Myint Phyo Kyaw

MD Robert N Luben

MRC Senior Scientist Paul G Surtees

MRC Scientist Nicholas W J Wainwright

Director Nicholas J Wareham

Professor Kay-Tee Khaw

Clinical Senior Lecturer Phyo K Myint
email: phyo.k.myint@uea.ac.uk

Paul G Bsc

Surtees

Mbbchir

Senior Research

Dr Phyo Kyaw Myint

coronary heart disease in the European Prospective Investigation into Cancer (EPIC)-Norfolk prospective population based study

Keywords: Coronary Heart Disease, SF-36, Functional

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Coronary heart disease (CHD) remains the number one cause of death in the Western societies and the global burden of cardiovascular disease is expected to rise. In the US alone, each year ~ 1.1 million persons have a myocardial infarction (MI) [START_REF]Heart and Stroke Facts: 2000 Statistical Supplement[END_REF]. Early identification and appropriate management of people who are at risk of developing CHD has huge potential in reducing global burden of cardiovascular diseases.

The Short-Form 36 item questionnaire (SF-36) is a widely used and well validated questionnaire [START_REF] Ware | SF-36 physical and mental health summary scales: a user's manual[END_REF] which assesses an individual's self-reported physical and mental functional health. The physical component summary (PCS) score or physical functional health of SF-36 measures an individual's capability to perform physical tasks such as carrying grocery, walking etc. Although it was originally designed to assess the efficacy of treatment/s provided from patients' perspective, recent evidence suggests that physical functional health may also serve as a predictor of objective health outcomes such as mortality [START_REF] Myint | Relation between self-reported physical functional health and chronic disease mortality in men and women in the European Prospective Investigation into Cancer (EPIC-Norfolk): a prospective population study[END_REF] and chronic disabling condition such as stroke [START_REF] Myint | Physical health-related quality of life predicts stroke in the EPIC-Norfolk[END_REF] independently of known risk factors.

In this study we examine the relationship between self-reported physical functional health measured by PCS scores of SF-36 and incidence of coronary heart disease in a large British prospective population based study.

Methods

Study sample

The participants were 14,222 men and women, aged 40-79 years at the baseline, drawn from the Norfolk, UK component of the European Prospective Investigation into Cancer (EPIC-Norfolk). The EPIC-Norfolk is a prospective population-based study, described in detail elsewhere [START_REF] Day | EPIC-Norfolk: Study design and characteristics of the cohort[END_REF]. Briefly, the participants were recruited from age-sex registers of general practices. As virtually 100% of people in the UK are registered with general practitioners through the National Health Service, the age-sex registers form a population-based sampling frame. From the inception of the EPIC-Norfolk cohort, data collection was broadened to enable the examination of a wider range of determinants of chronic diseases including coronary heart disease. The Norfolk cohort was comparable to national population samples with respect to characteristics including anthropometry, blood pressure, and lipids, but with a lower prevalence of current smokers [START_REF] Day | EPIC-Norfolk: Study design and characteristics of the cohort[END_REF]. The Norwich Local Research Ethics Committee approved the study.

There were a total of 30,445 who consented to participate (40% of the eligible general population). The majority (99.6%) are British White Caucasians. After excluding people who did not attend health check (4846), those who did not have SF-36 data [START_REF] Wareham | Validity and repeatability of the EPIC-Norfolk Physical Activity Questionnaire[END_REF]910)

Measurements

Study participants completed a baseline health and lifestyle questionnaire and attended a clinic at the study baseline during 1993-1997. Height, weight, and blood pressure (BP) were measured by trained staff using standardized protocols [START_REF] Lohman | Anthropometric standardization reference manual[END_REF]. Means of two measures of systolic BP was used. Non-fasting blood samples were taken.

Serum total cholesterol was measured on fresh samples with the RA 1000 (Bayer Diagnostics, Basingstoke, UK).

Participants were asked "Have you ever smoked as much as one cigarette a day for as long as a year?" and "Do you smoke cigarettes now?" in the baseline questionnaire and classified as current or ex-smokers, or those who had never smoked. A four-level physical activity index (level I = inactive, II = moderately inactive, III = moderately active, IV = active) was derived from the EPIC validated short physical activity questionnaire [START_REF] Khaw | Work and leisure time physical activity assessed using a simple, pragmatic, validated questionnaire and incident cardiovascular disease and all-cause mortality in men and women: The European Prospective Investigation into Cancer in Norfolk prospective population study[END_REF]. The detailed description of this physical activity questionnaire had been previously reported [8][9]. This index was validated against heart rate monitoring with individual calibration in two independent studies [START_REF] Wareham | Validity and repeatability of the EPIC-Norfolk Physical Activity Questionnaire[END_REF] [START_REF] Wareham | Validity and repeatability of a simple index derived from the short physical activity questionnaire used in the European Prospective Investigation into Cancer and Nutrition (EPIC) study[END_REF].

Alcohol consumption derived from the question "How many alcoholic drinks do you have each week?" with four separate categories of drinks. A unit of alcohol (approximately 8 g) was defined as a half pint of beer, cider, or lager; a glass of wine; a single unit of spirits (whisky, gin, brandy, or vodka); or a glass of sherry, port, At the baseline, they were also asked, "Has a doctor ever told you that you have any of the following?" followed by a list of conditions to obtain baseline prevalent illnesses which included stroke, heart attack, cancer and diabetes. Family history of myocardial infarction was ascertained from the question "Have any of your immediate family had any of the following conditions" which included "heart attack".

Social class was classified according to the Registrar General's occupation-based classification [START_REF] Elias | CASOC: Computer-Assisted Standard Occupational Coding[END_REF] (I= professionals, II =managerial and technical occupations, III =skilled workers (non-manual and manual), IV= partly skilled workers, V =unskilled manual workers). Social classes I, II, and III non-manual were re-categorised as "nonmanual" and III manual, IV and V as "manual" [START_REF] Shohaimi | Residential area deprivation predicts smoking habit independently of individual educational level and occupational social class. A cross sectional study in the Norfolk cohort of the European Investigation into Cancer (EPIC-Norfolk)[END_REF]. Aspirin use was ascertained by a question "Have you taken aspirin continuously for 3 months or more?".

Eighteen months later, the surviving participants were asked to complete by mail a psychosocial questionnaire [START_REF] Surtees | Obesity, confidant support and functional health: cross-sectional evidence from the EPIC-Norfolk cohort[END_REF] that included the anglicised version of SF-36 (UK SF-36). This assessment included eight dimensions (subscales) of health: physical functioning, social functioning, role limitation due to physical problems, role limitation due to emotional problems, mental health, energy/vitality, pain and general health perception.

