PhD, MFPH Cother Hajat

MD, MPH Peter U Heuschmann

MSc Catherine Coshall

MD Soundrie Padayachee

MD John Chambers

FRCP Anthony G Rudd

Professor Charles Wolfe
email: charles.wolfe@kcl.ac.uk

; Charles

MD, FFPH D A Wolfe

Incidence of aetiological subtypes in a multi-ethnic population based study:the South London Stroke Register

Keywords: Stroke, incidence, aetiology, ethnicity

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

Introduction

The risk of stroke varies significantly internationally and although estimating overall stroke risk is relevant to planning stroke care, more detailed assessment of the underlying risk factor profile in stroke populations is relevant to understanding the aetiology and how preventive strategies may need to be tailored in different sociodemographic groups. [START_REF] Feigin | Stroke epidemiology: a review of population-based studies of incidence, prevalence, and case-fatality in the late 20th century[END_REF] Population-based studies following key methodological criteria are able to deliver this information but registers that have investigated aetiological stroke subtypes using a mechanism based classification system, 2 such as the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) classification system, [START_REF] Adams | Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment[END_REF] are few and include studies in Germany, [START_REF] Kolominsky-Rabas | Epidemiology of ischemic stroke subtypes according to TOAST criteria -Incidence, recurrence, and long-term survival in ischemic stroke subtypes: A population-based study[END_REF] the US, [START_REF] White | Ischemic stroke subtype incidence among whites, blacks, and Hispanics: the Northern Manhattan Study[END_REF][START_REF] Petty | Ischemic stroke subtypes: a population-based study of incidence and risk factors[END_REF][START_REF] Schneider | Ischemic stroke subtypes: a population-based study of incidence rates among blacks and whites[END_REF][START_REF] Woo | Incidence rates of first-ever ischemic stroke subtypes among blacks: a population-based study[END_REF] New Zealand, [START_REF] Feigin | Ethnic disparities in incidence of stroke subtypes: Auckland Regional Community Stroke Study, 2002-2003[END_REF] and Chile [START_REF] Lavados | Incidence, case-fatality rate, and prognosis of ischaemic stroke subtypes in a predominantly Hispanic-Mestizo population in Iquique, Chile (PISCIS project): a community-based incidence study[END_REF] but none estimating risk in European multi-ethnic populations or in different groups in the Black population.

Overall there are higher incidence rates of ischemic stroke among Black, Hispanics, Maori/Pacific Islanders and Asian/other groups in the previously reported studies. The most common subtypes are CE, SVO, LAA in the White populations; SVO, CE, LAA in Black/ Hispanics; and CE, SVO, LAA in Maori/Pacific & Asian/Other groups in New Zealand. [START_REF] White | Ischemic stroke subtype incidence among whites, blacks, and Hispanics: the Northern Manhattan Study[END_REF][START_REF] Petty | Ischemic stroke subtypes: a population-based study of incidence and risk factors[END_REF][START_REF] Schneider | Ischemic stroke subtypes: a population-based study of incidence rates among blacks and whites[END_REF][START_REF] Woo | Incidence rates of first-ever ischemic stroke subtypes among blacks: a population-based study[END_REF][START_REF] Feigin | Ethnic disparities in incidence of stroke subtypes: Auckland Regional Community Stroke Study, 2002-2003[END_REF][START_REF] Lavados | Incidence, case-fatality rate, and prognosis of ischaemic stroke subtypes in a predominantly Hispanic-Mestizo population in Iquique, Chile (PISCIS project): a community-based incidence study[END_REF] In recent years, a number of epidemiological studies have also revealed substantial differences in overall stroke risk between different ethnic groups, e.g. in Black populations in the UK or in Black or Hispanic populations in the US. [START_REF] White | Ischemic stroke subtype incidence among whites, blacks, and Hispanics: the Northern Manhattan Study[END_REF][START_REF] Schneider | Ischemic stroke subtypes: a population-based study of incidence rates among blacks and whites[END_REF][START_REF] Woo | Incidence rates of first-ever ischemic stroke subtypes among blacks: a population-based study[END_REF][START_REF] Wolfe | Incidence and case fatality rates of stroke subtypes in a multiethnic population: the South London Stroke Register[END_REF] The aim of this paper is therefore to estimate the incidence of aetiology of first ever ischemic stroke in different ethnic groups within a multi-ethnic inner city population of South London to determine whether patterns of population risk vary with ethnicity.

Methods

Study population

Patients with first ever ischemic stroke were register within the South London Stroke Register (SLSR). The SLSR is a prospective, ongoing population-based stroke register recording since January 1995 all hospitalised and non-hospitalised first-ever strokes in patients of all age groups within in 22 electoral wards in Lambeth and Southwark. [START_REF] Wolfe | Incidence and case fatality rates of stroke subtypes in a multiethnic population: the South London Stroke Register[END_REF] Data on aetiological subtype commenced in 1999. The source population of the SLSR is 271,871 inhabitants (based on UK population census data and projections) comprising 63% White, 28% Black (9% black Caribbean (BA), 15% black African (BA) and 4% black other/mixed (BO)) and 9% other ethnic groups. The methods of case ascertainment and data collection have been previously described. [START_REF] Wolfe | Incidence and case fatality rates of stroke subtypes in a multiethnic population: the South London Stroke Register[END_REF] In brief, all patients with a suspected diagnosis of stroke or transient ischaemic attack documented in different hospital and community based information sources were investigated for study eligibility. Multiple sources of notification were used and capturerecapture models were developed to estimate completeness of case ascertainment. Case ascertainment was estimated to be 84% complete in 1995/1996 and 81% in 2003/2004. [START_REF] Heuschmann | Ethnic group disparities in 10-year trends in stroke incidence and vascular risk factors: the South London Stroke Register (SLSR)[END_REF]

Stroke classification

Stroke was defined according to WHO criteria. [START_REF] Hatano | Experience from a multicentre stroke register: a preliminary report[END_REF] Pathological stroke subtypes were classified using neuro-radiology or necropsy results into ischemic stroke, primary intracerebral haemorrhage (PICH) or subarachnoid haemorrhage (SAH). Ischaemic strokes were further investigated according to an investigation algorithm and categorised by a study clinician into aetiological subtypes according to the TOAST classification with local modifications to improve investigation rates in different ethnic groups. 14. This modified classification has excellent inter and intra operator agreement and has been adapted to enable conditions such as sickle cell disease to be classified specifically in 'Other'. Ischemic stroke subtypes included large artery atherosclerosis (LAA) (including extra-cranial large artery atherosclerosis and intra-cranial large artery atherosclerosis), cardioembolism (CE), small vessel occlusion (SVO), other aetiology (OTH) (including other vascular aetiology, other haemoglobinopathy aetiology, other hypercoaguable aetiology, migrainous stroke, other aetiology not previously mentioned), no aetiology identified (UND) and multiple or concurrent aetiology (CONC).

Data collection

Special trained study nurses and field workers collected all data prospectively. A study clinician verified stroke diagnosis. The following information on sociodemographic characteristics and prior-to-stroke risk factors was collected at initial assessment: self-defined ethnic origin (UK 1991 Census question), classified into White, Black (black-Caribbean, black African, and black Other), other ethnic group; socio-economic status (SES) (based on the Registrar General's codes of occupation), categorised into non-manual (I, II and III nonmanual) and manual (III manual, IV and V); hypertension (general practice or hospital records of high blood pressure >140 mm Hg systolic or >90 mm Hg diastolic) [START_REF] Heuschmann | Ethnic group disparities in 10-year trends in stroke incidence and vascular risk factors: the South London Stroke Register (SLSR)[END_REF] ; diabetes mellitus (self-reported); smoking; previous transient ischaemic attack (TIA).

Statistical Analyses

The t-test was used to test differences in continuous variables, and the chi-square test for differences in proportions. The denominators for incidence calculations were based on the 1991 and 2001 Office of National Statistic's Census data for the study area by assuming a linear trend (REF 11). In January 2004 the register area was extended after the revision of electoral ward boundaries by about 15%. The population estimates for 2004 onwards for the enlarged study area were estimated in a similar way. Crude incidence rates of ischemic stroke were calculated for age group, gender, ethnic groups, and stroke aetiology; specific incidence rates for gender, ethnic group and aetiological subtype were age adjusted to the standard European population. [START_REF] Doll | Summarizing indices for comparison of cancer incidence data[END_REF] 95% confidence intervals (CI) for the age-specific rates and ageadjusted rates were calculated using the Poisson distribution. Incidence rate ratios (IRRs) were calculated for each aetiological subtype to compare stroke incidence between different ethnic groups with the White group being the reference group; 95% CI for the direct standardized IRRs were calculated by the delta method. [START_REF] Daly | Simple SAS macros for the calculation of exact binomial and Poisson confidence limits[END_REF] Differences in proportions of prior-to-stroke risk factors among aetiological stroke subtypes were explored by running separate logistic regression models for each risk factor, adjusted for age, sex, ethnicity and SES as appropriate. Two-tailed probability values are reported and a p-value <0.05 was considered statistically significant in all analyses. Statistical analyses were performed with SAS software version 9.1 (SAS Institute Inc).

Ethics

Patients and/or their relatives gave written informed consent to participate in the study. The design of the study was approved by the ethics committees of Guy's and St Thomas' Hospital Trust, King's College Hospital, St George's Hospital, Queens Square, and Westminster Hospital (London).

Results

Between 1 st September 1999 and 31 st August 2005, 1181 patients with first ever ischemic stroke were registered in the SLSR and were assessed for aetiological subtype of stroke. In 12 of these patients, TOAST classification was not possible mainly because of missing or incomplete diagnostic information. No differences were found for age, sex and ethnic group between classified and unclassified patients. Unclassified patients were excluded from further analyses. Among the remaining 1169 patients included in subsequent analyses, mean age was 71. 4 OTH, 1 (1.5%); UND, 20 (30.3%); CON, 4 (6.1%). Table 2 contrasts the SES, demographic and risk factor between White, Black and Other ethnic groups. There are significant differences in age in White, Black and Other ethnic groups across aetiological subtypes and in Whites for SES. There were no significant differences across subtypes for risk factor prevalence in the Black and Other ethnic group but a significantly increased prevalence of smoking in White groups with LAA and SVO (P=0.0002).

