Early changes in arterial structure and function following statin initiation : Quantification by magnetic resonance imaging Effective LDL-cholesterol -LRB- LDL-C -RRB- reduction improves vascular function and can bring about regression of atherosclerosis . Alterations in endothelial function can occur rapidly , but changes in atherosclerosis are generally considered to occur more slowly . Vascular magnetic resonance imaging -LRB- MRI -RRB- is a powerful technique for accurate non-invasive assessment of central and peripheral arteries at multiple anatomical sites . We report the changes in atherosclerosis burden and arterial function in response to open label statin treatment , in 24 statin-naïve newly diagnosed stable coronary artery disease patients . Patients underwent MRI before , and 3 and 12 months after commencing treatment . Mean LDL-C fell by 37 % to 70.8 mg/dL -LRB- P < 0.01 -RRB- . The plaque index -LRB- normalised vessel wall area -RRB- showed reductions in the aorta -LRB- 2.3 % , P < 0.05 -RRB- and carotid -LRB- 3.1 % , P < 0.05 -RRB- arteries at 3 months . Early reductions in atherosclerosis of aorta and carotid observed at 3 months were significantly correlated with later change at 12 months -LRB- R2 = 0.50 , P < 0.001 ; R2 = 0.22 , P < 0.05 , respectively -RRB- . Improvements in aortic distensibility and brachial endothelial function that were apparent after 3 months treatment were sustained at the 12-month time point . 1 Introduction HMG CoA reductase inhibitors or ` statins ' reduce cardiovascular events by approximately 25 -- 30 % in patients with stable atherosclerotic disease -LSB- 1,2 -RSB- . Previous studies of ultrasound carotid intima media thickness -LRB- CIMT -RRB- have suggested relatively slow regression of atherosclerosis after 1 -- 2 years of high dose statin treatment -LSB- 3,4 -RSB- . More recently , intravascular ultrasound -LRB- IVUS -RRB- of the coronary arteries has demonstrated coronary plaque regression following intensive LDL-C reduction in over 300 patients treated with 40 mg rosuvastatin for 2 years -LSB- 5 -RSB- . Corti et al. -LSB- 6 -RSB- were among the first to use serial magnetic resonance imaging -LRB- MRI -RRB- , to demonstrate reduction in carotid and aortic atherosclerosis in 18 patients in response to 12 months of statin treatment . Subsequently , the same group demonstrated that more effective lipid lowering , to LDL-C < 100 mg/dL , was associated with a larger decrease in plaque size at 12 months -LSB- 7 -RSB- . Similarly , Saam et al. -LSB- 8 -RSB- have recently identified statin treatment as an independent predictor of reduced annual rate of carotid atheroma progression , measured using MRI . In the setting of acute coronary syndromes , early initiation of ` intensive ' statin treatment can rapidly reduce cardiovascular risk within 4 -- 16 weeks -LSB- 9,10 -RSB- . However , given the apparently slow changes in atheroma burden observed in previous studies -LSB- 6 -RSB- , it has been hypothesised that the early clinical benefits of statins can not reflect structural changes within arteries , but are due to ` pleiotropic ' effects of statins such as improvement in endothelial function and reduction in inflammation and thrombosis -LSB- 11 -RSB- . Indeed , numerous studies have demonstrated rapid improvement in endothelial function in response to statin treatment -LSB- 12 -- 14 -RSB- . However , there is also evidence that rapid changes in plaque size and composition might also be possible . Animal models of atherosclerosis indicate that potent correction of dyslipidaemia can result in prompt regression and favourable remodelling of plaques after only 9 weeks -LSB- 15 -RSB- . Furthermore , in humans , a recent study of treatment with intravenous apoAI-Milano induced modest but significant regression of coronary atherosclerosis after only 6 weeks -LSB- 16 -RSB- . We have previously shown that MRI can assess both central and peripheral vascular function , including measurements of arterial stiffness and endothelial function -LSB- 17,18 -RSB- . These parameters are of additional interest because they have been shown prospectively to predict cardiovascular risk -LSB- 19,20 -RSB- . By assessing both atherosclerosis and vascular function in the same patients we hoped to examine the extent to which changes in structural parameters might be anticipated by early functional changes . In this study , we used magnetic resonance imaging to evaluate , in vivo , the changes in structure and function in human aorta , carotid and brachial arteries at 0 , 3 and 12 months in response to statin therapy . Notably , the mean post-treatment LDL-C achieved in this study reached the contemporary target of 70 mg/dL -LSB- 21 -RSB- . 2 Methods 2.1 Study population Newly diagnosed patients -LRB- n = 32 -RRB- with coronary artery disease were recruited from the Cardiology Department of a single tertiary centre . Diagnosis was based on a history of typical symptoms of angina together with an exercise ECG that showed ischaemic-type ST segment changes or a stenosis of ≥ 50 % in at least one coronary artery at angiography . No patients had taken statins prior to study enrolment . No other cholesterol modifying agents were permitted , but no further restrictions were placed on concomitant cardiovascular medications . Patients with acute coronary syndromes or contra-indication to MRI were excluded . MRI was performed at baseline , 3 and 12 months . At each time point , venous blood samples were obtained after a 12-h fast . Statin treatment was withheld until immediately after the first MRI scan , when a statin was started according to the preference of the managing clinician . Decisions regarding cholesterol treatment and management of cardiac risk factors were taken by the responsible clinicians . Statin dose titration , whilst allowed , was not protocol-driven . The study protocol was approved by the local Research Ethics Committee and all subjects gave informed written consent . Of the 32 subjects initially recruited , 8 did not complete all three study time points : 1 patient died from an out of hospital cardiac arrest , 5 were unwilling to return for follow-up scans due to claustrophobia and 2 were lost to follow-up . The 8 patients who did not complete the study were not significantly different from the 24 who did in terms of either their risk factors -LRB- age , cholesterol , diabetes , hypertension and smoking -RRB- or MRI measures of atheroma or vascular function at baseline . The data reported below refer to the 24 patients , who completed the study protocol . 