Briefly, scores for each dimension are obtained by summing of scores from individual relevant responses concerned with that particular health dimension. For each health dimension (subscale) the raw scores were transformed into a scale from 0 to 100; To address the reverse causality issue we excluded all those who had CHD within the first two, four and six years of follow-up and constructed further models (models H, I and J) controlling for all of the above mentioned variables (as in model G).

All numerical data were entered as continuous variables and ordinal data as categorical variables. Initial analyses were performed using SF-36 PCS quartile categories. As there was continuous inverse dose-response relationship, the analyses were repeated using every 10 points (approximately 1 SD) increase in SF-36 PCS as the predictor variable. We also explored the association between SF-36 PCS and fatal and non-fatal CHD adjusting for confounders (model G). reported better physical functional health (those in higher quartile) were significantly younger, more likely to be men, had lower body mass index, cholesterol level and systolic blood pressure. The proportion of participants who smoke, those with history of diabetes and family history of MI, and prior aspirin usage were significantly lower in those participants who were in the top quartile compared to the bottom quartile.

People in the top quartile are more likely to be physically active and being less likely to be from lower occupational social class background. Weekly alcohol consumption differed statistically significantly between top and bottom quartiles. The average alcohol consumption, however, was well within the recommended weekly limit for UK population. The unadjusted rates for coronary heart disease were 4.5%, 3.0%, 2.1% and 1.2% during the follow up for quartile 1, 2, 3, and 4, respectively. 2 shows the relative risks and their corresponding 95% confidence interval for having a diagnosis of coronary heart disease by quartiles of physical component summary scores of SF-36, firstly for all and then for men and women separately. In all models the results were consistent. Participants with highest SF-36 PCS scores (quartile 4) compared to the lowest (quartile 1) had significant relative risk reduction (54%) for incident CHD during the follow-up (RR 0.46; 95% CI: 0.32-0.65) in the fully adjusted model controlling for age, sex, body mass index, cholesterol, systolic blood pressure, smoking, alcohol consumption, physical activity, diabetes, family history of MI, social class, and aspirin usage (model G & Figure 1). Exclusion of CHD diagnosed within first two years of follow-up (model H) attenuated the results only slightly (RR 0.48; 95%CI: 0.33-0.70). Sensitivity analysis after excluding people with known diabetes as the prevalence of diabetes was hugely different between physical functional quartiles (2.9% in Q1 and 0.8% in Q4), did not alter the results (RR 0.46; 95% CI: 0.32-0.66).

Table 3 shows the relative risks reduction and their corresponding 95% confidence interval for having coronary heart disease by every increase in 10 points of SF-36 PCS scores (1 SD equivalent) controlling for age, sex (in sex-combined analyses), body mass index, cholesterol, systolic blood pressure, smoking status, alcohol consumption, physical activity, history of diabetes and family history of myocardial infarction, social class and prior aspirin usage. Every increase in 10 points (1 SD equivalent) of SF-36 PCS was associated with significant relative risk reduction of 24% (RR 0.76; 95%CI: 0.69-0.84). The relationships between SF-36 PCS and CHD remained consistent when fatal and non-fatal CHD were examined separately. The RR In constructing SF-36, symptoms and problems that are specific to a particular condition were not included. The comparison with other longer measures such as Sickness Impact Profile and Health Insurance Experiment battery, however, showed that the SF-36 includes eight of the most frequently represented health concepts [START_REF] Ware | SF-36 Health Survey: Manual and Interpretation Guide[END_REF].

The plausible mechanisms by which baseline physical functional health predicts subsequent risk of coronary heart disease is unclear. Future studies should look for plausible explanations of the associations.

The relative risk reduction for every increase in 10 points of SF-36 PCS (1 SD increase) were 21% in men (RR 0.79, 95%CI: 0.70-0.89) and 27% in women (RR 0.73, 95%CI: 0.62-0.87) in the current study. There was some attenuation of relative risk in both men and women after excluding early coronary heart disease (18% and 25%, respectively). Interestingly, these findings are similar to our previous report for the relationship between physical functional health and stroke incidence [START_REF] Myint | Physical health-related quality of life predicts stroke in the EPIC-Norfolk[END_REF]. This consistent finding of relationship between physical functional health and subsequent It may be possible to attenuate the decline in physical functional health associated with chronological age. We have shown the relationships between modifiable lifestyle behaviours such as smoking, physical activity, and alcohol consumption and other potential determinants of SF-36 in the EPIC-Norfolk cohort [17][18][19] [START_REF] Myint | Modifiable lifestyle behaviors and functional health in the European Prospective Investigation into Cancer (EPIC)-Norfolk population study[END_REF]. In this study we also adjusted for these factors and the relationships observed appeared to be independent of these factors. It may be that SF-36, being a health profile measure reflects the overall health status of an individual, and captures subclinical stage of conditions before it can be detected by objective measures such as a diagnosis made based on a blood test result. Poor physical functional health may reflect underlying biological processes such as chronic inflammation which may relate to coronary heart disease. It is also possible that a poor score may reflect psychosocial factors such as stress which may also influence cardiovascular risk. The PCS score of SF-36 may therefore be a potential useful additional marker in assessing coronary heart disease risk. Our findings suggest that inclusion of physical functional health assessment in predicting cardiovascular risk may be a useful approach to identify better people at high risk of future coronary heart disease at the population level in an apparently coronary heart disease free population. However, further studies in different populations are required to replicate these findings.

Study limitations

Underestimation of incident coronary events results towards the null, i.e. underestimate the magnitude of the association. Using self-reported myocardial infarction to exclude people with prevalent disease may have missed some prevalent CHD cases. Reverse causality is a potential major issue. People who are already ill might be more likely to be physically inactive and change their lifestyle (e.g. smoking habit) as a result of prevalent disease. To address this we excluded those with prevalent myocardial infarction, stroke and cancer at the baseline and also adjusted for other potential indicators of ill health such as blood pressure, cholesterol concentration, body mass index, diabetes mellitus, and aspirin use, and also repeated the analyses excluding people with coronary heart disease diagnosed within the first two years of follow-up. People with angina have been shown to have impaired health related quality of life [START_REF] Marquis | Clinical validation of a quality of life questionnaire in angina pectoris patients[END_REF]. However, the exclusion of coronary heart disease incidents occurring within first two years of follow up did not alter the results significantly. The residual confounding with known or unknown factors is always possible.