The annual age adjusted incidence rate per 100,000 was for total ischemic stroke 101.2 (95% CI 82.4-122.9) in men and 75.1 (95% CI 59.1-94.1) in women; for LAA 10.4 (95% CI 5.1-18.9) in men and 6.8 (95% CI 2.7-14.2) in women; for CE 23.0 (95% CI 14.6-34.5) in men and 21.5 (95% CI 13.4-32.8) in women; for SVO 30.3 (95% CI 20.5-43.2) in men and 20.3 (95% CI 12.5-31.3) in women; for OTH 3.7 (95% CI 1.0-9.9) in men and 2.7 (95% CI 0.5-8.2) in women; for UND 24.8 (95% CI 16.0-36.7) in men and 18.8 (95% CI 11.2-29.4) in women; and for CONC 9.0 (95% CI 4.1-17.1) in men and 5.0 (1.6-11.7) in women. Table 3 reports on the annual, age adjusted incidence rates of the aetiological subtypes by ethnic group and sex.

The overall incidence rate ratio (IRR) for black patients was 1.25 (95% CI 1.07-1.46), for BC patients 1.31 (95% CI 1.09-1.58), for BA patients 1.22 (95% CI 0.93-1.61) and for other 1.24 (95% CI 0.96-1.61). Table 4 details the IRR and corresponding 95% CIs for Black, BC, BA and other ethnic groups for individual aetiological subtypes, using the White group as the reference. IRRs for Black ethnic groups as well as for BA and BC were significantly higher for SVO in both sexes and for OTH in females, for OTH in black females and in BA for males and females compared to white ethnic group. Figure 1

compares the incidence rates and

IRRs reported in the studies involving Black ethnic groups (African, African Caribbean). [START_REF] White | Ischemic stroke subtype incidence among whites, blacks, and Hispanics: the Northern Manhattan Study[END_REF][START_REF] Woo | Incidence rates of first-ever ischemic stroke subtypes among blacks: a population-based study[END_REF]

Discussion

This study represents the first European data on the incidence of aetiological stroke subtypes in different ethnic groups, including White, Black and Other ethnic groups, using a modified TOAST classification. It has demonstrated stark differences between the White, Black and Other ethnic groups such as the age at stroke onset and patterns of prior to stroke risk factors and aetiological subtypes in different ethnic groups. In this study, the risk of small vessel occlusion was increased for black African, black Caribbean and other ethnic groups in both sexes, and for Other aetiologies the risk was increased in black females and in black African compared to White ethnic groups. Age standardised incidence rates varied significantly between groups in SLSR, with cardioembolic stroke being more common in the White group and small vessel occlusion and Other aetiologies in the Black group. Variation between studies may potentially be explained by such factors as for diagnostic precision and the proportion classified as of undetermined aetiology. The proportion of ischemic stroke patients with undetermined aetiology was low in the SLSR (24%) compared with that reported in previous studies (24-54%), which is mainly explained by the fact, that in the present study ischemic stroke of undefined and of multiple aetiology (Concurrent) were reported separately whereas previous studies reported them combined. [START_REF] Kolominsky-Rabas | Epidemiology of ischemic stroke subtypes according to TOAST criteria -Incidence, recurrence, and long-term survival in ischemic stroke subtypes: A population-based study[END_REF][START_REF] White | Ischemic stroke subtype incidence among whites, blacks, and Hispanics: the Northern Manhattan Study[END_REF][START_REF] Feigin | Ethnic disparities in incidence of stroke subtypes: Auckland Regional Community Stroke Study, 2002-2003[END_REF][START_REF] Lavados | Incidence, case-fatality rate, and prognosis of ischaemic stroke subtypes in a predominantly Hispanic-Mestizo population in Iquique, Chile (PISCIS project): a community-based incidence study[END_REF][START_REF] Kleindorfer | The unchanging incidence and casefatality of stroke in the 1990s: a population-based study[END_REF] The distribution of subtypes in the SLSR is similar to a non population based study in South London that included data from the register. [START_REF] Markus | Differences in stroke subtypes between black and white patients with stroke: the South London Ethnicity and Stroke Study[END_REF] Two previous studies comparing the incidence and distributions of aetiological subtypes of stroke amongst non Black groups have reported in detail the differences in risk of the aetiological subtypes across studies to date. [START_REF] Feigin | Ethnic disparities in incidence of stroke subtypes: Auckland Regional Community Stroke Study, 2002-2003[END_REF][START_REF] Lavados | Incidence, case-fatality rate, and prognosis of ischaemic stroke subtypes in a predominantly Hispanic-Mestizo population in Iquique, Chile (PISCIS project): a community-based incidence study[END_REF] Their Tables and Figures illustrate the difficulty in comparisons as rates are not always standardised to World or European populations or reported in ways that are readily comparable. Additionally, the studies span 13 years and comparisons are compromised by potential changes in risk over the time period and differences in diagnostic procedures. Figure 1 compares, as far as is feasible given the ways risk and incidence rate ratios have been calculated, the incidence rate ratios for the different aetiological subtypes of stroke in the US and UK studies using Black groups over a 13 year time frame. Figure 1 summarises the main findings that in the studies to date there are consistently increased risks of small vessel occlusion in Black groups, with inconsistent differences for other groups.

There is evidence of diversity within different Black ethnic groups. [START_REF] Agyemang | Negro, Black, Black African, African Caribbean, African American or what? Labelling African origin populations in the health arena in the 21 st century[END_REF] Previous studies in the US have included significant numbers of Black ethnic minority individuals in stroke incidence studies but have not reported separately on these disparate groups. [START_REF] White | Ischemic stroke subtype incidence among whites, blacks, and Hispanics: the Northern Manhattan Study[END_REF][START_REF] Petty | Ischemic stroke subtypes: a population-based study of incidence and risk factors[END_REF][START_REF] Woo | Incidence rates of first-ever ischemic stroke subtypes among blacks: a population-based study[END_REF][START_REF] Bild | Multi-ethnic study of atherosclerosis: objectives and design[END_REF][START_REF]The Atherosclerosis Risk in Communities (ARIC) Study Investigators. The Atherosclerosis Risk in Communities (ARIC) Study: design and objectives. The ARIC investigators[END_REF][START_REF] Sacco | Race-ethnic disparities in the impact of stroke risk factors[END_REF] The SLSR has looked separately at Black ethnic groups by black African and black Caribbean ancestry although the power to assess risk across subtypes is limited due to the small number of patients particularly in the Black African group. Overall, risk patterns between these distinct ethnic groups are similar, except a higher rate of other aetiologies among black Africans, which in this study was associated with higher rates of sickle cell disease.

Comparing aetiological subtypes between ethnic groups, we have demonstrated much higher rates of small vessel occlusion in the Black population both in the black Caribbean and black African groups compared with the White ethnic group confirming previous reports (Figure 1). 5, 7, 8 Previous studies have also reported higher rates of large artery atherosclerosis overall, and for both intracranial large artery atherosclerosis and extra cranial large artery atherosclerosis but the confidence intervals are wide in the study reporting intra cranial and extra cranial incidence rate ratios. [START_REF] White | Ischemic stroke subtype incidence among whites, blacks, and Hispanics: the Northern Manhattan Study[END_REF][START_REF] Schneider | Ischemic stroke subtypes: a population-based study of incidence rates among blacks and whites[END_REF][START_REF] Woo | Incidence rates of first-ever ischemic stroke subtypes among blacks: a population-based study[END_REF] In the current study, although incidence rate ratios for individual subtypes corroborated the pattern expected from previous literature on large artery atherosclerosis subtype, the findings did not reach statistical significance. This may have been due to small numbers in the groups. It may also reflect a true difference between the US and UK Black populations due to differences in risk factors such as smoking, which has a great impact on the development of atherosclerosis. [START_REF] Hajat | Ethnic differences in risk factors for ischemic stroke: a European case-control study[END_REF] Differences in risk of stroke between different ethnic groups might be caused by differences in underlying risk factors. Age at onset of stroke is significantly lower in all ethnic groups compared to whites in our population and in other previously reported literature. [START_REF] Wolfe | Incidence and case fatality rates of stroke subtypes in a multiethnic population: the South London Stroke Register[END_REF] We have previously identified that there are significant variations in underlying risk factor patterns in the SLSR: The African Caribbean/African group have higher rates of prior-to-stroke hypertension and diabetes but reduced rates of smoking, history of transient ischaemic attack and obesity, [START_REF] Hajat | Ethnic differences in risk factors for ischemic stroke: a European case-control study[END_REF] and these finding mirror the results from another, non-population based, South London study assessing differences in stroke subtypes between ethnic groups. [START_REF] Markus | Differences in stroke subtypes between black and white patients with stroke: the South London Ethnicity and Stroke Study[END_REF] Comparing results with international studies contrasting ethnic groups, similar differences have been observed in the US with regard to increased hypertension and diabetes in Black Americans and Hispanics in Northern Manhattan and in Blacks in Greater Cincinnati. [START_REF] White | Ischemic stroke subtype incidence among whites, blacks, and Hispanics: the Northern Manhattan Study[END_REF][START_REF] Kleindorfer | The unchanging incidence and casefatality of stroke in the 1990s: a population-based study[END_REF] The increased rates of hypertension are linked to small vessel occlusion but in a parallel study in London, using the population of SLSR, even after adjustment for hypertension there was an excess of small vessel occlusion in the Black group. [START_REF] Kleindorfer | The unchanging incidence and casefatality of stroke in the 1990s: a population-based study[END_REF] This study reported a relative excess of small vessel disease and intracranial atherosclerosis in Black patients compared with an excess of extracranial atherosclerosis and cardioembolic stroke in White patients, independent of conventional risk factors and social class. [START_REF] Markus | Differences in stroke subtypes between black and white patients with stroke: the South London Ethnicity and Stroke Study[END_REF] Large artery atherosclerosis incidence rate ratios did not differ between ethnic groups unlike the study by Markus, and population-based studies in US. [START_REF] White | Ischemic stroke subtype incidence among whites, blacks, and Hispanics: the Northern Manhattan Study[END_REF][START_REF] Kleindorfer | The unchanging incidence and casefatality of stroke in the 1990s: a population-based study[END_REF][START_REF] Markus | Differences in stroke subtypes between black and white patients with stroke: the South London Ethnicity and Stroke Study[END_REF] We also observed higher incidence rate ratios for small vessel occlusion in females and for undetermined aetiology in males in other ethnic group compared to Whites. Thus, the increased risk of stroke in different ethnic groups compared to Whites might represent a non ethnic group specific "migration effect". This effect might be caused by variations in socioeconomic status, differences in access to health care services or different attitudes towards preventive measures between native and migrant populations.