2.2 Vascular MRI protocol Imaging was performed on a 1.5 T magnetic resonance scanner -LRB- Siemens Sonata , Erlangen , Germany -RRB- as previously described -LSB- 17,18 -RSB- . In brief , aortic imaging was performed using a combination of a two-element array surface coil placed on the chest and spine coil array . For carotid artery imaging , a two-element array surface coil was used -LRB- Machnet BV , Eelde , Netherlands -RRB- and for brachial artery imaging a surface coil was attached at the right elbow . Brachial artery blood pressure was monitored using a blood pressure cuff on the left arm . For quantification of aortic wall area , ECG-gated double-inversion recovery -LRB- black-blood -RRB- fast spin echo images were acquired during breath-hold -LRB- Figs. 1b and 2b -RRB- . Typical parameters were FOV 200 mm , TR 750 ms , TE 11 ms , in plane resolution 0.8 mm × 0.8 mm , slice thickness 5 mm . Using an oblique sagittal image of the aorta as a pilot , 11 serial axial images were acquired with 5 mm interslice gap to cover the entire descending thoracic aorta . The midpoint of the right pulmonary artery in cross section was used as the anatomical reference for the first slice in baseline and follow-up scans . For the carotid arteries axial ECG-gated T2 weighted , black blood images of the neck were acquired during free breathing -LRB- Fig. 2b -RRB- . Sequence parameters : FOV 150 mm , TR 2 R -- R intervals , TE 81 ms , resolution 0.5 mm × 0.5 mm in plane , slice thickness 3 mm , no interslice gap . Nine images of the common carotid artery were acquired using the common carotid bifurcation as the anatomical reference position for baseline and follow-up scans . Care was taken to place aortic and carotid image slices perpendicular to the long axis of the vessel on the pilot images in order to limit partial volume effects . For analysis of aorta and carotid plaque images all identifying data were removed apart from a code number so that observers were blinded to both patient identity and study time point . The external vessel boundary and vessel lumen were contoured manually by one of two experienced observers -LRB- J.L. , C.S. -RRB- using CMRtools software -LRB- Imperial College , London , UK -RRB- . Vessel wall area was calculated from the difference between these two contours , and then normalised to external vessel area to yield a plaque index -LRB- PI -RRB- , as previously described -LSB- 22,23 -RSB- . Plaque index for each patient was then expressed as the mean of all aortic or carotid slices . In keeping with previous studies of carotid atherosclerosis , a mean value was obtained for left and right carotid arteries combined -LSB- 3,4 -RSB- . A subset of images from four randomly selected patients -LRB- > 100 images -RRB- were analysed by both observers to assess inter-observer variability . Steady state free precession -LRB- SSFP -RRB- acquisitions were used to determine aortic distensibility and brachial artery reactivity , as previously described -LSB- 17,18 -RSB- . Post processing of aortic and brachial images was performed using semi-automated edge detection methods developed with Matlab software -LRB- Mathworks Inc. -RRB- -LSB- 24 -RSB- . Maximum and minimum aortic cross-sectional areas over the cardiac cycle were measured , from which distensibility was calculated as the relative change in area divided by the pulse pressure . To assess brachial artery flow mediated dilatation -LRB- FMD -RRB- , cross sectional images of the brachial artery were acquired at baseline and following release of a cuff inflated to 50 mmHg above systolic blood pressure on the forearm for 5 min . After 10 min , further brachial artery images were acquired following administration of 400 μg of sublingual glyceryl trinitrate to elicit maximal -LRB- endothelial-independent -RRB- dilatation . Maximum percentage change in cross sectional area was used to determine the response to each stimulus . 2.3 Serum and plasma assays Cholesterol and lipoprotein assays were performed using a Cobas-Mira Analyser -LRB- ABX Diagnostics , Shefford , UK -RRB- . Total cholesterol was assayed using the enzymatic CHOD-PAP method and triglycerides were assayed using the enzymatic GPO-PAP method . HDL-cholesterol was assayed using a homogenous second generation PEGME method -LRB- Roche Diagnostics , Burgess Hill , UK -RRB- . Apolipoprotein AI -LRB- apoAI -RRB- and apolipoprotein B -LRB- apoB -RRB- were assayed using immunoturbidimetric methods , using reagents supplied by ABX Diagnostics . C-reactive protein was analysed using ELISA -LRB- MP Biomedicals , UK -RRB- according to the manufacturer 's instructions . 2.4 Statistical analyses Statistical analysis was performed using SPSS 12.0 -LRB- SPSS Inc. , Chicago IL -RRB- . The Kolmogorov -- Smirnov test was used to assess whether data were normally distributed . Measurements at each time point were compared using repeated measures ANOVA for normally distributed data and Friedman analysis of variance by ranks for non-normally distributed data . Post-hoc analysis of paired time points was performed using a Bonferroni correction . Categorical data were analysed by the χ2 test . Data are presented as mean ± standard deviation or median and interquartile range as appropriate . Statistical significance was assigned at P < 0.05 . 3 Results 3.1 Clinical and biochemical measures Baseline characteristics of the 24 patients who completed all three study time points are shown in Table 1 . The most common statin dose used was simvastatin 40 mg daily -LRB- 63 % patients -RRB- ; other statin prescriptions are detailed in Table 1 . Mean baseline total cholesterol was 187.9 mg/dL -LRB- LDL-C 112.7 mg/dL -RRB- , however 3 months after commencing statins mean LDL-C was reduced by 37 % to 70.8 mg/dL -LRB- P < 0.01 -RRB- with a corresponding 24 % -LRB- P < 0.01 -RRB- reduction in apoB . Mean LDL-C at 12 months -LRB- 79.3 g/dL -RRB- appeared slightly greater than at 3 months , but this was not statistically significant . Triglycerides and CRP did not change significantly at 3 or 12 months , though HDL cholesterol did show significant increase by 12 months -- lipid and apolipoprotein data are shown in Table 2 . Compared to baseline , at 3 months there were no significant changes in the proportion of patients taking either angiotensin converting enzyme inhibitors -LRB- 12/24 versus 8/24 : χ2 = 0.77 ; P = 0.38 -RRB- or beta adrenoceptor blockers -LRB- 22/24 versus 18/24 : χ2 = 1.35 ; P = 0.25 -RRB- . By 12 months , the number of patients taking ACE inhibitors had increased significantly compared to baseline -LRB- 17/24 versus 8/24 : χ2 = 5.3 ; P < 0.05 -RRB- , though this would not have influenced the study findings at the early time point of 3 months . Beta blocker usage was not changed at 12 months -LRB- 19/24 versus 18/24 : χ2 = 0.11 ; P = 0.74 -RRB- . There was no significant change in either blood pressure or heart rate over the course of the study -LRB- Table 3 -RRB- . 