We used only one measure at one point in time to characterise individuals and did not take into account possible changes in lifestyles or other treatment effects on the variables we examined (e.g. antihypertensive medication or cholesterol lowering therapy during the study period). Nevertheless, random measurement error would probably attenuate any associations observed, so the estimated differences in risk are likely to be larger than those observed.

Because participants had to be willing to provide detailed information and participate in a long-term follow-up, the EPIC-Norfolk had modest participation rate of ~40% of the eligible population at the baseline despite 100% follow-up rate. However, EPIC-Norfolk sample is comparable with the other representative National Surveys in UK with only slight lower prevalence of smokers [START_REF] Day | EPIC-Norfolk: Study design and characteristics of the cohort[END_REF] [START_REF] Surtees | Obesity, confidant support and functional health: cross-sectional evidence from the EPIC-Norfolk cohort[END_REF]. The potential healthy responder bias resulting in truncation of sample distribution would probably only attenuate the findings and very unlikely to change the direction of the study results. We were not able to examine the relationship between the SF-36 PCS and incident CHD in the whole cohort. This is due to the fact that SF-36 survey was not completed by all participants and we also excluded anyone with missing variables which are included in the current report. Exclusion of these individuals, however, is unlikely to influence the internal relationship between physical functional health and incidence of coronary heart disease in the study population.

Conclusion

F o r R e v i e w O n l y 24

In both models adjustments were made for age at the time of completion of SF-36, sex (in sex-combined models only), body mass index, cholesterol, systolic blood pressure, smoking status, alcohol consumption, physical activity, history of diabetes and family history of myocardial infarction, social class and prior aspirin usage. Model A-including all over the whole length of the study follow-up; model B -after exclusion of those who had CHD within first two years of follow-up; model C -after exclusion of those who had CHD within first four years of follow-up; model D -after exclusion of those who had CHD within first six years of follow-up.

  , and prevalent stroke (455), MI (977) and cancer (1642) at baseline, the current report is based on 14,222 participants aged 40-79 years at the baseline. Not all who attended health check provided SF-36 data and vice versa. Comparison between participants who completed the SF-36 and those who did not, showed no material difference, though statistically significantly different due to large numbers age, sex, body mass index, systolic blood pressure and cholesterol level (appendix).

  liqueurs. Total alcohol consumption was estimated as the total units of drinks consumed in a week.

  poor health and 100 represents good health for each subscale.Scoring on the 0 to 100 metric provides with a score that represents the percentage of the total possible score that the participant received. This score is computed by: 1) subtracting the lowest possible raw score from the participant's actual raw score; 2) dividing this number by the possible raw score range; and 3) multiplying this number by 100.Physical Component Summary (PCS) scores were derived according to algorithms specified by the original developers[START_REF] Ware | SF-36 physical and mental health summary scales: a user's manual[END_REF][15]. They were created by aggregating across the eight SF-36 subscales after transforming to z-scores and multiplying by their respective factor score coefficients and standardised as T-scores with mean 50 and SD 10 [14]. Three scales (Physical Functioning, Role-Physical, and Bodily Pain) correlate most highly with the physical component and contribute most to the scoring of the PCS measure. Outcome measures Complete follow-up of the study sample was achieved using death certification at the UK Office of National Statistics and hospital record linkage with vital status ascertained for the whole cohort. Incident cases of coronary heart disease were identified by the Tenth Revision of the International Classification of Diseases (ICD 10) code I10-I79. The follow-up period commenced from time of completion of the SF-36 (18 months after baseline) and until date of myocardial infarction or 31st March 2008 as the end of follow up date. Statistical analyses were performed using SPSS for Windows Version 14.0 (SPSS Inc., Chicago, Illinois). We used Cox proportional hazards model [16] to determine the overall and sex-specific independent association of physical functional health with incident coronary heart disease adjusting firstly for age (model A); secondly for age, and sex (model B); thirdly age, sex, systolic BP, body mass index (BMI), cholesterol level (model C); fourthly additionally adjusting for lifestyle behaviours including smoking status, alcohol consumption and physical activity (model D); fifthly additionally adjusting for history of diabetes and family history of MI (model E); then including occupational social class (model F) and finally additionally adjusting for prior aspirin use (model G).

  ResultsAfter exclusion of participants with prevalent myocardial infarction, stroke and cancer, a total of 14,222 participants aged 40-79 years at the time of study enrolment were included in the current report. There were a total of 389 incident coronary heart disease cases (fatal = 102, 26.2%) during the follow-up (total person years 126,896 years, mean follow-up 8.9 years).

  78 and 0.75 for fatal and non-fatal CHD with every increase in 1SD (10 points) of SF-36 PCS showing risk reduction of 22% and 25%, respectively (table3). Similar to the above findings in table 2, exclusion of CHD diagnosed within first two years of follow-up only attenuated the results slightly and relative risk reduction remained highly significant (RR 0.79; 95% CI: 0.71-0.87). Further models excluding initial four and six years of follow-up (models C & D) showed similar results.

  physical functional health assessed by SF-36 was inversely related to risk of incident coronary heart disease independently of known risk factors in a general population free of known heart attack, stroke and cancer at the baseline. The strengths of our study include a prospective design, a study population drawn from the community, case ascertainment using objective death certificates and hospital record linkage data and the ability to adjust for known biological, social and lifestyle risks factors for both physical functional health and coronary heart disease.

  conditions raises an interesting hypothesis that maintaining or prevention of decline in physical functional health in ageing populations may have added benefit in prevention of cardiovascular diseases.

  Physical functional health-related quality of life measured as PCS scores of SF-36 predicts subsequent risk of coronary heart disease independently of known risk factors in a general population without history of ischemic heart disease at the baseline. The nature of the association between physical functioning and subsequent risk of CHD needs further exploration. Further studies could perhaps investigate different ethnic populations to reflect the current situation of increasing number of diverse populations in the UK. In the interim, regardless of the underlying mechanism, a poor physical function score may identify men and women in the apparently healthy general population at increased risk of coronary event independent of classical risk factors. People with low level of SF-36 PCS may benefit most from targeted preventive interventions such as management of known risk factors and increase uptake of positive lifestyle behaviours.

  age and sex adjusted (for sex-combined analyses only) Model C: age, sex (for all), body mass index, cholesterol level, and systolic blood pressure adjusted model; Model D: as in model C with additional adjustment of life style behaviours of smoking, alcohol consumption and physical activity; Model E = as in model D with additional adjustment for history of diabetes and family history of MI; Model F: as in model E with additional adjustment for social class; Model G = as in model F plus aspirin use -adjusted; Model H: as in the final model (model G) after exclusion of coronary heart disease occurring within initial 2 years of follow-up; Model I: as in the model G after exclusion of coronary heart disease occurring within first 4 years of follow-up and Model J: as in the model G after exclusion of coronary heart disease occurring within first 6 years of follow-up. For sex-specific analyses, sex (dichotomised) was not included in the model. P values indicate the level of significance between all quartiles.