There are strengths and limitations of our study. One limitation to the use of the TOAST classification is the proportion of cases classified as Undetermined. Several studies have estimated the risk and distribution of both the Bamford and TOAST classifications of stroke and identified variations in syndromes between ethnic groups and differences between the incidence rate ratios estimated using each classification. [START_REF] Feigin | Ethnic disparities in incidence of stroke subtypes: Auckland Regional Community Stroke Study, 2002-2003[END_REF][START_REF] Markus | Differences in stroke subtypes between black and white patients with stroke: the South London Ethnicity and Stroke Study[END_REF] The advantage of the Bamford classification is that it is less protocol driven yet its prime aim is not to classify aetiology and it does not phenotype the stroke in terms of underlying risk.

Selection bias resulting from differential uptake of diagnostic tests is a possibility. In the SLSR an investigation algorithm was devised as it was thought that the systematic selection of patients for investigation would increase the overall investigation rate. The investigation rates attained with this study are similar to those of other European and US studies. [START_REF] Kolominsky-Rabas | Epidemiology of ischemic stroke subtypes according to TOAST criteria -Incidence, recurrence, and long-term survival in ischemic stroke subtypes: A population-based study[END_REF][START_REF] White | Ischemic stroke subtype incidence among whites, blacks, and Hispanics: the Northern Manhattan Study[END_REF] We cannot confirm whether investigation rates have been improved upon by the use of this algorithm due to a lack of prior data. However, it seems that the limiting step in achieving high levels of investigation in acute stroke patients is the acutely debilitating nature of the condition itself rather than the availability of scanning facilities. There is also the possibility of information bias. No inter-rater reliability in diagnosis was formally assessed. However a small inter-rater analysis was undertaken on the first 45 consecutive patients recruited into this study. [START_REF] Hatano | Experience from a multicentre stroke register: a preliminary report[END_REF] In addition, all diagnoses were made by trained study clinicians and reviewed on a regular basis by an expert stroke physician (AR). No information on genetic and certain lifestyle factors, such as physical activity and body mass index were collected. The use of self-defined ethnicity is necessitated by the use of this for the denominator population (population Census question) but it is known to have problems with accuracy and fluidity over time. One way that this may be overcome for future studies is to record the place of birth and origin in order to further delineate ethnic groups and in order to glean information on genetic versus environmental influence on the risk of stroke. Although the SLSR population contains amongst the highest proportion of black Africans in the UK and probably Europe, numbers in this ethnic group are still small for the purposes of sub-group analyses resulting in lower estimate precisions compared with the other, larger ethnic groups. Some of the tools used in defining aspects of risk have been designed for use in the West European white population and are not validated or may not be meaningful in these particular ethnic groups, an example of which is the Registrar General Classification for socioeconomic status. [START_REF] Wolfe | Incidence and case fatality rates of stroke subtypes in a multiethnic population: the South London Stroke Register[END_REF]

Conclusion

Important differences in patterns of risk of subtypes of ischaemic stroke have been demonstrated between different ethnic groups which strengthen the case for assessing stroke risk strategies separately in these populations. Furthermore, the pattern of aetiological subtypes seen in these ethnic groups demonstrates the importance of studying individual subtypes in multi-ethnic populations. Reasons for the significant increased risk in Blacks and Other ethnic groups of small vessel occlusion and in black Africans of other aetiologies have to be explored in more detail. Further characteristics of populations with regard to genetic risk and other environmental and life course factors is required to develop tailor-made prevention programmes adapted to the needs of specific ethnic groups. Large artery atherosclerosis (LAA), cardioembolism (CE), small vessel occlusion (SVO), other aetiology (OTH), undetermined (UND) and multiple possible or concurrent aetiologies (CON)

  Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust (CW & AR). CW is anNIHR Senior Investigator. AR is funded by the Guy's & St Thomas' NHS Trust AHSC PA Scheme. PH receives funding from the Federal Ministry of Education and Research via the grant Center for Stroke Research Berlin (01 EO 0801).

Figure 1 .

 1 Figure1. Comparison of Incidence Rate Ratios with 95% confidence intervals in Black ethnic groups in SLSR, Northern Manhatten (NOMASS) and Greater Cincinnati (GCNKSS) studies (White as Reference).[START_REF] White | Ischemic stroke subtype incidence among whites, blacks, and Hispanics: the Northern Manhattan Study[END_REF][START_REF] Schneider | Ischemic stroke subtypes: a population-based study of incidence rates among blacks and whites[END_REF]

Table 1 .

 1 There were significant differences between aetiological subtypes for age, ethnicity, socioeconomic status, a history or transient ischemic attacks and smoking.The distribution of aetiological subtypes in Whites was: LAA, 91 (9.7%); CE, 257 (30.8%);

	years, 50.6% were female; 71.3% were of White, 19.9% of Black (12.8% black
	Caribbean, 6.7% black African, 0.5% black Other), 5.7% of Other and 3.1% of unknown
	ethnic origin; 86.6% of the patients had an ECG, 59.5% underwent a transthoracic or

SVO, 187 (22.4%); 21 (2.5%); UND, 205 (24.6%). CONC 83 (10.0%); in Blacks: LAA, 20 (8.6%); CE, 40 (17.2%); SVO, 98 (42.1%); OTH, 18 (7.7); UND, 49 (21.0%); CONC, 8 (3.4%); and in other ethnic groups: LAA, 7 (10.6%); CE, 11 (16.7%); SVO, 23 (34.9%);

Table 1 .

 1 Sociodemographic characteristic and risk factors of patients with first ischaemic stroke by aetiological subtype, Sept 1999-2005

		Total	LAA	CE	SVO	OTH	UND	CONC	p-value#
	N	1169	109 (9.3)	325 (27.8)	316 (27.0)	40 (3.4)	283 (24.2)	96 (8.2)
	Age, y, mean (SD)	71.35 (13.51)	72.15 (10.52)	74.47 (13.63) 68.77 (12.71)	54.18 (16.24)	71.80 (13.04)	74.30 (12.08)
	Age group, y, n (%)								<.0001
	<65	327 (27.97)	30 (27.52)	59 (18.15)	107 (33.86)	30 (75.00)	80 (28.27)	21 (21.88)
	65-74	311 (26.60)	31 (28.44)	76 (23.38)	99 (31.33)	5 (12.50)	79 (27.92)	21 (21.88)
	75-84	340 (29.08)	33 (30.28)	117 (36.00)	80 (25.32)	5 (12.50)	70 (24.73)	35 (36.46)
	85+	191 (16.34)	15 (13.76)	73 (22.46)	30 (9.49)	0	54 (19.08)	19 (19.79)
	Female, n (%)	591 (50.56)	51 (46.79)	190 (58.46)	143 (45.25)	18 (45.00)	145 (51.24)	44 (45.83)	0.12
	Ethnic group, n (%)								<.0001
	White	834 (71.34)	81 (74.31)	257 (79.08)	187 (59.18)	21 (52.50)	205 (72.44)	83 (86.46)
	Black	233 (19.93)	20 (18.34)	40 (12.31)	98 (31.01)	18 (45.00)	49 (17.31)	8 (8.33)
	Other	66 (5.65)	7 (6.42)	11 (3.38)	23 (7.28)	1 (2.50)	20 (7.07)	4 (4.17)
	Unknown	36 (3.08)	1 (0.92)	17 (5.23)	8 (2.53)	0	9 (3.18)	1 (1.04)
	Socioeconomic status, n (%)
	Non-manual	306 (26.18)	26 (23.85)	88 (27.08)	74 (23.42)	17 (42.50)	76 (26.86)	25 (26.04)	0.0016
	Manual	664 (56.80)	64 (58.72)	176 (54.15)	211 (66.77)	19 (47.50)	144 (50.88)	50 (52.08)
	Unknown	199 (17.02)	19 (17.43)	61 (18.77)	31 (9.81)	4 (10.00)	63 (22.26)	21 (21.88)
	Risk factors pre stroke, n (%)*
	Hypertension	729 (63.67)	74 (69.16)	209 (65.52)	203 (64.86)	15 (39.47)	165 (60.00)	63 (67.74)	0.09
	Diabetes	236 (20.67)	23 (22.12)	60 (18.52)	65 (21.17)	6 (15.38)	58 (21.17)	24 (25.53)	0.50
	Previous TIA	149 (12.93)	24 (22.43)	45 (14.02)	32 (10.19)	3 (7.69)	30 (10.83)	15 (15.96)	0.07
	Current smoking	330 (30.44)	39 (37.86)	61 (20.68)	116 (37.79)	10 (27.78)	82 (32.16)	22 (25.00)	0.0001
	*restricted to patients without missing values; #p-value for differences between aetiological stroke subtypes; adjusted for age, sex, ethnicity, social class as appropriate

Table 2 .