3.2 Atherosclerosis regression All 24 patients had aortic plaque images of sufficient quality for analysis . Four patients had carotid plaque images at one or more time points of insufficient quality for analysis ; therefore they were excluded from statistical analysis . Images -LRB- total > 100 -RRB- from four randomly selected patients were analysed by both observers yielding inter-observer coefficients of variation for plaque index of 4.8 % in the aorta , and 2.9 % in the carotid . In both the aorta -LRB- Fig. 1a -RRB- and the carotid arteries -LRB- Fig. 2a -RRB- , there were statistically significant reductions in plaque index after 3 months of statin treatment . Mean aortic plaque index decreased from 0.303 ± 0.024 at baseline to 0.296 ± 0.022 at 3 months -LRB- P < 0.05 versus baseline -RRB- and 0.288 ± 0.024 at 12 months -LRB- P < 0.01 versus baseline -RRB- . Plaque regression in the aorta between 3 and 12 months was also significant -LRB- P < 0.05 -RRB- . In the carotid artery , plaque index fell from 0.446 ± 0.053 at baseline to 0.432 ± 0.046 at 3 months -LRB- P < 0.05 versus baseline -RRB- and 0.416 ± 0.032 at 12 months -LRB- P < 0.01 versus baseline -RRB- . Plaque index for the carotid artery between 3 and 12 months did not show significant reduction -LRB- P = 0.09 -RRB- . The number of patients with early regression in the aorta at 3 months compared to baseline was 18/24 , similar to that at 12 months where 20/24 showed regression -LRB- χ2 = 0.126 , P = 0.72 -RRB- -LRB- Fig. 1c -RRB- . The number of patients with measurable early regression at 3 months in the carotid was 14/20 , which was not significantly different to the 18/20 observed at 12 months -LRB- χ2 = 1.4 , P = 0.24 -RRB- ; see Fig. 2c . Furthermore within patients , the early -LRB- 3 months -RRB- change in plaque index of aorta and carotid arteries showed significant correlation with the final change observed at 12 months -LRB- Figs. 1c and 2c -RRB- . Mean aortic lumen area was 431 ± 77 mm2 at baseline , 434 ± 77 mm2 at 3 months -LRB- P = 1.0 versus baseline -RRB- and 442 ± 85 mm2 at 12 months -LRB- P = 0.09 versus baseline -RRB- . Mean lumen area in the carotid arteries was 44 ± 9 mm2 at baseline , 44 ± 8 mm2 at 3 months -LRB- P = 1.0 versus baseline -RRB- and 43 ± 8 mm2 at 12 months -LRB- P = 0.55 versus baseline -RRB- . 3.3 Physiological measures After 3 months of statin treatment , aortic distensibility increased by > 20 % at each of the three locations along its length . This effect was sustained but did not increase further at 12 months -LRB- Table 3 -RRB- . Flow-mediated dilatation of the brachial artery , a measure of endothelial function , also improved after 3 months by > 30 % -LRB- Table 3 -RRB- . Endothelial independent relaxation induced by GTN was not significantly changed after 3 months , but did show a significant increase by 12 months . 3.4 Relationship between variables Within individual patients there was no correlation between MRI quantification of atheroma burden in the aorta and carotid at baseline . Although at a group level , endothelial function and aortic compliance improved and atheroma burden diminished , there was no correlation of these changes within individual patients . Furthermore , there were no significant associations between measures of vascular function or atheroma burden and any of : attained LDL-C ; change in LDL-C , HDL-C , apoB , apoA-I , or CRP . 4 Discussion In this study , we have observed that regression of atherosclerosis in response to statin treatment can occur earlier than previously appreciated in both the aorta and carotid arteries . The robustness of this observation is enhanced by the finding that , within individual patients , regression at the early time point of 3 months was closely related to the magnitude and direction of change at 12 months . Patients also showed early and sustained improvement in aortic distensibility and in flow mediated vasodilatation of the brachial artery . The magnitude of atheroma regression observed in our study after 3 months is consistent with previous longer-term studies of regression in response to statin treatment -LSB- 6,7,25 -RSB- . A lesser LDL-C reduction than that achieved in our study was not associated with early plaque regression after 6 months statin treatment -LSB- 6 -RSB- . Intensive LDL-C reduction has been reported to achieve greater regression , but this study did not include an early time point -LSB- 7 -RSB- . Thus , the early regression observed here is a new finding that accords with the analysis of Nissen et al. -LSB- 5 -RSB- in which atheroma regression , assessed by intravascular ultrasound in the coronary arteries , was predicted by attained mean LDL-C < 70 mg/dL . A recent observational study reported overall carotid wall area progression by 2.2 % per year -LSB- 8 -RSB- , although individual cases of regression of up to 7.9 % were reported . Importantly , the population in that study differed by the inclusion of older patients with a higher prevalence of hypertension and selected only patients with carotid plaques of ≥ 50 % stenosis on a prior duplex scan . Mean lumen size of the aorta and carotid appeared to show slight increase by 12 months , this did not reach statistical significance implying that regression was occurring predominantly by reversal of ` positive ' vessel remodelling as suggested by Corti et al. -LSB- 6 -RSB- . There was significant change in plaque index between 3 and 12 months in the aorta , whilst the carotid artery showed a trend that did not reach significance -LRB- P = 0.09 -RRB- over the same time period . This could reflect the smaller size of the carotid artery compared to the carotid , making a small change harder to detect . Alternatively the plaques in the aorta and carotid may differ in composition , with certain elements of the plaque likely to be more susceptible to removal than others . For instance , lowering plasma LDL-C could slow the rate of lipoprotein deposition in the arterial wall , allowing reverse cholesterol transport mechanisms to predominate , culminating in net regression -LSB- 26 -RSB- . Different effects on individual plaque components is suggested by MRI studies of advanced carotid atherosclerosis in which patients treated with aggressive lipid lowering therapy showed a reduction in size of lipid rich areas -LSB- 27,28 -RSB- . This study was not designed to examine plaque composition , though initiation of study to address this question has recently been reported -LSB- 29 -RSB- . New developments such as lipid selective contrast agents or higher field strength -LRB- 3 T -RRB- imaging with improved resolution might further establish the degree to which plaque regression involves lipid removal -LSB- 30,31 -RSB- . Early clinical benefits of statins have also been ascribed to anti-inflammatory effects -LSB- 32 -RSB- . The degree of change in coronary atheroma volume measured using intravascular ultrasound has been related to the magnitude of change in both LDL and CRP -LSB- 33 -RSB- . However , in the present study , the early changes in plaque size occurred in the absence of significant change in CRP . Lack of measurable change in CRP probably reflects both the relatively small sample size , and the low baseline CRP level in this stable CAD population , comparable to the post treatment CRP levels in other studies -LSB- 34 -RSB- . We