Figure 1 Q1=quartile 1 ,Figure 1

 111 Figure 1 Showing the Relative Risks (95% confidence interval) by quartiles of Short-Form 36 Physical Component Summary (PCS) score in fully adjusted model (model G)

Table 1

 1 shows the sample characteristics by quartiles of SF-36 physical component summary scores. Quartile 1 represents the bottom 25% and quartile 4 represents the top 25%. The corresponding SF-36 PCS scores for quartile 1, 2, 3 & 4 were 5.20-43.50, 43.51-51.00, 51.01-55.10, and 55.11-72.10, respectively. Participants who

Table 1

 1 sample characteristics of 14,222 men and women by quartile of Short Form-36 Physical Component Summary (PCS) Age at the time of completion of SF-36. P values indicate the level of significance between all quartiles.

	Q1	Q2	Q3	Q4	P
	5.20-43.50	43.51-51.00	51.01-55.10	55.11-72.10
	3555	3586	3558	3523

Acknowledgements

We would like to thank participants and general practitioners who took part in the study. We also thank the staff of EPIC-Norfolk and our funders.

Funding EPIC-Norfolk is supported by research programme grant funding from Cancer Research UK and the Medical Research Council. The EPIC-Norfolk HLEQ research programme is supported by a programme grant from the Medical Research Council UK (G0300128).

Competing interests

None

Ethics Approval

The Norwich Local Research Ethics Committee approved the study. The corresponding address for the Norwich LREC is Clinical Governance Department, Aldwych House, 57 Bethel Street, Norwich.

Contributors

KTK and NJW are principal investigators in EPIC-Norfolk population study. PGS is the principal investigator of EPIC-Norfolk HLEQ programme. RNL is responsible for data management, computing and data linkages. PKM and NWJW conducted the analyses. PKM prepared the draft manuscript and all co-authors contributed in writing of this paper. KTK is the guarantor.

O n l y 21


N R Hussein
email: nawfal.hussein@nottingham.ac.uk

The association of dupA and Helicobacter pylori-related gastroduodenal diseases

Helicobacter pylori is associated with the development of ulceration and gastric cancer. Recently, a novel virulence factor, duodenal ulcer promoting gene A (dupA), has been identified and found to associate with disease in some populations but not others. We investigated the relationship of dupA genotypes and H. pylori-related clinical outcomes by meta-analysis using previous reports of 2,358 patients from around the world. dupA-positive genotypes was found in 48% and was associated with duodenal ulcer (p=0.001, odds ratio [OR]=1.4, confidence interval [CI]=1.1-1.7). The prevalence of dupA-positivity and its association with disease differed among the various regions around the world. In South America, the highest prevalence was recorded (Colombia and Brazil) and a significant relationship was found between dupA-negative strains and both gastric ulcer (GU) and gastric cancer (GC) (for GU, p=0.001, OR=0.2, CI=0.1-0.4 and for GC, p=0.001, OR=0.3, CI=0.2-0.6). In China, a significant correlation between dupA-positive strains and GU (p= 0.001, OR=5.5, CI=2.4-12.4) and GC (p=0.009, OR=2, Cl=1.1-3.1) was found. To conclude, dupA promotes duodenal ulceration in some populations and GU and GC in others. This is typical of other virulence factors, such as cagA. Hence, it was concluded that the H. pylori virulence factor, dupA, is a true virulence factor.

Introduction

Helicobacter pylori infects approximately half of the world's population and is the major cause of gastric ulcer (GU), duodenal ulcer (DU) and gastric cancer (GC). H. pylori has a wide genetic diversity amongst isolates from different subjects. Among infected persons, who develops disease depends on the bacterial strain's virulence, the host's genetic susceptibility and environmental factors. H. pylori virulence factors associated with the development of ulceration and GC include VacA, CagA, BabA and OipA (see review by [START_REF] Atherton | The pathogenesis of Helicobacter pyloriinduced gastro-duodenal diseases[END_REF] [START_REF] Atherton | The pathogenesis of Helicobacter pyloriinduced gastro-duodenal diseases[END_REF]).

More recently, a novel virulence factor, dupA, has been identified that may also play a role in IL-8 secretion [START_REF] Lu | Duodenal ulcer promoting gene of Helicobacter pylori[END_REF]. The duodenal ulcer promoting gene A (dupA) gene has homology to virB4 and comprises two overlapping genes (jhp0917 and jhp0918) in genome sequence strain J99, but Lu et al. showed that dupA formed a single open reading frame (ORF) in H. pylori strain C142 and 23 other strains, due to a single nucleotide insertion (at position 1386). They showed that the presence of dupA was significantly associated with duodenal ulceration, but negatively associated with gastric cancer, in populations from South Korea, Japan and Colombia [START_REF] Lu | Duodenal ulcer promoting gene of Helicobacter pylori[END_REF].

Although it has been 5 years after the first dupA publication, it is still unclear whether the dupA gene is associated with an increased risk for gastrointestinal disease or not because of the small sample size used in some reports. Therefore, this study was designed to elucidate the relationship between the dupA gene and GC and peptic ulcer susceptibility in H. pylori-infected patients using systematic analyses that avoids type 2 error. [START_REF] Lu | Duodenal ulcer promoting gene of Helicobacter pylori[END_REF] Iran [11] South Africa [START_REF] Argent | The presence of dupA in Helicobacter pylori is not significantly associated with duodenal ulceration in Belgium, South Africa, China, or North America[END_REF] China

Materials and methods

Previous publications studying the dupA gene in H. pylori strains and its association with clinical outcomes were enrolled in this study. All eligible studies were identified by searching the PubMed database for manuscripts written in English and published up to January 2010 using the following search criterion: "dupA H. pylori". A metaanalysis was conducted to determine the association of dupA with the risk of developing gastrointestinal diseases.

Data analysis

Statistical differences in the frequencies of dupA among the individual countries were determined by one-way analysis of variance (ANOVA) or the Chi-square test using Minitab 15 software. The effects of dupA on the risk of gastric cancer and peptic ulcer development were expressed as odds ratios (ORs) with 95% confidence intervals (CIs) with reference to non-ulcer dyspepsia (NUD) subjects with H. pylori infection using Excel 2007. NUD was regarded as the control group in this study. All p-values were two-tailed and p-values less than 0.05 were considered to be statistically significant.

