased motivation. This effect might positively extrapolate to patients in a BCI communication setting.A third limitation of this study is that only relatively young healthy subjects without neurological impairments participated in the current study. The average patient will probably be older, and might have altered somatosensory representations due to motor impairment, which could lead to different imagery representations. Also, patients might have shorter attentional spans leading to more variable imagery quality. However, the current approach does not rely on a particular imagery representation, it only relies on the presence of Finally, it is important to acknowledge that fMRI-based brain-computer interfacing is unlikely to ever become a viable technique for bedside use. However, due to its high reliability and the fact that little pre-training is needed, it can be a very useful technique to communicate with patients about important questions, e.g., related to the continuation of treatment. This is especially relevant in hospital settings where fMRI is usually readily available. Also, it could be a crucial adjuvant method to identify and train patients on promising strategies for subsequent use with fNIRS, a less expensive and more mobile imaging technique that is also based on sampling of hemodynamic signals, albeit at a lower spatial resolution. By first using real-time fMRI-based brain-computer interfacing, clinicians can be faster at weeding out the unsuccessful strategies to subsequently implement those that were shown to be successful in fNIRS brain-computer interfacing. Finally, real-time fMRI communication/control BCIs could also be useful to assess the level of consciousness of patients , Maastricht University, Maastricht, the Netherlands. The patients/participants provided their written informed consent to participate in this study.AK, RG, and BS contributed to the conception and design of the study. AK performed the preprocessing. AK and GV performed the multivariate analyses. AK wrote the first draft of the manuscript. GV and BS wrote sections of the manuscript. All authors contributed to manuscript revision, and read and approved the submitted version.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.