also found no relationship between the LDL-C attained and change in plaque size , which may again reflect the sample size and a clustering of LDL-C levels in the lower range precluding observation of a quantitative effect . However , the absence of changes in biochemical parameters despite plaque regression highlights the complementary role of MRI as an imaging biomarker . Rapid improvements in endothelial vasomotor function have previously been demonstrated in patients with atherosclerosis within weeks -LSB- 12,14 -RSB- or even days -LSB- 13 -RSB- of starting statin treatment . Thus our finding of increased flow mediated dilatation at 3 months was not surprising . By contrast , timing of statin effects on central arterial stiffness is less established . Improvements in large artery stiffness have been demonstrated after a year of statin treatment -LSB- 35,36 -RSB- , although a shorter study of 8 weeks pravastatin treatment in patients with familial hypercholesterolemia did not show any improvement -LSB- 37 -RSB- . Hypertensive patients without coronary artery disease treated with high dose atorvastatin -LRB- mean LDL of < 70 mg/dL -RRB- did show improvements in large artery stiffness after 3 months -LSB- 38 -RSB- . Our finding of rapid improvement in aortic stiffness was of similar magnitude and extends this potentially beneficial effect of statin treatment to a coronary artery disease population . We also observed that GTN mediated -LRB- endothelium independent -RRB- dilatation of the brachial artery was increased after 12 months statin treatment , as has been previously reported -LSB- 39,40 -RSB- and which might reflect downstream statin effects e.g. on smooth muscle cell sensitivity to nitric oxide . We initially hypothesised that changes in arterial function might predict changes in structural parameters , but in this study we found no correlation on an individual patient basis between changes is structure and function . Endothelial function , aortic stiffness and atheroma burden all represent different aspects and stages of disease . Although at a population level all these parameters may change in a favourable direction in response to treatment , individual patients may show variable response in each according to their stage of disease . As a result , it is likely that measures of vascular structure and function will provide complementary insights into vascular disease -LSB- 18 -RSB- . The capability of MRI to perform non-invasive assessment of changes in the arterial wall using relatively small numbers of patients is highlighted by this and other studies -LSB- 6,22 -RSB- . Accurate and reproducible assessment of the vessel wall is key to detection of atherosclerosis progression or regression -LSB- 41 -RSB- . The rationale for this approach has been illustrated by previous carotid intima-media thickness or coronary IVUS studies -LSB- 42 -- 44 -RSB- . An early appreciation of effects on both structure and function , as provided by MRI , could be used to guide selection of novel agents prior to investment in major Phase III trials -LSB- 45 -RSB- . MRI is an emerging technique and unlike ultrasound measures of IMT and FMD , has not yet been validated as a means to predict future events in large studies . However , recent data shows correlation of aortic plaque burden by MRI with clinical risk scores -LSB- 46 -RSB- . As our findings suggest MRI potentially offers additional value through the assessment of arterial structure in multiple locations and complementary measures of vascular function . Therefore , it seems increasingly likely MRI will play a key role in evaluation of new and existing therapies and may even become part of individual patient risk assessment . 4.1 Study limitations A potential limitation of this study is the absence of a control group . However , given strong evidence of the benefits of early and intensive treatment with statins , a placebo controlled arm or even low intensity statin-treatment arm was not considered ethical . In common with several recent studies -LSB- 5,6 -RSB- , we therefore conducted a longitudinal study of changes compared to baseline . As a result , it is not possible to exclude the possibility that the changes observed were due to an unknown factor , though in context , this is improbable . These patients were newly diagnosed with coronary artery disease , some of whom were taking vasoactive drugs such as ACE inhibitors and beta adrenoceptor blockers for hypertension prior to study enrolment . However , the increase in use of such medications between study baseline and the 3-month time point was small and not statistically significant and so the vascular changes observed over that period are not likely to be confounded by changes in these other medications . The 2 -- 3 % reductions in plaque index observed in this study at 3 months appear relatively modest when compared to decreases in lesion size of greater than 30 % observed over a similar time frame in some animal studies . However , the interventions in animal models of atherosclerosis usually involve much more extreme changes in lipid levels than those achievable by statin treatment in humans . Thus , whilst statin treatment to achieve effective LDL-C reduction appears important for plaque regression , additional HDL-C based interventions in order to enhance reverse cholesterol transport may prove even more effective -LSB- 26 -RSB- . The patient population studied was overwhelmingly male and Caucasian therefore the findings may not be applicable across all population groups . 5 Conclusions This study shows that in a population of statin naïve , clinically stable but otherwise unselected coronary artery disease patients , cholesterol reduction using statins to mean LDL-C of approximately 70 mg/dL was associated with rapid regression of atheroma at 3 months . Early changes were highly correlated with changes after 12 months . These rapid structural changes were accompanied by early improvements in arterial stiffness and endothelial function that were sustained to 12 months . Use of multi-modal vascular MRI to detect early changes in atheroma and vascular function in small numbers of patients could prove to be an efficient strategy to screen novel anti-atherosclerotic agents .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

statin
magnetic resonance imaging
aorta
carotid
atherosclerosis