Results and discussion

Ten studies investigating the dupA gene and its relationship with clinical outcomes in different populations were found. Reports from Lu et [START_REF] Argent | The presence of dupA in Helicobacter pylori is not significantly associated with duodenal ulceration in Belgium, South Africa, China, or North America[END_REF]. There was no significant association between the presence of dupA and clinical outcomes in any individual population. However, when they combined the Belgian and South African populations, a higher proportion of dupA-positive genotypes was found in gastric cancer patients than that which was found among patients with gastritis alone [START_REF] Argent | The presence of dupA in Helicobacter pylori is not significantly associated with duodenal ulceration in Belgium, South Africa, China, or North America[END_REF] (Table 1).

In this study, it was found that, worldwide, dupApositive genotypes were found in 48% (1,137/2,358) of the samples and significantly increased the risk of DU (p= 0.001, OR=1.4, CI=1.1-1.7) (Table 2). The prevalence of dupA-positivity differed significantly among the various regions around the world, with highest prevalence found in South Africa (84%) and Brazil (89%) (p<0.001 when the South African and Brazilian populations were compared with other populations) (Table 1). In South Africa, no significant correlation between dupA and disease could be found. Probably, the dupA gene's ubiquity makes it not a good disease indicator. Two reports studied samples from Brazil, but neither of them showed any significant relationship with clinical outcome [8,9]. When we combined samples from both studies, dupA-positivity in NUD patients was much higher than that of GU, which reflects protectivity from GU (p = 0.001, OR = 0.16, CI=0.05-0.5). The sample size, probably, may have been too small to reveal a true effect in the individual studies. In South America (Colombia and Brazil), a significant relationship was found between dupA-negative strains and both GU and GC (for GU, p = 0.001, OR = 0.2, CI=0.1-0.4 and for GC, p=0.001, OR 0.3, CI=0.2-0.6) (Table 2).

The dupA-positivity rate in South Africa and South America was significantly higher than that of East Asia (40%, p=0.0001), such as Japan, China and Korea, where the cancer rate is high. Three reports studied samples from China [START_REF] Argent | The presence of dupA in Helicobacter pylori is not significantly associated with duodenal ulceration in Belgium, South Africa, China, or North America[END_REF]5,7] and two from Japan [START_REF] Lu | Duodenal ulcer promoting gene of Helicobacter pylori[END_REF]10]. When we combined the Chinese samples, we found a significant correlation between dupA-positive strains and GU (p =0.001, OR =5.5, CI =2.4-12.4) and GC (p =0.009, OR=2, Cl=1.1-3.1). In Japan, no significant relationship was found between dupA and clinical outcomes, even after the combination of both studies. This may be partly attributable to the overall lower prevalence of dupApositive strains (25%) in this population. The prevalence of dupA-positivity in the Middle East and India was 45 and 31%, respectively, and was significantly lower than that of South America and South Africa (p=0001). Among the USA and European samples, 45 and 49% of samples, respectively, carried dupA-positive strains, and signifi-cantly less than South Africa and South America (p=0.001). In Europe, the USA and the Middle East, the positivity rate of dupA was almost similar. This is in agreement with other studies that showed a similarity in H. pylori virulence factor between Western countries and the Middle East. No significant relationships between dupA and disease were found in these populations. There is a general trend of low dupA-positive rates in countries where the gastric cancer rate is high.

Although five reports studied the association of dupA with other virulence factors, there were no detailed data of each patient [START_REF] Lu | Duodenal ulcer promoting gene of Helicobacter pylori[END_REF]4,[6][7][8]. In Brazil, the presence of dupA was associated with cagA status in the group of adults with DU [8]. In China and Iraq, the presence of dupA was not associated with the other H. pylori virulence factors [4,7]. In Iran, it was found that 86% of strains with more than three phosphorylation motifs were dupA-positive, which is significantly more than the 36% of strains with only three phosphorylation motifs [4].

Conclusion

As summarised in Table 1, the sample number of some studies was small and insufficient to clarify the significance of the correlation between the duodenal ulcer promoting gene A (dupA) of Helicobacter pylori and gastroduodenal diseases. Therefore, by meta-analysis using many patients, the association between dupA and disease risk worldwide needed to be clarified. The prevalence of dupA-positivity differed significantly among the various regions around the world. We speculated that differences in dupA prevalence between populations could be associated with the selected nature of the dyspeptic populations studied or there may be genetic differences between versions of the dupA gene in different strains, similar to the difference found in vacA alleles and cagA phosphorylation motifs. To conclude, dupA promotes duodenal ulceration in some populations and gastric cancer in others. This is typical of other virulence factors, such as the cagA gene, which, like dupA, is thought to induce disease largely through inducing gastric inflammation.

Table 1

 1 The dupA status studies used in the meta-analysis

	%
	Positive
	No. of cases
	Disease
	%
	Positive
	No. of cases
	Disease
	%
	Positive
	No. of cases
	Disease
	%
	Positive
	No. of cases
	Disease	Japan

  al., Argent et al., Hussein et al. and Schmidt et al. included more than one population in their studies [2-5]. Of ten studies, six did not demonstrate the

Table 2

 2 Summary of dupA status in different regions around the world. The * indicates significant correlations

	Diseases	No. of samples	No. of dupA-positive samples	%	p-value (G versus other clinical outcomes)
	Worldwide
	G	938	421	44.88
	DU	682	366	53.67	0.001*
	GU	252	104	41.27	0.097
	GC	441	219	49.66	0.305
		2,313	1,110	47.99
	East Asia (Japan, China and Korea)
	G	355	130	36.62
	DU	285	123	43.16	0.093
	GU	191	79	41.36	0.277
	GC	216	83	38.43	0.665
		1,047	415	39.64
	South America (Colombia and Brazil)
	G	202	160	79.21
	DU	184	143	77.72	0.7
	GU	44	20	45.45	0.001*
	GC	131	77	58.78	0.001*
		561	400	71.30
	Middle East (Iraq and Iran)
	G	97	39	40.21
	DU	45	24	53.33	0.143
	GU	28	11	39.29	0.9
	GC	36	20	55.56	0.113
		206	94	45.63
	Europe (Sweden and Belgium)
	G	96	42	43.75
	DU	51	27	52.94	0.288
	GU	0	0
	GC	40	23	57.50	0.144
		187	92	49.20