 2 Sociodemographic characteristic and risk factors of patients with first ischaemic stroke by aetiological subtype stratified for White (W) and Black (B) ethnic group

			Total			LAA			CE			SVO			OTH			UND			CONC			p-value#
		W	B	O	W	B	O	W	B	O	W	B	O	W	B	O	W	B	O	W	B	O	W	B	O
	N	834	233	66	81	20	7	257	40	11	187	98	23	21	18	1	205	49	20	83	8	4
	Age, y, mean (SD)	73.69	64.85	65.02	73.25	66.85	74.71	76.09	65.73	67.73	71.65	64.74	62.83	56.76	51.50	48	74.05	67.12	62.55	74.72	73.13	69.75
		(12.74)	(13.46)	(13.07)	(10.50)	(10.72)	(6.18)	(12.32)	(16.20)	(13.64)	(12.84)	(10.81)	(14.25)	(14.62)	(18.30)		2.41)	(12.54)	(12.89)	(12.47)	(11.44)	(4.11)
	Age group, y, n (%)																						0.0022	0.0258	0.05
	<65	192	94	32	20	10	0	38	13	5	53	40	12	15	14	1	47	15	14	19	2	0
		(23.02)	(40.34)	(48.48)	(24.69)	(50.00)		(14.79)	(32.50)	(45.45)	(28.34)	(40.82)	(52.17)	(71.43)	(77.78)	(100)	(22.93)	(30.61)	(70.00)	(22.89)	(25.00)
	65-74	192	90	17	23	4	3	57	14	2	46	41	7	2	3	0	50	25	2	14	3	3
		(23.02)	(38.63)	(25.76)	(28.40)	(20.00)	(42.86)	(22.18)	(35.00)	(18.18)	(24.60)	(41.84)	(30.43)	(9.52)	(16.67)		(24.39)	(51.02)	(10.00)	(16.87)	(37.50)	(75.00)
	75-84	282	38	13	25	5	3	97	11	4	61	15	3	4	1 (5.56)	0	62	5	2	33	1	1
		(33.81)	(16.31	(19.70)	(30.86)	(25.00)	(42.86)	(37.74)	(27.50)	(36.36)	(32.62)	(15.31)	(13.04)	(19.05)			(30.24)	(10.20)	(10.00)	(39.76)	(12.50)	(25.00)
	85+	168	11	4	13	1	1	65	2	0	27	2	1	0	0	0	46	4	2	17	2	0
		(20.14)	(4.72)	(6.06)	(16.05)	(5.00)	(14.29)	(25.29)	(5.00)		(14.44)	(2.04)	(4.35)				(22.44)	(8.16)	(10.00)	(20.48)	(25.00)
	Female, n (%)	435	115	22	40	10	1	155	18	6	80	49	10	8	10	0	111	25	5	41	3	0	0.24	0.95	0.44
		(52.16)	(49.36)	(33.33)	(49.38)	(50.00)	(14.29)	(60.31)	(45.00)	(54.55)	(42.78)	(50.00)	(43.48)	(38.10)	(55.56)		(54.15)	(51.02)	(25.00)	(49.40)	(37.50)
	Socioeconomic status, n (%)																						0.0015	0.47	0.39
	Non-manual	231	49	16	21	3	1	73	5	5	47	21	3	9	8	0	58	12	5	23	0	2
		(27.70)	(21.03)	(24.24)	(25.93)	(15.00)	(14.29)	(28.40)	(12.50)	(45.45)	(25.13)	(21.43)	(13.04)	(42.86)	(44.44)		(28.29)	(24.49)	(25.00)	(27.71)		(50.00)
	Manual	459	153	37	50	12	2	135	27	5	124	70	15	11	7	1	98	31	12	41	6	2
		(55.04)	(65.67)	(56.06)	(61.73)	(60.00)	(28.57)	(52.53)	(67.50)	(45.45)	(66.31)	(71.43)	(65.22)	(52.38)	(38.89)	(100)	(47.80)	(63.27)	(60.00)	(49.40)	(75.00)	(50.00)
	Unknown	144	31	13	10	5	4	49	8	1	16	7	5	1	3	0	49	6	3	19	2	0
		(17.27)	(13.30)	(19.70)	(12.35)	(25.00)	(57.14)	(19.07)	(20.00)	(9.09)	(8.56)	(7.14)	(21.74)	(4.76)	(16.67)		(23.90)	(12.24)	(15.00)	(22.89)	(25.00)
	Risk factors pre stroke, n (%)*
	Hypertension	511	160	43	52	16	5	170	27	7	117	70	13	9	6	0	111	34	14	52	7	4	0.20	0.24	0.87
		(62.32)	(70.48)	(67.19)	(65.00)	(84.21)	(71.43)	(66.93)	(69.23)	(63.64)	(62.90)	(72.16)	(59.09)	(42.86)	(37.50)		(55.78)	(70.83)	(73.68)	(65.00)	(87.50)	(100)
	Diabetes	34	73	23	15	5	3	39	16	2	27	28	9	3	3	0	33	19	5	17	2	4	0.83	0.68	0.70
		(16.42)	(32.44)	(35.38)	(18.99)	(27.78)	(50.00)	(15.23)	(40.00)	(18.18)	(14.84)	(29.79)	(39.13)	(14.29)	(17.65)		(16.75)	(39.58)	(25.00)	(20.99)	(25.00)	(100)
	Previous TIA	123	15	6	20	3	0	39	4	1	24	5	2	3	0	0	23	3	2	14	0	1	0.11	0.80	0.84
		(14.95)	(6.52)	(9.23)	(25.00)	(15.79)		(15.23)	(10.26)	(9.09)	(12.97)	(5.10)	(8.70)	(14.29)			(11.50)	(6.12)	(10.53)	(17.28)		(25.00)
	Current smoking	243	57	17	34	4	0	48	7	1	75	28	9	7	2	1	60	14	5	19	2	1	0.0002	0.39	0.81
		(31.52)	(25.45)	(27.42)	(44.74)	(20.00)		(20.08)	(18.92)	(11.11)	(41.44)	(29.17)	(39.13)	(38.89)	(11.76)	(100)	(32.97)	(30.43)	(26.32)	(25.33)	(25.00)	(25.00)

*restricted to patients without missing values; # p-value for differences between aetiological stroke subtypes; adjusted for age, sex, social class as appropriate

Table 3 .

 3 Annual incidence rates per 100,000 and corresponding 95% confidence intervals by stroke subtype and ethnic group, categorised as white, black (stratified into black Caribbean (BC) and black African (BA)) and Other ethnic group; incidence rates age adjusted to the Standard European population

		White		Black		BC		BA		Oth
		M	F	M	F	M	F	M	F	M	F
	Total	92.6	65.5	107.1	92.2	117.0	95.5	102.9	90.1	132.5	64.7
		(74.7-113.5)	(50.6-83.4)	(87.8-129.4)	(74.4-113.1)	(96.8-140.2)	(77.3-116.7)	(84.0-124.8)	(72.5-110.7)	(110.9-157.1)	(49.9-82.5)
	LAA	9.9	6.7	9.8	9.1	9.8	10.4	7.0	12.1	21.5	3.6
		(4.7-18.2)	(2.6-14.0)	(4.6-18.1)	(4.2-17.2)	(4.7-18.1)	(5.1-18.9)	(2.8-14.5)	(6.3-21.1)	(13.4-32.7)	(0.9-9.7)
	CE	22.9	21.3	18.0	16.0	21.7	17.1	17.3	16.1	14.8	18.0
		(14.5-34.4)	(13.3-32.5)	(10.6-28.4)	(9.2-26.0)	(13.6-33.0)	(10.0-27.3)	(10.2-27.6)	(9.3-26.2)	(8.3-24.5)	(10.7-28.5)
	SVO	24.8	13.2	45.1	37.3	49.1	39.5	53.0	31.4	34.3	29.4
		(16.1-36.7)	(7.1-22.5)	(32.9-60.3)	(26.3-51.3)	(36.3-64.8)	(28.2-53.9)	(39.7-69.3)	(21.4-44.5)	(23.8-47.9)	(19.8-42.1)
	OTH	3.0	1.7	6.5	5.1	2.1	4.0	13.5	6.9	2.6	0
		(0.6-8.8)	(0.2-6.8)	(2.5-13.7)	(1.7-11.8)	(0.3-7.5)	(1.1-10.2)	(7.3-22.8)	(2.7-14.3)	(0.5-8.2)
	UND	22.3	16.9	21.7	21.6	25.7	21.2	12.1	21.6	47.1	13.6
		(14.1-33.7)	(9.8-27.1)	(13.6-33.0)	(13.5-32.9)	(16.8-37.8)	(13.2-32.3)	(6.3-21.1)	(13.5-32.8)	(34.6-62.6)	(7.4 -23.0)
	CONC	9.6	9.6	6.1	6.1	8.5	3.4	0	2.0	12.1	0
		(4.5-17.8)	(4.5-17.8)	(2.3-13.2)	(2.3-13.2)	(3.8-16.5)	(0.8-9.3)		(0.2-7.2)	(6.3-21.1)

Table 4 .

 4 Incidence Rate Ratios and corresponding 95% confidence intervals of ischaemic stroke subtype by ethnic groups (White as reference)*

		Black		BC		BA		Oth
		M	F	M	F	M	F	M	F
	Total	1.16	1.41	1.26	1.46	1.11	1.38	1.43	0.99
		(0.93-1.44)	(1.13-1.75)	(0.97-1.64)	(1.12-1.89)	(0.76-1.63)	(0.93-2.04)	(1.04-1.98)	(0.63-1.54)
	LAA	0.99	1.36	0.99	1.55	0.71	1.81	2.17	0.54
		(0.49-1.99)	(0.66-2.79)	(0.40-2.46)	(0.67-3.58)	(0.21-2.38)	(0.50-6.54)	(0.89-5.29)	(0.07-3.98)
	CE	0.79	0.75	0.95	0.80	0.76	0.76	0.65	0.85
		(0.49-1.27)	(0.45-1.26)	(0.53-1.69)	(0.44-1.46)	(0.28-2.01)	(0.24-2.40)	(0.25-1.66)	(0.37-1.95)
	SVO	1.82	2.83	1.98	2.99	2.14	2.38	1.38	2.23
		(1.28-2.58)	(1.94-4.12)	(1.29-3.04)	(1.94-4.61)	(1.24-3.69)	(1.29-4.38)	(0.76-2.53)	(1.13-4.40)
	OTH	2.17	3.00	0.70	2.35	4.50
		(0.85-5.53)	(1.08-8.31)	(0.15-3.23)	(0.58-9.59)

(1.24-16.29) 4.06 (1.04-15.82)

								0.87	NR
								(0.11-6.78)
	UND	0.97	1.28	1.15	1.25	0.54	1.28	2.11	0.80
		(0.59-1.62)	(0.81-2.01)	(0.67-1.99)	(0.73-2.18)	(0.21-1.42)	(0.62-2.65)	(1.21-3.70)	(0.31-2.08)
	CONC	0.64	0.57	0.89	0.61	NR	0.36	1.26	NR
		(0.22-1.80)	(0.17-1.97)	(0.32-2.43)	(0.14-2.70)		(0.05-2.58)	(0.44-3.58)
	*NR -not reported as no observation in the respective field

Acknowledgements

We wish to thank all the patients and their families and the health care professionals involved.

Particular thanks to all the Register team and the fieldworkers who have collected data for the South London Stroke Register.