New taxa of Neosartorya and Aspergillus in Aspergillus section Fumigati Three new species of Neosartorya and one new Aspergillus of section Fumigati are proposed using a polyphasic approach based on morphology , extrolite production and partial β-tubulin , calmodulin , and actin gene sequences . The phylogenetic analyses using the three genes clearly show that the taxa grouped separately from the known species and confirmed the phenotypic differences . Neosartorya denticulata is characterized by its unique denticulate ascospores with a prominent equatorial furrow ; N. assulata by well developed flaps on the convex surface of the ascospores which in addition have two distinct equatorial crests and N. galapagensis by a funiculose colony morphology , short and narrow conidiophores and ascospores with two wide equatorial crests with a microtuberculate convex surface . Aspergillus turcosus can be distinguished by velvety , gray turquoise colonies and short , loosely columnar conidial heads . The four new taxa also have unique extrolite profiles , which contain the mycotoxins gliotoxin and viriditoxin in N. denticulate ; apolar compounds provisionally named NEPS in N. assulata and gregatins in N. galapagensis . A. turcosus produced kotanins . N.denticulata sp. nov. , N. assulata sp. nov. , N. galapagensis sp. nov. , and A. turcosus sp. nov. are described and illustrated . Introduction Aspergillus section Fumigati and its teleomorph Neosartorya include many species which are important because they can be pathogenic or allergenic to man -LRB- Brakhage and Langfelder 2002 -RRB- , cause food spoilage and produce mycotoxins -LRB- Cole and Cox 1981 -RRB- . Certain species also produce interesting bioactive extrolites that are potential drug candidates -LRB- Turner and Aldridge 1983 -RRB- . Section Fumigati currently includes now 26 Neosartorya species and nine anamorph species -LRB- Pitt et al. 2000 ; Samson 2000 ; Horie et al. 2003 ; Hong et al. 2005 , 2006 -RRB- . During a survey of Aspergillus and Penicillium species from Korea , many isolates belonging to section Fumigati were isolated . These isolates were compared to known taxa and those present at the CBS and IBT culture collections which were atypical or unidentified , using a polyphasic approach -LRB- Frisvad and Samson 2004 -RRB- . We have examined the macro - and micromorphology , extrolite profiles and β-tubulin , calmodulin , and actin gene sequences of the isolates , and based on the above data , here we describe four new species in Aspergillus section Fumigati . Materials and methods Morphological examinations For macro-morphological observations , isolates were cultivated on Czapek yeast autolysate -LRB- CYA -RRB- , malt extract agar -LRB- MEA -RRB- , CZ agar -LRB- CZA -RRB- , and oatmeal agar -LRB- OA -RRB- -LRB- Samson et al. 2004 -RRB- . The isolates were inoculated at three points on each plate of each medium and incubated at 25 °C in the dark for 7 days , and additionally at 37 °C on CYA . For microscopic observations , mounts were made in lactic acid from MEA colonies ; a drop of alcohol was added to remove air bubbles and excess conidia . Scanning Electron Microscopy -LRB- SEM -RRB- was performed using a Hitachi S570 electron microscope . For SEM , mature cleistothecia were transferred to aluminum stubs with double sided adhesive tape . A small drop of 10 mM ACES buffer containing 0.05 % Tween-80 was added and the cleistothecia crushed . The suspension was air dried and coated with platinum . DNA analyses Isolates used for sequence analyses are listed in Table 1 . Genomic DNA was extracted according to the procedure described by Lee and Taylor -LRB- 1990 -RRB- . The 5 ′ portion of the β-tubulin gene -LRB- benA -RRB- was amplified using primers bt2a and bt2b -LRB- Glass and Donaldson 1995 -RRB- . Amplifications of the partial calmodulin and actin genes were set up as described previously -LRB- Hong et al. 2005 -RRB- . The amplified DNA fragments were purified by QIAquick PCR purification kits -LRB- Qiagene , Hilden , Germany -RRB- . DNA sequences were determined using BigDye Terminator v3 .1 Cycle Sequencing kit -LRB- ABI 0401041 , Foster , CA , USA -RRB- and ABI 3100 DNA sequencer . Both strands of each fragment were sequenced using the same primers . Table 1Aspergillus section Fumigati isolates used in this studySpeciesIsolate numberGenBank accession numberSourceβ-tubulinCalmodulinActinA . brevipesCBS 118.53 TAF057311Soil , AustraliaA . duricaulisCBS 481.65 TAF057313Soil , Buenos Aires , ArgentinaA . fumigatiaffinisIBT12703TDQ094885Soil , USAA . fumigatusCBS 133.61 AY685150Chicken lung , USAA . fumisynnematusIFM 42277TAB248076Soil , VenezuelaA . lentulusCBS 117887AY738513Man , USAA . novofumigatusIBT 16806TDQ094886Soil , EcuadorA . unilateralisCBS 126.56 TAF057316AY689366DQ094847Rhizosphere , AustraliaA . viridinutansCBS 127.56 TAF134779Rabbit dung , AustraliaA . turcosus sp. nov.KACC 42090 = IBT 27920DQ534142DQ534147DQ534178Air conditioner , Inchen , South KoreaKACC 42091T = IBT 27921DQ534143DQ534148DQ534179Air conditioner , Seoul , South KoreaKACC 41955 = CBS 117265 = IBT 3016DQ534144DQ534149DQ534180Car air conditioner , Seoul , South KoreaN . assulata sp. nov.KACC 41691TDQ114123DQ114131DQ534189Tomato soil , Buyeo , North KoreaN . aurataCBS 466.65 TAF057318AY870685DQ534112Jungle soil , BruneiN . aureolaCBS 105.55 TAF057319Soil , Tafo , GhanaN . coreanaKACC 41659TAY870758Tomato soil , Buyeo , North KoreaN . denticulata sp.nov.CBS 652.73 T = KACC 41183DQ114125DQ114133DQ534181Soil under Elaeis guineensis , SurinameCBS 290.74 = KACC 41175DQ114126DQ114134DQ534182Acer pseudoplatanus , NetherlandsN . fennelliaeCBS 598.74 TDQ114127DQ114135DQ534121Eye ball of Oryctolagus cuniculus , USAN . fischeriCBS 544.65 TAF057322Canned applesN . galapagensis sp. nov. . CBS 117522T = IBT 16756 = KACC 41935DQ534145DQ534151DQ534190Soil , EcuadorCBS 117521 = IBT 16763 = KACC 41936DQ534146DQ534152DQ534191Soil , EcuadorN . glabraCBS 111.55 TAY870734AY870693DQ534183Rubber scrab from old tire , Iowa , USAN . hiratsukaeCBS 294.93 TAF057324AY870699DQ534184Aloe juice , Tokyo , JapanN . laciniosaKACC 41657TAY870756Tomato soil , Buyeo , North KoreaN . multiplicataCBS 646.95 TDQ114129DQ114137DQ534185Soil , Mouli , TaiwanN . nishimuraeIFM 54133AB201360Forest soil , KenyaN . nishimuraeCBS 116047DQ534075DQ534150DQ534186Cardboard , NetherlandsN . pseudofischeriCBS 208.92 TAY870742AY870702DQ534187Human vertebrate , USAN . quadricinctaCBS 135.52 TAF057326Cardboard , York , UKN . spathulataCBS 408.89 TAF057320Soil under Alocasia macrorrhiza , TaiwanN . spinosaCBS 483.65 TAF057329Soil , NicaraguaN . strameniaCBS 498.65 TAY870766AY870726DQ534188Soil from maple-ash-elm forest , Wisconsin , USAN . tatenoiCBS 407.93 TDQ114130DQ114139Soil of sugarcane , Timbauba , BrazilN . udagawaeCBS 114217TAF132226Soil , BrazilCBS Centraalbureau voor Schimmelcultures , Utrecht , the Netherlands , IBT Institute for Biotechnology , Lyngby , Technical University of Denmark , IFM Institute for Food Microbiology -LRB- at present , the Research Center for Pathogenic Fungi and Microbial Toxicoses , Chiba University -RRB- , Chiba , Japan , KACC Korean Agricultural Culture Collection , Suwon , South Korea , T type strain DNA sequences were edited with the DNASTAR computer package . Sequence alignments were performed by using CLUSTAL W -LRB- Thompson et al. 1994 -RRB- and improved manually . The neighbor-joining -LRB- NJ -RRB- method was used for the phylogenetic analysis . Evolutionary distances between the sequences were calculated by Kimura 