  4. Hussein NR, Mohammadi M, Talebkhan Y et al (2008) Differences in virulence markers between Helicobacter pylori strains from Iraq and those from Iran: potential importance of regional differences in H. pylori-associated disease. J Clin Microbiol 46:1774-1779 5. Schmidt HM, Andres S, Kaakoush NO et al (2009) The prevalence of the duodenal ulcer promoting gene (dupA) in Helicobacter pylori isolates varies by ethnic group and is not universally associated with disease development: a case-control study. Gut Pathog 1:5 6. Arachchi HS, Kalra V, Lal B et al (2007) Prevalence of duodenal ulcer-promoting gene (dupA) of Helicobacter pylori in patients with duodenal ulcer in North Indian population. Helicobacter 12:591-597 7. Zhang Z, Zheng Q, Chen X et al (2008) The Helicobacter pylori duodenal ulcer promoting gene, dupA in China. BMC Gastroenterol 8:49 8. Gomes LI, Rocha GA, Rocha AM et al (2008) Lack of association between Helicobacter pylori infection with dupA-positive strains and gastroduodenal diseases in Brazilian patients. Int J Med Microbiol 298:223-230 9. Pacheco AR, Proença-Módena JL, Sales AI et al (2008) Involvement of the Helicobacter pylori plasticity region and cag pathogenicity island genes in the development of gastroduodenal diseases. Eur J Clin Microbiol Infect Dis 27:1053-1059 10. Nguyen LT, Uchida T, Tsukamoto Y et al (2009) Helicobacter pylori dupA gene is not associated with clinical outcomes in the Japanese population. Clin Microbiol Infect (in press) 11. Douraghi M, Mohammadi M, Oghalaie A et al (2008) dupA as a risk determinant in Helicobacter pylori infection. J Med Microbiol 57:554-562


Manuel A González

Pedro P Olea

Luis Robles

Vicente Ena

The Mediterranean Quercus pyrenaica oak forest: a new habitat for the Capercaillie?

Keywords: conservation, leks, Mediterranean, Quercus pyrenaica, Tetrao urogallus cantabricus, plasticity

We described an extension of the known distribution range of capercaillie into an atypical area and habitat for the species. Nine Cantabrian capercaillie Tetrao urogallus cantabricus leks and 14 cocks were registered in Mediterranean Quercus pyrenaica forests in an area of 1,500 km 2 , of which 4,500 forest hectares were surveyed. At present this population represents both the southern-most distribution for capercaillie and the only one inhabiting Mediterranean Q. pyrenaica forests, what suggests a wider adaptation of this (sub)species than previously thought. This capercaillie population and its habitat need to be better studied, as well as to be considered in conservation planning for Cantabrian capercaillie.

INTRODUCTION

Populations across distribution range show different adaptations to local conditions from the core areas to the periphery of their distribution (Guo et al. 2003). In the case of capercaillie, the vast majority of populations inhabit boreal and montane coniferous forests with abundant bilberry Vaccinium myrtillus understory [START_REF] Storch | Habitat selection by capercaillie in summer and autumn: Is bilberry important?[END_REF]2000a;[START_REF] Storch | Status Survey and Conservation Action Plan 2000-2004[END_REF][START_REF] Storch | Capercaillie[END_REF]). However, the Cantabrian subspecies Tetrao urogallus cantabricus (Cantabrian Mountains, NW Spain) resides mainly in pure deciduous forests of beech Fagus sylvatica, birch Betula pubescens and sessile oak Quercus petraea, as well as mixed forests of beech and oaks (Quercus robur, Q. petraea, and Q. pyrenaica) [START_REF] Bañuelos | Update of the situation of the Cantabrian capercaillie Tetrao urogallus cantabricus: an ongoing decline[END_REF].

In these forests holly Ilex aquifolium provides a key, evergreen winter food resource for Cantabrian capercaillie [START_REF] Storch | Capercaillie[END_REF][START_REF] Blanco-Fontao | Diet and habitat selection in Cantabrian capercaillie: ecological differentiation of a rearedge population[END_REF] whereas it relies on bilberry in summer-autumn [START_REF] Blanco-Fontao | Diet and habitat selection in Cantabrian capercaillie: ecological differentiation of a rearedge population[END_REF].

Previous studies showed, however, a negative selection to Quercus pyrenaica forests by Cantabrian capercaillie at the northern slope of the Cantabrian range (Quevedo et al. 2006b).

This endemic capercaillie subspecies is isolated and separated more than 300km from the nearest capercaillie population in the Pyrenees. After severe population decline in the last three decades (from 2,000 to 400 adults), only 108 leks remain occupied in the northern watershed and 85 leks in the southern, corresponding to an overall occupancy rate of 32% of all known leks [START_REF] Bañuelos | Update of the situation of the Cantabrian capercaillie Tetrao urogallus cantabricus: an ongoing decline[END_REF]. Consequently, Cantabrian capercaillie subspecies qualifies to be listed as "Endangered" according to the IUCN criteria [START_REF] Storch | Subspecies Cantabrian capercaillie Tetrao urogallus cantabricus endangered according to IUCN criteria[END_REF].

Capercaillie populations have so far been generally considered to be distributed in the Eurosiberian biogeographic region, outside Mediterranean region (but see [START_REF] Handrinos | The birds of Greece[END_REF]Akriotis 1997, Storch 2001; discussion section).

Unlike the Eurosiberian region, the Mediterranean region is characterized by a period of drought during hot summer which determines the existing vegetation (e.g., [START_REF] Penas | Atlas del Medio Natural de la provincia de León[END_REF][START_REF] Blondel | Biology and Wildlife of the Mediterranean Region[END_REF][START_REF] Rivas-Martínez | Mapa de series, geoseries y geopermaseries de vegetación de España[END_REF]. This Mediterranean environment is thought to be unsuitable for a species primarily adapted to boreal environments. However, there are historical records suggesting capercaillie presence during 17th-19th centuries in Mediterranean areas in Spain [START_REF] Madoz | Diccionario geográfico-estadístico-histórico de España y sus posesiones de ultramar[END_REF][START_REF] Castroviejo | El urogallo en España[END_REF][START_REF] Martínez | Contribución al conocimiento de la eco-etología del urogallo cantábrico[END_REF]), but the first sighting record in Q. pyrenaica forests in this Mediterranean area was unnoticed until May 1998 [START_REF] Pollo | Aumentan los urogallos "locos" en la cordillera Cantábrica[END_REF]) (No. 1 in Fig. 1).