Dr C Hajat was the lead investigator for the first two years of data collection and was involved in designing and setting up the study, study conduct and management, data collection, data analysis, interpretation of the data and writing of the manuscript. Professor P Heuschmann led on the data handling and data. Ms C Coshall was involved in the methodology, data handling and data analysis, had full access to all of the data in the study and takes responsibility for the integrity of the data. Dr S Padayachee was involved in study design and carotid imaging. Dr J Chambers was involved in study design and cardiac imaging. Dr A Rudd was involved in the study design, review of all diagnoses and review of the manuscript. Professor C Wolfe is the guarantor of the work. He was involved throughout in designing and setting up the study, study conduct and management, interpretation of the data, writing up and review of the manuscript and is the corresponding author.

Funding


J Kačmarčík

Z Pribulová

P Szabó

P Samuely

C Marcenat

T Klein

D-J Jang

H-G Lee

H-S Lee

S I Lee

H-G Lee

H-S Lee

Superconducting energy gap in MgCNi 3 single crystals

Even though MgCNi 3 contains large molar volume of magnetic Ni atoms it clearly shows superconductivity. The origin of superconductivity in this material has not yet been clarified. There is a lot of discrepancy in experimental results and physical interpretation where even unconventional pairing or a two-band model have been proposed for the system. All that is based on results obtained on polycrystalline samples. Here we present the first point-contact spectroscopy and ac-calorimetry measurements on the MgCNi 3 single crystals of very good quality.

Measurements have been performed in the temperature range from 1.5 to 10 K and in magnetic fields up to 8 T. The temperature dependence of the energy gap of the system is presented and compared to the BCS model.

A c c e p t e d m a n u s c r i p t 1. Introduction

Despite the fact that MgCNi 3 contains also magnetic Ni ions it shows superconductivity around 7 K [1]. The origin of superconductivity remains unclear. Mao et al. [2] have discussed unconventional pairing based on their point-contact data showing the zero-bias peak. Another point-contact experiments on MgCNi 3 [3] have also found the zero-bias anomaly in the tunneling conductance data but explained it not as a consequence of unconventional pairing mechanism but due to an extrinsic origin, namely due to a presence of weak links near the point contact. With this assumption the BCS s-wave gap has been obtained from fitting of the data. The measurements of the penetration depth [4] have indicated non-s-wave superconductivity. The heat capacity and the upper critical field data [5] have been described by a single-band/single-gap s-wave model with strongly coupled holes. In order to explain the Hall conductivity and thermopower data published in the same paper the two-band/two gap model of phonon mediated s-wave superconductivity partly suppressed by an electron-paramagnon interaction has been proposed. To resolve this controversial situation measurements on single crystals of good quality are highly desirable.

Recently, the MgCNi 3 crystals of high quality have been grown [6]. Here we present the first pointcontact spectroscopy and ac-microcalorimetry data on these crystals with aim to study the superconducting energy gap in the system. Although certain scattering of the gap value is found in our point-contact spectra, which is probably due to the surface inhomogeneity, these results together

A c c e p t e d m a n u s c r i p t

with the analysis of our heat-capacity measurements suggest single gap s-wave superconductivity with the medium coupling in the system.

Experimental

Difficulties in preparation of MgCNi 3 single crystals have been overcome using a high-pressure closed system as described elsewhere [6]. X-ray analysis proved that carbon deficiencies from stoichiometry are negligible. Contrary to the polycrystalline MgCNi 3 samples, which have usually local carbon deficiency, in the single crystals the Ni sites were partly deficient. This leads to a critical temperature T c ~ 6.7 K (obtained from transport measurements) which is lower than the highest T c of 7.3 K of polycrystalline samples. The samples used for our measurements have typical dimensions of few hundred microns.

The point-contact spectroscopy measurements were performed on several samples from the same batch. A standard lock-in technique was used to measure the differential resistance as a function of applied voltage on the point contacts. The point contacts were prepared in situ by mechanically pressing platinum tips on the surface of the crystals. The approaching system allows for both lateral and vertical movements of the tip enabling a formation of the junction as well as the probing of different places on the sample surface. The point-contact differential conductances (calculated from differential resistance) have been normalized to the conductance background found at the voltages above the gap. The normalized curves were fitted to the standard point-contact model accounting for the Andreev reflection as well as the tunneling contributions to the point-contact current. This

A c c e p t e d m a n u s c r i p t

allows obtaining information about the superconducting energy gap(s) ∆, the spectral broadening Γ and the barrier transparency z [7].

Although an ac calorimetry is not capable to measure the absolute values of the heat capacity it is very sensitive technique for measurements of relative changes on minute samples. The ac microcalorimeter [8] which uses a diode as the heater has been used. An optical fiber is used to drive the heating power onto the sample. The absence of a contact between the heater and the sample reduces total addendum. The temperature of the sample is measured by a very sensitive thermocouple.

The point-contact and ac heat capacity measurements of MgCNi 3 single crystals were performed in the temperature range between 2 and 10 K and in magnetic fields up to 8 T.

Results and Discussion

Tens of the point-contact spectra and few sets of their temperature dependences have been recorded on several crystals from the same batch. The point contacts revealed intermediate barrier strength with both Andreev reflection and tunneling contributions. The spectra have shown a well defined pair of peaks indicating the size of the superconducting energy gap. By positioning the tip at different places on the surface of the samples a certain scattering of the gap peaks was observed in line with the previous observations [3]. The scattering could be due to local inhomogeneities, e.g.

local phases with slightly different T c 's, so it was important to know the local transition temperature at the junction. For this, the temperature dependence of the point-contact spectrum was measured.

Then, the point-contact spectra were fitted to the point-contact s-wave formula accounting also for The superconducting energy gap extrapolated to zero temperature ∆(0) was found to be scattered between 1.0 and 1.2 meV for different measurements although the local T c equal to 6.6 ±0.1 K was quite homogeneous. Figure 1 shows such an example with the inset showing the temperature dependence of the gap together with the BCS prediction. The resulting coupling ratio of 2∆(0)/k B T c in the range between to 3.5 and 4.2 was found. In some cases a significantly smaller size of the gap ∆(0), about 0.7 meV was also detected. Beside the small size of this gap, its temperature dependence revealed a significant deviation from the BCS shape. Namely, although the local T c has been again in the range of 6.6 ±0.1 K, the measured gap undershooted the BCS prediction in the temperature range between 3 K and T c . Such behavior is indicative for a proximity effect on the surface with somewhat degraded superconductivity. There, the degraded surface layer has a lower T * c and smaller ∆ * (0) which has a tendency to close at T * c (undershooting ∆(T) with the bulk T c ), but due to an increase of the coherence length near the bulk T c the superconductivity is kept until T c where ∆ * is finally closed.

Another possible interpretation of observing smaller and larger gaps would be a two gap scenario.

We remark that the two-gap behavior can be effectively unveiled when magnetic field is applied on point contacts [7]. Details of the gap measurements on MgCNi 3 at different crystallographic directions as a function of temperature and magnetic field will be published elsewhere [10] but these results point again to the single gap scenario [10].

Thus, from the point-contact spectroscopy we can conclude that MgCNi 3 behaves like an s-wave single-gap superconductor with weak up to moderate coupling strength.

A c c e p t e d m a n u s c r i p t

Since the point-contact spectroscopy is a surface sensitive technique it is important to compare its results with the bulk thermodynamic measurements such as the heat capacity. The samples have shown a well-defined superconducting transition in the heat capacity. The anomaly represents around 40% of the total signal including addenda. It is sharper (∆T c ~ 0.2 K) than the specific heat anomaly observed on the polycrystalline samples [6], indicating the quality and homogeneity of our samples. To clarify the superconductivity in MgCNi 3 , it is of interest to derive the electronic heat capacity by subtracting the normal state heat capacity C n i.e. ∆C(T)/T=C(T)/T-C n (T)/T. A magnetic field of 8 T was enough to suppress the superconductivity down to 3 K in our case. At low temperature region the high field heat capacity C(8T) with subtracted addenda has followed well the usual formula C(8T)/T=C n /T= A + B.T 2 . This dependence was extrapolated to the lowest temperatures. The value of ∆C(T c )/γ n T c can be used to measure the strength of the electron coupling. With the heat capacity data measured down to 2.4 K γ n could not be determined directly.

To estimate γ n a plot of ∆C(T)/T dependence on temperature down to 0 K was made following the entropy conservation law, demanding that the areas between the superconducting and normal part below and above the crossing point must have equal surfaces. This procedure will be verified by the experiments in He-3 cryostat which are in progress.

The resulting ∆C(T)/T at H=0 is shown in Fig. 2. From an entropy conserving construction (see the dotted line), the transition temperature T c was estimated to be 5.86 K. We remark that this T c is lower than that deduced from the transport and point-contact measurements on the samples from the same batch.

The obtained ratio of ∆C(T c )/γ n T c estimated from our data is equal to 1.71. This value is larger than the BCS weak coupling limit (1.43) indicating stronger coupling. Another possibility to estimate

A c c e p t e d m a n u s c r i p t

the coupling strength of MgCNi 3 is to compare the measured dependence of ∆C(T) to the so called alpha model [9] based on the BCS theory. In this model the only adjustable parameter is the gap ratio 2∆(0)/k B T c . In our case the comparison yields a ratio 2∆(0)/k B T c ~ 3.8, which is higher than the BCS weak coupling limit 3.52 in agreement with the large value of ∆C(T c )/γ n T c . Thus, the heat capacity measurements values also suggest that the MgCNi 3 may be a superconductor with a moderate coupling strength. The measurements at lower temperatures capable to resolve the symmetry of the superconducting gap are in progress.

Conclusions

We have performed the point-contact spectroscopy and highly sensitive ac-microcalorimetry studies on MgCNi 3 single crystals. Both methods point to the conventional s-wave single-gap superconductivity with the moderate coupling in the system.

A c c e p t e d m a n u s c r i p t

  the temperature smearing. The gap value as a function of temperature was resulting from the fit.

A c c e p t e d m a n u s c r i p tFig. 1

 1 Fig. 1 Pt-MgCNi 3 point-contact spectra (solid lines) measured at indicated temperatures and fit to the single gap swave point-contact formula (circles). Inset: Temperature dependence of the fitted energy gap with ∆(0) = 1 meV and the BCS prediction (line).

A c c e p t e d m a n u s c r i p tFig. 2

 2 Fig. 2 Electronic heat capacity in relative units. Dotted lines indicates the transition temperature as determined by conservation of entropy.