's formula -LRB- Kimura 1980 -RRB- using the program DNADIST of the PHYLIP program package -LRB- Felsenstein 1995 -RRB- . Phylogenetic trees were prepared by the NJ method -LRB- Saitou and Nei 1987 -RRB- using the program NEIGHBOR of the PHYLIP package . Bootstrap values were calculated from 1,000 replications of the bootstrap procedure using programs SEQBOOT , DNADIST , NEIGHBOR , and CONSENSE of the package -LRB- Felsenstein 1995 -RRB- . For parsimony analysis , the PAUP * Version 4.0 software was used -LRB- Swofford 2002 -RRB- . Alignment gaps were treated as a fifth character state and all characters were unordered and of equal weight . Maximum parsimony analysis was performed for all data sets using the heuristic search option with 100 random taxa additions and tree bisection and reconstruction -LRB- TBR -RRB- as the branch-swapping algorithm . Branches of zero length were collapsed and all multiple , equally parsimonious trees were saved . The robustness of the trees obtained was evaluated by 1,000 bootstrap replications -LRB- Hillis and Bull 1993 -RRB- . An Aspergillusclavatus isolate was used as outgroup in these experiments . The β-tubulin , calmodulin and actin gene sequences , determined in this study , have been deposited in GenBank and the accession numbers are listed in Table 1 . Analysis for extrolites The isolates were grown at 25 °C for 1 week on CYA and YES in the dark and extracted according to Smedsgaard -LRB- 1997 -RRB- . Extrolites were analyzed by HPLC using alkylphenone retention indices and diode array UV-VIS detection as described by Frisvad and Thrane -LRB- 1987 -RRB- , as modified by Smedsgaard -LRB- 1997 -RRB- . Results and discussion Molecular studies For the phylogenetic analysis of β-tubulin sequences , most accepted species in section Fumigati except Neosartorya indohii , N. sublevispora , and N. tsurutae were included to determine the phylogenetic positions of the putatively new species -LRB- Fig. 1 -RRB- . For the calmodulin and actin datasets only sequences of closely related species were included -LRB- Suppl . Figs 1 , 2 -RRB- . 468 nucleotides of the β-tubulin gene were analyzed . Among the 225 polymorphic sites , 126 were found to be phylogenetically informative . The topology of the NJ tree is the same as one of the 28 most parsimonious trees inferred by the PAUP program -LRB- length : 441 steps , consistency index : 0.7143 , and retention index : 0.6993 -RRB- . The calmodulin data set included 538 characters , with 87 parsimony informative characters -LRB- tree length : 272 , consistency index : 0.8051 , and retention index : 0.7706 -RRB- . The actin data set included 394 characters , with 66 parsimony informative characters -LRB- tree length : 200 , consistency index : 0.7900 , and retention index : 0.7801 -RRB- . Fig. 1Taxonomic position of some new species in Aspergillus section Fumigati inferred from Neighbor-Joining analysis of partial β-tubulin gene sequences . The numbers above/below the nodes represent bootstrap values of > 60 % -LRB- out of 1,000 bootstrap replications -RRB- . The number of nucleotide changes is represented by branch length The cladograms based on β-tubulin , calmodulin , and actin gene sequences revealed that isolates CBS 652.73 and CBS 290.74 , which had identical sequences at each loci , were related to the heterothallic species N. fennelliae , but the similarity between this species and the two isolates was quite low -LRB- 96.5 % in the β-tubulin gene partition and 97.8 -- 98.4 % in the calmodulin gene partition -RRB- . These two strains had unique ascospore ornamentations , with denticulate convex surfaces and a prominent equatorial furrow -LRB- Fig. 2 -RRB- and could be easily microscopically differentiated from any other Neosartorya species -LRB- Samson et al. 1990 ; Horie et al. 2003 -RRB- . Both isolates produced gliotoxin , while CBS 652.73 also produced viriditoxin . Gliotoxin is also produced by A. fumigatus and N. pseudofischeri , but there were several differences in the profile of extrolites in N. pseudofischeri and these isolates -LRB- data not shown -RRB- . A. fumigatus and N. pseudofischeri are among the most divergent species in the group -LRB- Geiser et al. 1998 ; Horie et al. 2003 ; Hong et al. 2005 , 2006 ; Varga et al. 2000 -RRB- , yet they share the production of this mycotoxin . Here we describe CBS 652.73 and CBS 290.74 as N. denticulata sp. nov. . Fig. 2Neosartoryadenticulata sp. nov. -LRB- A -RRB- colonies on OA after 28 days of incubation , -LRB- B -RRB- aspergillum , -LRB- C -RRB- ascoma , -LRB- D -RRB- ascospores under a light microscope , -LRB- E -RRB- and -LRB- F -RRB- ascospores by SEM Isolate KACC 41691 did not show a clear relationship to any species in the β-tubulin phylogeny , but was closest to CBS 116047 based on calmodulin and actin sequence data -LRB- Suppl . Figs 1 , 2 -RRB- . CBS 116047 is best accommodated as N. nishimurae . However , isolate KACC 41691 is homothallic , whereas N. nishimurae is heterothallic . This isolate has similar morphological characteristics to N. pseudofischeri , but our genotypic analyses indicate that they are phylogenetically distinct . KACC 41691 produces ascospores with several large flaps and two distinct equatorial crests -LRB- Fig. 3 -RRB- . These characteristics are similar to those of N. pseudofischeri in which the ascospores also have triangular flaps on a convex surface -LRB- Peterson 1992 -RRB- , but in KACC 41691 the flaps are more pronounced . Furthermore , KACC 41691 differs from N. pseudofischeri by its growth rates on MEA and CZA -LRB- after 7 days at 25 °C colonies were 49 -- 58 and 24 -- 42 mm , respectively , for KACC 41691 , and 90 , and 60 -- 70 mm for N. pseudofischeri -RRB- . The ascomatal initials in N. pseudofischeri are characterized by many coiled hyphae whereas the initial in KACC 41691 is simpler . Ascospores are larger -LRB- 5.1 -- 6.0 μm in KACC 41691 , while 4.5 -- 5 μm in N. pseudofischeri -RRB- . We could not detect any known extrolites in KACC 41691 but it produced partially characterized apolar compounds in common with several Neosartorya species . Here we propose the name N.assulata sp. nov. for isolate KACC 41691 . Fig. 3Neosartoryaassulata sp. nov. -LRB- A -RRB- colonies on CYA after 7 days of incubation , -LRB- B -RRB- aspergillum , -LRB- C -RRB- ascoma , -LRB- D -RRB- ascospores under a light microscope , -LRB- E -RRB- and -LRB- F -RRB- ascospores by SEM Isolates CBS 117522 and CBS 117521 , both isolated from soil from the Galapagos Islands , were phylogenetically distinct from all other species within section Fumigati -LRB- Fig. 1 , Suppl . Figs 1 , 2 -RRB- . The colony texture of these two isolates is funiculose with conidiophores which arise from bundles of aerial hyphae . These conidiophore structures resemble as described by Horie et al. -LRB- 1993 -RRB- for A. fumisynnematus , but in this species the conidophores are longer , up to 210 μm , with larger vesicles -LRB- 16 -- 20 -LRB- 