In this study we present information on the number of cocks and characteristics of leks located in an atypical area and habitat for the capercaillie, i.e., Mediterranean Q. pyrenaica forests with virtual lack of bilberry and holly.

This study is the first showing capercaillie widely present in this forest type, suggesting a wider ecological plasticity of this (sub)species than previously thought. We also discuss implications that this new capercaillie site may have in the conservation of Cantabrian capercaillie.

MATERIAL & METHODS

Study area

The study area is located on the southern slope of the Cantabrian Mountains in León province (NW Spain). It is centred at 42° 39'N and located in the Mediterranean biogeographical region bordering the Eurosiberian region (Atlantic) (Rivas- [START_REF] Rivas-Martínez | Biogeographic Map of Europe[END_REF], European Environment Agency, 2008) (Fig. 1). The study area covers approximately 1,500km² below the putative line separating the two biogeographical regions in a landscape slightly mountainous (elevation ranges from 800 to 1,700 m.a.s.l.). It belongs to the biogeographical unit Carpetan-Leonese Subprovince from the Mediterranean region with supramediterranean bioclimate [START_REF] Penas | Atlas del Medio Natural de la provincia de León[END_REF][START_REF] Rivas-Martínez | Mapa de series, geoseries y geopermaseries de vegetación de España[END_REF], del Río et al 2007). A meteorological station from our study area (Villameca) clearly shows two dry months (P<2T; [START_REF] Blondel | Biology and Wildlife of the Mediterranean Region[END_REF] in summer: July (P=26; T=19.9) and August (P=19; T=19.1) (data based on 37 years). Dominant forests are supramediterranean natural (more than 50 years old) and post-fire Q. pyrenaica forests frequently occurring interspersed with Scots pine Pinus sylvestris plantations (less than 50 years old). Both forest types are largely monoespecific in the study area. Q. pyrenaica forests cover 27,000ha (19% of the study area). Scots pine plantations occupy 11,000ha (8%).

Q. pyrenaica (Willd.) is a marcescent oak tree distributed widely in Spain as well as the western part of France and the northwest of Marocco. This tree develops under siliceous soils in Eurosiberian region as well as in the Mediterranean region with an extensive representation in this biogeographic region. In Spain, Q. pyrenaica represents the dominant species of different vegetation series (del Río et al 2007).

Forest fragments of the study area are embedded in a matrix mainly composed of heather Erica australis 1,560ha (1%), brooms Genista florida and Cytisus scoparius (hereafter C. scoparius) 3,640ha (3%), meadows 3,460ha (3%) and riparian lowland forest of Populus nigra, Fraxinus excelsior and Alnus glutinosa 5,340ha (4%). Understory cover is mainly dominated by heath Erica arborea (hereafter E. arborea) and broom C. scoparius while bilberry and holly is completely absent or very scarce (<0.5% of the forest ground cover) in the study area. The remaining 62% surface corresponds with anthropogenic landscapes such as villages, roads, opencast mines, valley bottoms, which are not suitable for capercaillie habitat. Human population is approximately 1,000 people (0.6 people/km²) and it has declined by 80% since the 1950's. Livestock rearing is the main economic activity in the study area, followed by coal mining industry, agriculture, forestry and hunting.

Methods

No systematic sampling of the species was performed for the whole study area. We gathered information on capercaillie presence from 2002-2009 from two sources: i) questionnaires and reports sent by forest wardens, hunters and local people to the León environmental agency from the regional government (Consejería de Medio Ambiente of the Junta de Castilla y León), and ii) field surveys. Samples were recorded over the whole year. Data from the first source were validated within two weeks after receipt of the sample through field surveys carried out by an experienced observer (L. Robles). Field surveys consisted of a 3-4 hours systematic zig-zag walk looking for presence signs of capercaillie (e.g., direct sightings, footprints, droppings or feathers), especially focusing on sites known to be highly used by the species (e.g., forest paths, dead trunks, big stones, oldest forest sites, etc.).

In forest patches bigger than 1km² (i.e., more than 3-4 hours of sampling required) we conducted surveys over consecutive days until covering the whole forest patch (i.e., a continuous forest isolated by a non-forest matrix). Direct sightings, footprints, droppings or feathers recorded in a site were considered as signs of capercaillie presence. Each forest patch was completely surveyed 6-10 times throughout one year, 1-2 months each between. If no sign of capercaillie presence was found, the forest patch was labelled unoccupied and no further surveys were conducted in that specific patch.

Additionally, we surveyed the occupied patches in order to find leks.

Selected sites were locations where survey after survey we found capercaillie signs. The visits were performed throughout April and May (i.e., display season) after 12 a.m. to avoid disturbing birds. A lek is defined as a site where one or more cocks consistently display for hens, plus the adjacent surrounding forest habitat. It has traditionally been used as capercaillie occurrence measure in the Cantabrian range (see [START_REF] Obeso | El urogallo (Tetrao urogallus cantabricus) en la Cordillera Cantábrica[END_REF].

During April-May where fresh capercaillie signs were found, 2-3 observers (forest wardens and L. Robles) visited the site at night as many as four times until the display finished, or until well past dawn. A site was considered as an occupied lek when at least one cock was heard calling or seen displaying.

Every detected lek was surveyed each year to label it as occupied or unoccupied just looking for presence signs after 12 a.m. (see above). In April-May 2009 every occupied lek was surveyed at night to get the number of displaying cocks.

Nine forest structure variables (Table 1) were measured in each lek consisting of twelve circular plots with 5m radius. Three plots separated by 10 meters among them were arranged in each cardinal direction from the lek centroid (i.e., the point where previously here a cock was recorded displaying).

RESULTS

A total of nine leks, including that discovered in 1998 [START_REF] Pollo | Aumentan los urogallos "locos" en la cordillera Cantábrica[END_REF], were registered in the study area, seven of them remained occupied in 2009 (Fig. 2). The highest occupancy was in 2006 with eight occupied leks (Fig. 2). Since 2006 no new lek was found, despite an increase of around 2,000 additional hectares surveyed (60% of the total surveyed surface, Fig. 2). Leks were grouped into three subareas according to the watersheds: one subarea had three leks, other had four and the last subarea had two (Fig. 1). Mean nearest neighbour distance between leks was 2.07 ± 0.74 kilometers (mean ± SE).

Leks were in Q. pyrenaica forests ranging from 1,132 to 1,398 m.a.s.l.