Acknowledgements

This work has been supported by the EC Framework Programme MTKD-CT-2005-030002, the Slovak Research and Development Agency under the contracts No. APVT-51-016604 and LPP-0101-06. The CLTP is operated as the Centre of Excellence of the Slovak Academy of Sciences and P.J. Safarik University. Liquid nitrogen for the experiments has been sponsored by the U.S. Steel Kosice. s.r.o.

A c c e p t e d m a n u s c r i p t


López De Hierro

Maria Dolores

De Neve

Liesbeth

Pigment limitation and female reproductive characteristics influence egg shell spottiness and ground colour variation in the house sparrow (Passer domesticus)

Keywords: Biliverdin, Protoporphyrin, laying sequence, clutch size, Age

One of the hypotheses to explain egg colour variation in birds lays in the context of sexual selection where egg colour may signal the female"s physical condition and antioxidant capacity. We tested one of the assumptions following from this hypothesis that depositing eggshell pigments should be limited for females. The study was conducted in a house sparrow (Passer domesticus) captive population, over several years under constant environmental conditions. This multi brooded species lays eggs which vary in ground colour (biliverdin pigment) and in the intensity and distribution of brownish-red spots (protoporphyrin pigment). Spot darkness, spread and ground colour diminished along the laying sequence, suggesting that the deposition of both pigments was limiting for females at short term. Also the proportion of eggs with biliverdin diminished in consecutive clutches laid by the same female over the breeding season, suggesting a long term cost of biliverdin deposition. On the other hand, spots were darker at the end of the breeding season, indicating that protoporphyrin deposition was probably not limited at long term. This result could indicate a lower capacity of calcium deposition at long term which was compensated for by darker spots. Also female age affected significantly the proportion of bluish eggs and spot patterns. Egg pigmentation decreased with age, indicating that senescing passerine females lay less pigmented eggs.

Clutch size, was positively related to the proportion of bluish eggs and to spot patterns (more evenly and darker spotted). These results are in accordance with assumptions for the sexual selection hypothesis.

Introduction

The great variation in avian egg-shell pigmentation has always received much attention from evolutionary biologists and numerous adaptive hypotheses on the subject have been proposed in recent decades (reviewed in [START_REF] Underwood | Adaptive significance of egg coloration In: Deeming DC (eds) Avian incubation, behaviour, environment and evolution Oxford UnivPress[END_REF][START_REF] Kilner | The evolution of egg colour and patterning in birds[END_REF]. For different bird taxa, this variation has been explained in the context of egg mimicry to face predation risk (e.g. [START_REF] Bakken | Avian eggs: thermoregulatory value of very high near-infrared reflectance[END_REF][START_REF] Lloyd | Nest-site selection, egg pigmentation and clutch predation in the ground-nesting Namaqua Sandgrouse Pterocles namaqua[END_REF]Blanco and Bortellotti 2002), to permit egg recognition in the case of brood parasitism [START_REF] Stokke | Rejection of artificial cuckoo (Cuculus canorus) eggs in relation to variation in egg appearance among reed warblers (Acrocephalus scirpaceus)[END_REF][START_REF] Stokke | Obligate brood parasites as selective agents for evolution of egg appearance in passerine birds[END_REF][START_REF] Davies | Approximate F-tests of Multiple Degree of Freedom Hypotheses in Generalized Least Squares Analyses of Unbalanced Split-plot Experiments[END_REF][START_REF] Soler | Host recognition of parasites eggs and the physical appearance of host eggs: the magpie and its brood parasite the great spotted cuckoo[END_REF]Lathi 2005Lathi , 2006)), or more recently also in the context of sexual selection where egg colour may signal the female"s physical condition and antioxidant capacity (e.g. Moreno andOsorno 2003, Soler et al. 2005;[START_REF] Siefferman | Female condition is correlated with egg coloration in eastern bluebirds[END_REF]Hanley et al. 2008).

Biliverdin (an antioxidant, responsible for blues and greens) and protoporphyrins (a pro-oxidant, responsible for reds and browns) are the principal pigments responsible for egg colour in birds [START_REF] Kennedy | A survey of avian eggshell pigments[END_REF][START_REF] Miksik | Avian eggshell pigments and their variability[END_REF][START_REF] Underwood | Adaptive significance of egg coloration In: Deeming DC (eds) Avian incubation, behaviour, environment and evolution Oxford UnivPress[END_REF]. [START_REF] Moreno | Avian egg colour and sexual selection: does eggshell pigmentation reflect female condition and genetic quality[END_REF] suggested that blue green egg pigmentation acts as a sexually selected, condition-dependent signal of female quality. They reasoned that since biliverdin has been shown to have antioxidant properties [START_REF] Kaur | Interaction of bilirubin and biliverdin with reactive nitrogen species[END_REF], females should balance the use of biliverdin for protection against free radicals and for deposition into eggshells. The sexual signalling hypothesis proposes that only highquality females can afford the costs of depositing large amounts of biliverdin during the laying period, a time of high oxidative stress [START_REF] Moreno | Avian egg colour and sexual selection: does eggshell pigmentation reflect female condition and genetic quality[END_REF]. Support for this hypothesis however has been ambiguous up till now. A growing number of studies corroborated that the deposition of the antioxidant pigment biliverdin in the eggshell may depend on the higher antioxidant capacity of laying females (Hanley et al. 2008), and be indicative of female quality in terms of immune system [START_REF] Moreno | Evidence for the signaling function of egg color in the pied flycatcher Ficedula hypleuca[END_REF][START_REF] Jagannath | Eggshell pigmentation indicates pesticide contamination[END_REF] or physical condition [START_REF] Moreno | Experimental evidence that egg color indicates female condition at laying in a songbird[END_REF][START_REF] Siefferman | Female condition is correlated with egg coloration in eastern bluebirds[END_REF][START_REF] Krist | Are blue eggs a sexually selected signal of female collared flycatchers? a cross-fostering experiment[END_REF][START_REF] Soler | Sexually Selected Egg Coloration in Spotless Starlings[END_REF]. However, some studies in other bird species did not find such clear support for the signalling function of the biliverdin pigment [START_REF] Hargitai | Eggshell coloration in relation to male ornamentation, female condition and egg quality in the collared flycatcher Ficedula albicollis[END_REF], Lopez-Rull et al. 2008;Hanley et al. 2008;[START_REF] Hanley | Egg coloration in ring-billed gulls (Larus delawarensis): a test of the sexual signaling hypothesis[END_REF], stressing the need for more studies on this subject.

On the other hand, a possible relation between porphyrin pigmentation (spot patterns) and female physical condition or reproductive parameters has received much less attention up till now [START_REF] Miksik | Quantification and variability of eggshell pigment content[END_REF][START_REF] Miksik | Avian eggshell pigments and their variability[END_REF]Martinez-de la Puente et al. 2007;[START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF]. Protoporphyrins are synthesized in the blood as an intermediate metabolite of haemoglobulin, and are transferred via epithelial cells to the shell gland during egg formation [START_REF] Baird | Localisation and characterisation of egg shell porphyrins in several avian species[END_REF]. Due to the pro-oxidant properties of protoporphyrins [START_REF] Afonso | Protoporphyrin IX and oxidative stress[END_REF][START_REF] Shan | Induction of the Heme Oxygenase-1 gene by metalloporphyrins[END_REF], it has been proposed that they could either signal female quality because increased pigmentation would indicate oxidative tolerance [START_REF] Moreno | Avian egg colour and sexual selection: does eggshell pigmentation reflect female condition and genetic quality[END_REF] or, alternatively, be an indicator of poor condition, since high levels of protoporphyrins produce physiological stress (Martínezde la [START_REF] Martínez-De La Puente | Are eggshell spottiness and colour indicators of health and condition in blue tits Cyanistes caeruleus?[END_REF]). Surprisingly, there has been found support for both hypotheses in blue tits (Cyanistes caeruleus). Martínez-de la [START_REF] Martínez-De La Puente | Are eggshell spottiness and colour indicators of health and condition in blue tits Cyanistes caeruleus?[END_REF] found support for the latter hypothesis since females that laid more spotted eggs showed poorer body condition and higher levels of a stress protein. [START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF] found support for the sexual selection and signalling hypothesis of spot patterns, since spotting was positively related to some indicators of female quality (tarsus length, clutch size) and paternal investment.

Nevertheless, as with any other phenotypic characteristic, the variation in egg pigmentation between females is due to a combination of genetic, environmental and maternal influences and the understanding of the source of variation is important for testing the various hypotheses advanced to explain egg colour variation. For example, both egg shell pigments have been shown to be influenced by environmental factors. In the case of biliverdin, evidence has emerged of the importance of local environmental factors such as dietary intake (e.g. [START_REF] Soler | Sexually Selected Egg Coloration in Spotless Starlings[END_REF] or rainfall and temperature [START_REF] Avilés | Environmental conditions influence egg color of reed warblers Acrocephalus scirpaceus and their parasite, the common cuckoo Cuculus canorus[END_REF]. Previous work showed how also the quantity of protoporphyrins may vary according to diet (e.g. [START_REF] Joseph | Shell quality and color variation in broiler breeder eggs[END_REF]). More recently, [START_REF] Gosler | Why are birds" eggs speckled?[END_REF] have proposed that protoporphyrins are used to compensate for localised shell thinness arising from a lack of calcium in the environment, thereby reducing the permeability and water loss during incubation [START_REF] Higham | Speckled eggs: water-loss and incubation behaviour in the great tit Parus major[END_REF][START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF]. Furthermore, it is also important to distinguish between spot patterns and eggshell ground colour, since the mechanisms determining eggshell ground colour and spot patterns are not necessarily the same (e.g. [START_REF] Underwood | Adaptive significance of egg coloration In: Deeming DC (eds) Avian incubation, behaviour, environment and evolution Oxford UnivPress[END_REF][START_REF] Moreno | Avian egg colour and sexual selection: does eggshell pigmentation reflect female condition and genetic quality[END_REF]Lopez-Rull et al. 2008).