25 -RRB- μm in diam . -RRB- . Ascomata were produced in 2 weeks-old colonies and ascospores were released after about 3 weeks . Ascospores resemble those of N. glabra and N. laciniosa , and have two conspicuous equatorial crests with a microtuberculate convex surface -LRB- Fig. 4 -RRB- . Isolates CBS 117522 and 117521 produced gregatins and several other extrolites not yet found in other Neosartorya or Aspergillus species , and appeared to be chemically unique . Gregatins have previously been found in A. panamensis in section Sparsi -LRB- Anke et al. 1980 , 1988 ; Peterson 2000 -RRB- . Fig. 4Neosartoryagalapagensis sp. nov. -LRB- A -RRB- colonies on CYA after 7 days of incubation , -LRB- B -RRB- ascoma , -LRB- C -RRB- and -LRB- E -RRB- stipes and conidial heads arisen from hyphal bundle , -LRB- D -RRB- conidia under a light microscope , -LRB- F -RRB- ascospores under a light microscope , -LRB- G -RRB- ascospores by SEM Here we describe isolates CBS 117522 and CBS 117521 as N.galapagensis sp. nov. . Isolates KACC 42090 , KACC 42091 , and KACC 41955 also showed a distinct taxonomic position within section Fumigati in the three gene phylogenies . The closest taxon to these three isolates in the β-tubulin and calmodulin gene phylogenies was the heterothallic species N.nishimurae -LRB- Fig. 1 , Suppl . Fig. 1 -RRB- . However , the similarity of β-tubulin sequences between the two species was only 96.9 % which is close to that observed between N. fischeri and N. spinosa -LRB- data not shown -RRB- . Although A. fumigatus , A. lentulus , A. viridinutans , A. fumigatiaffinis , and A. novofumigatus share similar morphological characteristics with these three isolates , these species showed comparatively low β-tubulin gene sequence similarities of 89.8 , 91.6 , 93.6 , 92.3 , and 92.7 % , respectively . Isolates KACC 42090 , 42091 , and 41955 did not produce any teleomorph structure after incubation for 28 days on CYA , MEA , CZA , and OA at 25 °C . During mating experiments , all of the pairings with N.fennelliae , N.nishimurae , N. spathulata , N. udagawae , and between conidial strains failed to yield cleistothecia . Some conidiophore characters suggest a similarity to A. fumigatus and A. lentulus , but these isolates are different from A. fumigatus by the vesicles which are fertile over the upper two-thirds and has short , loosely columnar conidial heads . These isolates are different from A. lentulus by their velvety and gray turquoise colonies . These isolates grow at 10 and 50 °C on MEA and CZA . On the contrary , A. fumigatus does not grow at 10 °C , while A. lentulus , A. fumigatiaffinis , and A. novofumigatus are unable to grow at 50 °C -LRB- Hong et al. 2005 -RRB- . Isolates KACC 42091 , 42090 , and 41955 were also chemically unique . The extrolites produced by the isolates described here are typical for Aspergillus section Fumigati -LRB- data not shown -RRB- . The three isolates also produced kotanins , previously found in species in less obviously related groups of Aspergillus , such as A. niger from section Nigri and A. clavatus from section Clavati -LRB- Turner and Aldridge 1983 -RRB- . Here we describe isolates KACC 42090 , KACC 42091 , and KACC 41955 as A. turcosus sp. nov. . The list of 26 known species of Neosartorya and nine anamorph species from the section Fumigati -LRB- Horie et al. 2003 ; Hong et al. 2005 , 2006 -RRB- is still expanding . With the species proposed here , there are now 29 Neosartorya species and 10 Aspergillus species in this group , 39 species in total . Unfortunately , some of the recently described species are not available for the scientific community , such as N. indohii , N. nishimurae , N. otanii , N. sublevispora , N. takakii , and N. tsurutae . Taxonomy Neosartoryadenticulata Samson , S.B. Hong and Frisvad . sp. nov. -LRB- Fig. 2 -RRB- . Species homothallica ; ascomata superficialia , luteo-alba vel dilute lutea , globosa vel subglobosa , 140 -- 230 μm in diam. , hyphis hyalinis vel luteolis laxe textis circumdata . Asci 8-spori , globosi vel subglobosi , 12 -- 14 μm diam . Ascosporae lenticulares , 4 -- 5 μm diam , denticulatae . Conidiophora ex hyphis aeriis oriunda , 3 -- 4.5 μm lata ; conidiorum capitula columnaria sed brevia , uniseriata , vesiculae spathulatae vel subclavatae , 7 -- 12 μm diam ; phialides 7.5 -- 9 × 2 -- 3 μm , didimidium superius vesiculae occupantes . Conidia subglobosa vel late ellipsoidea , levia , 2 -- 3 μm diam . Holotype of N.denticulata , here designated as CBS 652.73 T -LRB- dried culture -RRB- , isolated from soil in Suriname . Homothallic , cleistothecia superficial , yellowish white to pale yellow , globose to subglobose , 140 -- 230 μm in diam. , surrounded by a loose covering of hyaline to yellowish white hyphae . Asci 8-spored , globose to subglobose 12 -- 14 μm , evanescent at maturity . Ascospores , 4 -- 5 μm , denticulate with a prominent equatorial furrow . Mycelium composed of hyaline , branched , septate , smooth-walled hyphae . Conidial heads short , columnar . Conidiophores arising from aerial hyphae , uniseriate , stipes 3 -- 4.5 μm wide ; vesicles spathulate to subclavate , 7 -- 12 μm in diam. ; phialides 7.5 -- 9 × 2 -- 3 μm , covering the upper half of vesicle . Conidia subglobose to broadly elliptical , smooth , 2 -- 3 μm . Colonies on MEA growing rapidly , 35 -- 40 mm in 7 days at 25 °C , white . Conidial heads produced only in colony margins . Colonies on CYA , 22 -- 24 mm in 7 days at 25 °C , 35 -- 38 mm in 7 days at 37 °C , white , loosely overgrown by aerial hyphae in center , weakly sulcate in marginal area . Conidial heads few in number . Reverse yellowish white to pale yellow -LRB- 12A23 -RRB- -LRB- Kornerup and Wanscher 1978 -RRB- . Extrolites : The two isolates produced the mycotoxin gliotoxin . CBS 652.73 was a particularly strong producer , and also produced the mycotoxin viriditoxin . Furthermore , the two isolates produced some unique , yet unelucidated secondary metabolites . Additional isolates : CBS 290.74 = KACC41175 , from Acer pseudoplatanus , The Netherlands . Distinguishing features : Denticulate ascospores with a prominent equatorial furrow and the production of gliotoxin . Neosartorya assulata S.B. Hong , Frisvad and Samson . sp. nov. -LRB- Fig. 3 -RRB- . Species homothallica ; ascomata superficialia , alba vel luteo-alba , globosa vel subglobosa , 150 -- 250 μm diam , hyphis hyalinis vel luteolis laxe textis circumdata . Asci 8-spori , globosi vel subglobosi , 14 -- 16 μm diam . Ascosporae lenticulares , 5 -- 6 μm diam , duabus cristis distantibus praeditae , valvis nonnullis distinctis longis intumescentii ornamentatae . Conidiophora hyalina , 3 -- 7.5 μm lata ; conidiorum capitula columnaira , brevia , uniseriata ; vesicula subclavata , 10 -- 18 μm diam . Phialides 7 -- 9 × 2 -- 3 μm , didimidium superius vesiculae occupantes . Conidia subglobosa vel late ellipsoidea , levia , 2 -- 3 μm diam . Holotype of N. assulata , here designated as KACC 41691T -LRB- dried culture -RRB- , isolated from soil , tomato field , Buyeo , North Korea . Homothallic , cleistothecia superficial , white to yellowish white , globose to subglobose , 150 -- 250 μm in diam . Asci 8-spored , globose to subglobose 14 -- 16 μm , evanescent at maturity . Ascospores lenticular , spore body 5.0 -- 6.0 μm , with two well-separated equatorial crests and convex surface decorated with several large , round flaps . Mycelium composed of hyaline , branched , septate , smooth-walled hyphae . Conidial heads short , columnar . Conidiophores arising from aerial hyphae and substrate , 3 -- 7.5 μm wide ; vesicles subclavate , 10 -- 18 μm in diam. , uniseriate , phialides 7 -- 9 μm , covering the upper half of vesicles . Conidia , subglobose to broadly elliptical , ovoid , smooth , 2 -- 3 μm . Colonies on MEA , 49 -- 58 mm in 7 days at 25 °C , white , radially weak sulcate . Conidial heads aerial , numerous . Colonies white on CYA , 37 -- 41 mm at 25 °C , 64 -- 68 mm at 37 °C in 7 days . Radially and roundly sulcate , with some clear exudates . Conidial heads aerial , abundant . Reverse yellowish white -LRB- 1A2 -RRB- to pale yellow -LRB- 1A3 -RRB- -LRB- Kornerup and Wanscher 1978 -RRB- . Extrolite profile : This species is characterized by relatively weak production of secondary metabolites . It does produce some indole alkaloids and some apolar metabolites . Distinguishing features : Large , round flaps on convex surface of ascospores with two distinct equatorial crests . Neosartorya galapagensis Frisvad , S.B Hong and Samson . sp. nov. -LRB- Fig. 4 -RRB- . Species homothallica ; ascomata luteo-alba , globosa vel subglobosa , 90 -- 200 μm diam , . hyphis hyalinis vel luteolis laxe textis circumdata . Asci 8-spori , globosi vel subglobosi , 12 -- 15 μm diam ; ascosporae late lenticulates , ca. 5 μm diam , duabus cristis distantibus 1 -- 2 μm latis praeditae , valvis exigue tuberculatis . Conidiophora singula vel funiculosa , levia , 2 -- 4 μm lata ; conidiorum capitula columnaria , brevia , uniseriata ; vesiculae subclavatae , 4 -- 11 μm diam . Phialides lageniformes , 5 -- 7 × 2 -- 3 μm , dimidium superius vesiculae occupantes . Conidia globosa vel subglobosa , levia vel exigue asperulata , 2.3 -- 3.0 μm diam . Holotype of N.galapagensis , here designated as CBS 117522T -LRB- IBT 16756 = KACC 41935 -RRB- -LRB- dried culture -RRB- , isolated from soil , Galapagos Islands , Ecuador , D. Mahooney . Colonies on MEA 28 -- 35 mm in diam . after 7 days at 25 °C and more than 70 mm after 7 days at 37 °C , funiculose in texture , yellowish white -LRB- 3A12 -RRB- . Conidiophores sparse , cleistothecial initials produced after ca. 10 days of incubation . Colonies on CYA 27 -- 40 mm in diam . after 7 days at 25 °C and 61 -- 65 mm after 7 days at 37 °C , strongly funiculose in texture and white with a golden yellow -LRB- 5B78 -RRB- reverse without diffusible pigment . Conidial heads columnar . Conidiophores arising from bundles of aerial hyphae or the basal mycelium , smooth walled , up to 100 μm long , 2 -- 4 μm in width ; vesicles 4 -- 11 μm -LRB- sub -RRB- clavate with 5 -- 7 μm flask-shaped phialides which are fertile on the upper half to two-thirds of the surface . Conidia 2.3 -- 3.0 μm , globose to subglobose and the surface usually smooth . Cleistothecia yellowish white -LRB- 4A2 -RRB- , globose to subglobose , 90 -- 200 μm in diam. , surrounded by a loose covering of aerial hyphae . Peridium consisting of angular cells , 3 -- 8 μm in diam. ; asci 8-spored , globose to subglobose , 12 -- 15 μm in diam. ; ascospores broadly lenticular , spore body ca. 5 μm in diam . with two distinct equatorial crests 1 -- 2 μm wide , convex surface of ascospores microtuberculate . Extrolite profile : All isolates examined in the species produce several gregatins and several partially characterized secondary metabolites . This species is chemically very distinct and different from the other species in section Fumigati or Neosartorya species . The gregatins have also been found in A. panamensis -LRB- Anke et al. 1980 , 1988 -RRB- . The latter species was placed in Aspergillus section Usti by Raper and Fennell -LRB- 1965 -RRB- . Additional isolates : CBS 117521 = IBT 16763 = KACC 41936 , ex soil , Galapagos Islands , Ecuador . Distinguishing features : The Aspergillus anamorph arising in bundles of aerial hyphae and the ascospores with two wide conspicuous equatorial crests and microtuberculate convex surface . Aspergillus turcosus S.B. Hong , Frisvad and Samson . sp. nov. -LRB- Fig. 5 -RRB- . Fig. 5Aspergillus turcosus sp. nov. -LRB- A -RRB- and -LRB- B -RRB- colonies on MEA -LRB- A -RRB- and CYA -LRB- B -RRB- after 7 days at 25 °C , -LRB- C -RRB- conidia under a light microscope , -LRB- D -RRB- conidial heads Coloniae in agaro maltoso ad 42 -- 51 mm diam post-seven dies 25 °C , 70 mm diam 37 °C . Coloniae velutinae , griseo-glaucae vel griseo-virides , plerumque planae ; reversum luteo-aurantium vel griseo-aurantium . Conidiophora levia , 4 -- 7 μm lata . Conidiorum capitula columnaria , brevia , uniseriata ; vesiculae globosae vel subclavatae , 15 -- 25 μm diam . Phialides lageniformes , 6 -- 8 × 2 -- 3 μm , duo tertia superiora vesiculae occupantes . Conidia subglobosa vel ovoidea , levia , 2.5 -- 3.5 μm diam . Holotype of A. turcosus , here designated as KACC 42091T -LRB- = IBT 27921 -RRB- -LRB- dried culture -RRB- isolated from home air conditioner , Seoul , South Korea . Colonies on MEA 42 -- 51 mm in diam . after 7 days at 25 °C and more than 70 mm after 7 days at 37 °C . Colony texture velvety , gray-turquoise to gray-green -LRB- 24-25B3-5 -RRB- and usually plane . In reverse , colonies are yellowish orange -LRB- 4B6 -RRB- to grayish orange -LRB- 5B6 -RRB- . Colonies on CYA attain a diam . of 38 -- 41 mm after 7 days at 25 °C and more than 70 mm after 7 days at 37 °C . Colony texture is velvety . Colony texture and color similar to that on MEA . In reverse , colonies are deep yellow -LRB- 4A78 -RRB- . Conidial heads loose and short columnar . Conidiophores smooth-walled , up to 80 μm long and 4 -- 7 μm wide . Vesicles are 15 -- 25 μm in diam. , flask-shaped to globose , bearing 6 -- 8 μm flask-shaped phialides over two-thirds of the surface . Conidia are subglobose , ovoid and smooth , 2.5 -- 3.5 μm in diam . Extrolite profile : Kotanins and several unique compounds but not yet elucidated secondary metabolites . Additional isolates : KACC 42090 = IBT 27920 , KACC 41955 = IBT 3016 . Distinguishing features : Velvety colony , gray-turquoise -LRB- green -RRB- color on CYA , phialides over two-third of the vesicle and growth at 10 and 50 °C are distinctive characteristics of the species . Electronic supplementary material Below is the link to the electronic supplementary material . -LRB- DOC 19 kb -RRB-

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

actin
aspergillus turcosus
calmodulin
extrolites
neosartorya denticulata

dna sequencing
n. assulata
n. galapagensis
β-tubulin