(mean ± SE: 1275 ± 100.93, n=9) and mainly faced in a northerly direction with two exceptions facing south (Table 1). They showed heterogeneity in the forest structure, ranging from sites with young trees (perimeter 0.19 ± 0.005 meters) with high canopy cover (0.81 ± 0.06%) to mature trees (perimeter 0.90 ± 0.62 meters) with low canopy cover (0.28 ± 0.07%). The maximum tree height in leks varied from 7.75 ± 0.86 meters to 16.50 ± 3.20 meters (see Fig. 3).

Understory cover ranged from low (0.004 ± 0.86%) to high (0.62 ± 0.27%). In six of the nine leks the dominant understory species was E. arborea while C. scoparius dominated the remaining one (Table 1).

In 2009 twelve cocks were recorded at the seven occupied leks. Moreover, 1 and 2). These northern leks are mainly located in birch and sessile oak forests with understory dominated by bilberry (see Table 2) in a Eurosiberian bioclimatic environment (superior montane thermotype and inferior humid ombrotype) [START_REF] Penas | Atlas del Medio Natural de la provincia de León[END_REF]. Nonetheless lek 4, although in Q. pyrenaica forest, has an eurosiberian bioclimatic environment [START_REF] Penas | Atlas del Medio Natural de la provincia de León[END_REF], probably because of its most northerly relative position.

Despite an increase in the surveyed area, no new leks were found after 2006. However, the fact that we only surveyed 12% of the forest surface within our study area (Fig. 2), suggests new leks may be found in the future. Although the number of capercaillie in the study area is small, it represents approximately 7% of the global Cantabrian population, highlighting the conservation value of this site.

All the nine leks were located in Q. pyrenaica forests. Conversely no lek was located in pure Scots pine plantations, although these were available in the study area. In addition, two of four radio-tracked (one hen and one cock) capers used all year round Q. pyrenaica forests of the studied area (authors, unpublished data) and two capercaillie nests were discovered in these forests. All of this suggests that Q. pyrenaica forests are suitable year-round habitat for the Cantabrian capercaillie. This is particularly important because the potential habitat for this endangered subspecies could thus be greatly increased, as Q. pyrenaica forests are highly represented with > 100,000 hectares at the southern slope of the Cantabrian Mountains (Gil-Sanchez & Torre-Antón 2007). Additionally, despite the idea that Q. pyrenaica forests are not considered as suitable habitat (Quevedo et al. 2006b) and neither are included in the recovery plans for the Cantabrian capercaillie, our study highlights the need to take into account these forests for the conservation of this subspecies. Nonetheless, the role of the pine plantations for capercaillie in the study area should be further assessed.

Finally, this study supports what is suggested by Quevedo et al. (2006a;2006b) and [START_REF] Blanco-Fontao | Diet and habitat selection in Cantabrian capercaillie: ecological differentiation of a rearedge population[END_REF] that many general considerations for the species overall are not fully useful for the Cantabrian capercaillie, and suggests a wider adaptation and plasticity of this species than previously thought. For example, the near absence of bilberry and holly in the studied area suggest that capercaillie can maintain viable populations without this important summer and winter food resource for Cantabrian population and elsewhere [START_REF] Storch | Capercaillie[END_REF][START_REF] Blanco-Fontao | Diet and habitat selection in Cantabrian capercaillie: ecological differentiation of a rearedge population[END_REF]). Future research could address a detailed comparison of capercaillie suitable habitat between the two different biogeographical regions.

Studying the ecology and genetic variability of this local population living in this atypical habitat would be of a great value to further understand and conserve this subspecies in particular and Tetrao urogallus in general.

Zusammenfassung

  two additional cocks not referred to any lek were detected during the spring 2009 fieldwork. Two nests with five and seven eggs each (both in Q. pyrenaica forest) were found in early June 2009 (Fig. 1, No 5 and 8). DISCUSSION So far, capercaillie always occurred in the Eurosiberian biogeographical region with the exceptions of the probably introduced birds of Mount Athos (Greece; Handrinos and Akriotis 1997), currently extinct (S. Xirouchakis per. comm.), and two other unclear data in Mount Grammos and Lailias Forest Reserve in Greece (Storch 2001). Therefore, the capercaillie population we described here represents both the southern-most distribution for capercaillie and the only one inhabiting Mediterranean Q. pyrenaica forests. These forests are different from the nearest ones occupied by capercaillie leks located north of the putative line separating Eurosiberian and Mediterranean regions (Fig 1, Table

Der

  Wir beschreiben eine Ausdehnung des bekannten Verbreitungsgebietes des Auerhuhns in ein atypisches Gebiet und Habitat für diese Art. In einem 1,500 km 2 großen Pyrenäen-Eichenwald Gebiet, wovon 4,500 ha Wald untersucht wurden, konnten neun Balzarenen des Kantabrischen Auerhuhns Tetrao urogallus cantabricus und 14 Hähne festgestellt werden. Aktuell repräsentiert diese Population die sowohl südlichste Verbreitung von Auerhühnern als auch die einzige, die mediterrane Pyrenäen-Eichenwälder bewohnt. Dieses weist auf eine größere Anpassung dieser (Unter-)Art hin, als vorher vermutet. Diese Auerhuhn-Population und ihr Lebensraum müssen jedoch noch besser untersucht werden und sie sollte auch in Schutzstrategien für das Kantabrische Auerhuhn berücksichtigt werden. APPENDIX Appendix 1. Description of the leks (n=9) in the study area (see • in the map of the study area). Mean ± SE are shown for forest structure variables of the leks. Footnote: Lek = lek number by discovery date; Date = year when the lek was first-time recorded (April-May); Cocks = No. of capercaillie cocks recorded in each lek in 2009; m. a. s. l. = elevation (meters above the sea level); Exp. = exposure in the slope; Tree Perimeter = mean trees perimeter measured in DBH (diameter at breast height). Tree Height = mean trees height; Cover = mean canopy cover (%); Species = tree dominant species; Height = mean understory height (meters); Cover = mean understory cover (%); Species = understory dominant specie.

  FIGURE LEGENDS

  FIG.1

ACKNOWLEDGEMENTS

The forest wardens Fernando Gonzalo, Álvaro Ortiz and Ramón Balaguer showed us many little known aspects of the Q. pyrenaica forests. We wish to thank to María Angeles Osorio and César Pollo from the Consejería de Medio Ambiente de León of the Junta de Castilla y León. Particular thanks are due to Beatriz Blanco-Fontao, Patricia Mateo, Andy Dean and two anonymous reviewers whose revisions largely improved the manuscript. Manuel A. González is supported by a PhD scholarship of the Universidad de León.