In the present study, we collected data over a four-year period from various clutches laid by the same females in a captive house sparrow (Passer domesticus) population, which was kept under the same environmental conditions. This multiple brooded species lays eggs which vary both in ground colour and in the intensity and distribution of brownish-red spots [START_REF] Seel | Clutch-size, incubation and hatching success in the house sparrow and tree sparrow Passer spp at Oxford[END_REF][START_REF] Lowther | Spotting pattern of the last laid egg of the house sparrow[END_REF]. First, we explored within female constancy in egg shell patterns. Furthermore, the sexual selection hypothesis explaining variation in egg colour and patterns assumes that pigment deposition should be limiting for females [START_REF] Moreno | Avian egg colour and sexual selection: does eggshell pigmentation reflect female condition and genetic quality[END_REF][START_REF] Morales | Pigment allocation to eggs decreases plasma antioxidants in a songbird[END_REF]. We analysed variation in spot patterns and ground colour according to egg laying sequence and a negative relationship would reflect short term limitation in pigment deposition. In addition, we also explored if pigment deposition may be limiting for females along the breeding season (long time span) and analysed egg colouration according to the number of clutches laid by the same female in a breeding season. Finally, we studied if clutch size and female age influenced variation in egg ground colour and spot patterns. In house sparrows, as in many other species, clutch size is a plastic trait that varies with age and that shows significant variation between females [START_REF] Hatch | Age-related patterns of reproductive success in house sparrows Passer domesticus[END_REF][START_REF] Nakagawa | Does the badge of status influence parental care and investment in house sparrows? An experimental test[END_REF][START_REF] Westneat | Complex interactions among temporal variables affect the plasticity of clutch size in a multi-brooded bird[END_REF]). Since clutch size can be considered to be an indicator of investment in the present reproductive attempt, a positive relationship between clutch size and egg ground colour is predicted [START_REF] Moreno | Avian egg colour and sexual selection: does eggshell pigmentation reflect female condition and genetic quality[END_REF]. If spot patterns in the sparrow are indicative for poorer quality females (Martínez-de la [START_REF] Martínez-De La Puente | Are eggshell spottiness and colour indicators of health and condition in blue tits Cyanistes caeruleus?[END_REF], a negative relation between clutch size and spot patterns is expected. Alternatively, if spot patterns are indicative for high quality females, larger clutches should be more spotted [START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF]. With respect to age, most studies have reported that reproductive traits improve after the first year of life (e.g. review in Christians 2002) which could also be reflected in egg pigmentation (e.g. [START_REF] Gosler | Why are birds" eggs speckled?[END_REF]Sieffereman et al. 2006). However, there is also evidence that elderly females may display poorer physical condition and/or reproductive traits than those of intermediate age (e.g. [START_REF]Reproductive success University of[END_REF][START_REF] Newton | Lifetime reproduction in birds[END_REF][START_REF] Stearns | The evolution of life histories[END_REF][START_REF] Robertson | A long-term study of reproductive performance in tree swallows: the influence of age and senescence on output[END_REF] and then senescence may also be reflected on egg pigmentation [START_REF] Moreno | Evidence for the signaling function of egg color in the pied flycatcher Ficedula hypleuca[END_REF].

Methods

The study species

The house sparrow nests in cavities or builds enclosed nests on tree branches and performs between one and seven reproductive attempts during each breeding season in Europe [START_REF] Cramp | The Birds of the Western Palearctic[END_REF]Anderson 2006). The species is chiefly monogamous and has a low frequency of extra-pair copulations [START_REF] Veiga | Low frequency of extra-pair fertilisations in house sparrows breeding at high density[END_REF]. The ground colour of house sparrow eggs varies from pure white to bluish and the eggs are covered with spots of diverse size and pigmentation. The distribution of spots varies considerably, from a large patch at the blunt end to a uniform spot pattern across the entire eggshell and patterns of the last-laid egg in a clutch tends to be different from the others [START_REF] Seel | Clutch-size, incubation and hatching success in the house sparrow and tree sparrow Passer spp at Oxford[END_REF][START_REF] Yom-Tov | Intraspecific nest parasitism among Dead Sea sparrows Passer moabiticus[END_REF][START_REF] Lowther | Spotting pattern of the last laid egg of the house sparrow[END_REF]).

Characteristics of the study population

The study population has been kept in a 45m 3 aviary in the Science Faculty of Granada University since 1999. Due to significant mortality, especially of fledged juveniles in the post-breeding period, the population size was more or less constant (mean 75 ± SE 10 individuals) throughout the study years, with a mean sex ratio of 1:1. The aviary provided 40 nest boxes, of which about 50% were occupied each breeding season. The birds were provided ad libitum with water, seed mix, fly maggots, vitamins, powdered calcium, mineral salts, apple, lettuce and nestling food. They were also provided with a sand tray for their habitual dust-baths. Cotton wool and plant material was provided for nest construction during the breeding season. The artificial illumination was regulated by a timer which kept the birds at the same photoperiod as the external environment.

Data collection

The study was carried out during the breeding seasons of 2003-2006 inclusive. Daily observations were done from the start of each breeding season to identify breeding females and their nest boxes as well as possible changes in nest box use for consecutive clutches.

All birds born in the captive population were colour-ringed as nestlings with a combination of 4 rings to allow individual identification. Hence, the exact age was known for breeding females that were born in captivity. We used minimum ages of females that were not born in captivity, i.e. the number of years they spent in captivity, plus one year. Female age ranged from 1 to 6 years, and was divided in 3 age classes (1 = first-time breeders; 2 = middle aged, two and three years old and 3 = advanced age, ≥ four years old). Since individuals were not captured again during the course of the study, we do not have information on physical quality variables of breeders. Therefore, possible influences of male quality on female reproductive investment were not taken into account.

The nest boxes were examined daily to obtain information on the process of nest building, laying order, clutch size and the number of clutch. Throughout the study there were no instances of two eggs being laid in the same day in any of the nest boxes, so there is practically no chance that any of the eggs in the studied-clutches was deposited by another female [START_REF] Petrie | Laying eggs in other"s nest: intraspecific brood parasitism[END_REF][START_REF] Jackson | Estimating conspecific nest parasitism in the northern masked weaver based on within-female variability in egg appearance[END_REF]. Other experiments after clutch completion and during the nestling stage were performed in most of the studied broods, for which the number of clutches laid by a female could not be considered as a reliable possible indicator of female quality.

Evaluation of egg characteristics

During the course of the study 206 complete clutches, laid by 33 different females, were photographed against a neutral grey card, incorporating a colour chart, using a Minolta Dimage 7 digital camera. Photographs were taken indoor and under constant light conditions.

Assessments of egg characteristics were carried out for each egg from the photographs (n = 830 eggs). Clutches of 19 females were photographed in only one year, either because the birds died during the next winter or because they laid their first clutch in 2006, the final year of study. However, for these females, except for three, we had information on at least two consecutive clutches during the same breeding season.

Information from two or more years was available for 14 females. With respect to firsttime breeders, we obtained photos of clutches of two females in 2005 and seven in 2006. Clutches laid by first-time breeders in 2004 were used in a parallel experiment which did not permit complete clutches to be photographed and so these were not used to contribute first-time breeder data to the present study. Thus, in total we obtained data from 9 first-time breeders and 25 adults females, from which one of the first-time breeders past to adult in the following breeding season.

The same observer assessed the photos of each clutch against the following parameters, following the criteria and methodology of [START_REF] Gosler | Inheritance and variation in eggshell patterning in the great tit Parus major[END_REF][START_REF] Gosler | Why are birds" eggs speckled?[END_REF]; see also [START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF] with principal classes: spot intensity (I: scored from 1 for palest spots to 5 for the darkest); average spot size (S: scored from 1 for small spots to 3 for large spots) and spot distribution (D: scored from 1 for > 90% spots concentrated in one end, to 5 for an even spot distribution over the surface). Unspotted eggs received a 0-quote for all the spot-parameters. Shell ground colour was divided in four classes: white, brownish-white, bluish-white, or bluish. Within the study sample of 830 eggs, 69.6 % were white, 1.4 % were brownish-white, 3.8 % bluish-white, and 25.1 % were bluish. Given this very low percentage of brownish-white and bluish-white eggs, these were excluded from further analyses to avoid strong unbalanced models.

Repeatability of the observer"s assessments was estimated by evaluating the characteristics of 280 eggs from the 63 clutches of 16 females in 2003 a second time 15 days after the first [START_REF] Lessells | Unrepeatable repeatabilities: a common mistake[END_REF] and was highly significant for all variables (P < 0.001 in all cases).

Statistical analyses

Because some of the variables of eggshell spot patterns were significantly intercorrelated (I and S: r 830 = 0.379, P < 0.001; I and D: r 830 = 0.033, P = 0.337; S and D: r 830 = 0.166, P < 0.001), we used a Principal Component Analysis (PCA) of the correlation matrix of the original I, S and D values [START_REF] Gosler | Inheritance and variation in eggshell patterning in the great tit Parus major[END_REF][START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF]. Two Principal Components explained 80.7 % of the variation in spots pattern. The PC1 describes variation in pigment intensity and spot size and it was referred to as the "darkness" of the egg (see [START_REF] Gosler | Inheritance and variation in eggshell patterning in the great tit Parus major[END_REF][START_REF] Gosler | Why are birds" eggs speckled?[END_REF][START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF]. Eggs with larger and more intense spots showed higher PC1 values (factor loadings: I: 0.780, D: 0.325, S: 0.842). PC1 expresses 47.5 % of the total variation in I, D and S. The second principal component (PC2) describes the level of spot aggregation and shows higher values for eggs with more widely distributed spots over their surface (factor loadings: I: 0.377, D: 0.928, S: 0.009). PC2 then represents the "spread" of maculation (see [START_REF] Gosler | Inheritance and variation in eggshell patterning in the great tit Parus major[END_REF][START_REF] Gosler | Why are birds" eggs speckled?[END_REF][START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF]. PC2 expresses a further 33.2 % of the total variation in I, S and D.

To test which variables best explained variation in eggshell spot patterns (darkness and spread) General Linear Mixed Models (GLMM) were constructed with PC1 and PC2 as the dependent variables. For ground colour a Generalized Linear Mixed Model (GLIMMIX) was used (binomial distribution: 0 = white ground colour, 1 = bluish ground colour, logit link function). In all analyses laying order, clutch size and the number of clutch were considered as covariables. The three age classes (see Data collection) were included as a fixed factor using Bonferroni adjustment. The variables female identity and year were random factors to determine whether differences existed in egg colour patterns between females and to avoid pseudo replication, and to take into account possible random variation due to study year.

The model residuals did not differ from a normal distribution (Kolmogorov-Smirnov: all P > 0.2). The degrees of freedom of the GLMM and GLIMMIX models were calculated using the Satterthwaite method (Fai and Cornelius 1996) and were performed with SAS (SAS 1989-96 Institute Inc., Cary, NC, USA; [START_REF] Littell | SAS System for Mixed Models Cary[END_REF].

Furthermore, the same models were included in a Variance Components Analysis to obtain the % of variance explained by female identity in spot darkness and spread (STATISTICA 7.0, StatSoft, Inc. 2004).

Results

Female identity had a significant effect on all egg colour characteristics but the year had no significant effect on any of them (Tables 1 and2). Variance Components Analyses revealed that the variance attributable to female identity in pigment spread was higher (26.6%) than for pigment darkness (12.7%).

Spot darkness (PC1) was significantly influenced by egg laying order, clutch size, clutch number and female age (Table 1). The first eggs within a given clutch were darker spotted than the final ones and spot darkness increased with clutch number and clutch size (Table 1). In addition, clutches from older females (> four years) were less dark spotted compared to those of first time breeders (GLMM Bonferroni corrected: P = 0.005) and middle-aged ones (GLMM Bonferroni corrected: P < 0.001, Table 1, Fig. 1). Spot darkness did not differ significantly between first time breeders and middle-aged ones (GLMM Bonferroni corrected: P = 0.76, Fig. 1).

On the other hand, pigment spread (PC2) diminished significantly according to laying sequence and clutch number, but increased with clutch size (Table 1). No significant differences existed between age classes (Table 1).

Ground colour (white vs bluish) was significantly influenced by laying order, clutch number, clutch size and female age (Table 2). Within a clutch, the last-laid eggs were mainly white ones and the last clutches of the season had more white eggs than the earlier ones. Eggs from small clutches were more white and older females laid a higher proportion of white eggs (first time breeders = 67.9%, middle-aged = 64.8%, older females = 85.0%).

Discussion

House sparrow egg colour was significantly determined by female identity. However, the variance in spot patterns attributable to female identity was larger for pigment spread (26.6%) than for pigment darkness (12.7%), suggesting that pigment spread is less flexible within females than pigment darkness. The fact that pigment darkness was affected by female age, but pigment spread was not, supports this idea. Furthermore, and in agreement with the sexual selection prediction, our results indicate that house sparrows females experience certain limitation in pigment deposition for both pigments (ground colour and spots) and at least short term. With respect to ground colour, the last laid eggs within a clutch and the last clutches within the breeding season had a higher proportion of white eggs. Previous findings on free-living bird populations of pied flycatchers (Ficedula hypoleuca) and collared flycatchers (Ficedula albicollis) also pointed at a short time limitation or cost in the deposition of the biliverdin pigment since egg darkness decreased along the laying sequence (Moreno et al. 2005;[START_REF] Krist | Are blue eggs a sexually selected signal of female collared flycatchers? a cross-fostering experiment[END_REF]. However, other studies found the opposite relationship where last laid eggs were the most bluish ones [START_REF] Siefferman | Female condition is correlated with egg coloration in eastern bluebirds[END_REF][START_REF] Hargitai | Eggshell coloration in relation to male ornamentation, female condition and egg quality in the collared flycatcher Ficedula albicollis[END_REF]. This suggests different reproductive strategies across species. While a decrease in egg pigmentation indicates female limitation in pigment deposition; an increase in egg pigmentation may indicate higher investment in last laid eggs, facilitating survival of last-hatched young ("brood survival hypothesis", [START_REF] Slagsvold | On the adaptive value of intra-clutch egg size variation in birds[END_REF]. Our results, in addition, suggest also a possible cost of maintaining the same level of investment in biliverdin deposition throughout the breeding season (long term) in female house sparrows, even when food is freely available. With respect to spot patterns, results suggest that at least at short term the deposition of protoporphyrin would be limited for females because both spot darkness and spread decreased along the laying sequence.

Contrary to these findings, [START_REF] Gosler | Why are birds" eggs speckled?[END_REF] found a linear increase through the clutch in pigment darkness in great tits (Parus major). However, this increase was not predicted by the observed changes in eggshell mass, egg size or shape (i.e. structural hypothesis: spots compensate for eggshell thinning) and the authors suggest that some additional physiological programme may be operating through the clutch. Although we cannot test eggshell thickness in relation to laying order and pigmentation in the present study, our results support the idea that different mechanisms may operate across species or under different environmental conditions. More studies are necessary to attain more general patterns explaining within clutch egg pigmentation patterns. With respect to long term effects on protoporphyrin deposition, at least three other studies have examined this along the breeding season. In the great tit, pigment darkness was negatively related to laying date [START_REF] Gosler | Inheritance and variation in eggshell patterning in the great tit Parus major[END_REF]. In the closely related blue tit, females laid less spotted (% spots), but brighter and more reddish clutches at the end of the breeding season (Martínez de la [START_REF] Martínez-De La Puente | Are eggshell spottiness and colour indicators of health and condition in blue tits Cyanistes caeruleus?[END_REF]). Nevertheless, in the same species [START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF] found that laying date had no effect on pigmentation patterns (darkness and spread). So, again, these previous findings point at no general relationship between pigmentation and the course of the breeding season.

Still, all these studies were conducted on single brooded species while the present study analyzed the possible limitation in spot patterns in different clutches of the same female during the same breeding season and under constant environmental conditions. Our results showed that first clutches had eggs where spots were more spread, but less dark.

Then, given that spots were darker at the end of the breeding season, protoporphyrin deposition was probably not limited at long term. However, the less dark spots and more evenly spread at the beginning at the breeding season could indicate a lower capacity of calcium deposition for females in last clutches; so at long term. This lower capacity could then be compensated for by darker spots [START_REF] Gosler | Why are birds" eggs speckled?[END_REF]. However, in the present study we have no information to test possible effects of other egg properties or female condition variables on spot patterns.

Female age affected significantly the proportion of bluish eggs and spot patterns: the proportion of bluish eggs and spot darkness declined in females older than four years. Some studies failed to find effects of age on egg ground colour [START_REF] Hargitai | Eggshell coloration in relation to male ornamentation, female condition and egg quality in the collared flycatcher Ficedula albicollis[END_REF] or spot patterns [START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF]. Other studies found that adults compared to first-time breeders laid more pigmented [START_REF] Siefferman | Female condition is correlated with egg coloration in eastern bluebirds[END_REF], brighter (Martínez de la [START_REF] Martínez-De La Puente | Are eggshell spottiness and colour indicators of health and condition in blue tits Cyanistes caeruleus?[END_REF] or less dark spotted eggs [START_REF] Gosler | Why are birds" eggs speckled?[END_REF].

Nevertheless all these studies distinguished only two age classes and had no data on older age classes to evaluate senescence on egg pigmentation. One study however did evaluate the effect of an older age class on egg pigmentation in pied flycatchers, and also found that older females (> 4 years) laid lighter and less pigmented eggs [START_REF] Moreno | Evidence for the signaling function of egg color in the pied flycatcher Ficedula hypleuca[END_REF]. These results, together with results of the present study, suggest that senescing passerine females lay less pigmented eggs.

On the other hand, larger clutches had a higher proportion of bluish eggs and eggs were more evenly and darker spotted compared to small clutches. These findings are in accordance with those of [START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF] in blue tits where larger clutches also had more evenly distributed spots. Since clutch size is likely a reliable indicator of investment in the present reproductive attempt (e.g. [START_REF] Christians | Avian egg size: variation within species and inflexibility within individuals[END_REF]), these results rather support the hypothesis that red-brownish pigmentation and bluish pigmentation of eggs reflect a higher maternal investment in sparrows [START_REF] Moreno | Avian egg colour and sexual selection: does eggshell pigmentation reflect female condition and genetic quality[END_REF] and therefore may be involved in the sexual selection theory explaining egg colouration. However, further studies are needed to determine the relationship between female condition/quality and age and clutch size in the house sparrow.

In conclusion, both ground colour and spot darkness seem to be limited egg characteristics for house sparrow females, at least short term (along the laying sequence). Also females that laid larger clutches laid more bluish and more evenly and darker spotted eggs and female age had a negative effect on egg pigmentation. These findings are rather in agreement with predictions proceeding from the sexual selection hypothesis explaining egg colour variation in house sparrows.

Zusammenfassung

  Grenzen derPigmenteinlagerung und Merkmale der weiblichen Fortpflanzungsorgane beeinflussen Fleckung und Hintergrundfarbvariation von Eierschalen in Haussperlingen (Passer domesticus). In der vorliegenden Arbeite haben wir Merkmale von Eifärbung innerhalb individueller Weibchen einer in Gefangenschaft gehaltenen Haussperlingspopulation (Passer domesticus) über mehrere Jahre hinweg untersucht. Ziele waren (1) die Bedeutung der genetischen Einflüsse zu schätzen, (2) zu untersuchen, ob Pigmenteinlagerung eierlegender Weibchen limitiert ist, und (3) ob Indikatoren individueller Qualität von Weibchen (z. B. Gelegegröße, Alter) die Eifärbung beeinflussen. Unsere Ergebnisse zeigen das Vorhandensein einer genetischen Basis, welche die Eifärbung individueller Weibchen bestimmt. Darüber hinaus nahm die Grundfarbe und die Intensität und Größe der Flecken über die Legereihenfolge ab, was auf begrenzte Kapazitäten der Pigmenteinlagerung pro Reproduktionsereignis hinweist. Junge Weibchen und Weibchen mit größeren Gelegen legten dichter gefleckte Eier mit intensiveren und größeren Flecken, sowie einen größeren Anteil Eier mit bläulicher Grundfarbe. Dies kann darauf hindeuten, dass beide Pigmentierungen mit der weiblichen Kondition zusammenhängen. Unsere Ergebnisse zeigen außerdem, dass die Verteilung der Flecken jenes Merkmal von Eiern ist, welches anscheinend am wenigsten von Umweltfaktoren beeinflusst wird.Table1. GLMM models which best explained variation in spots darkness and spots spread. The "Variable Type" column indicates whether a variable was regarded as a covariable (C), fixed factor (F) or random factor (R). F values are given for covariables and fixed factors and Z values for random factors. The sign of the estimator indicates a positive or negative relation between covariables and the dependent variable.

(

  binomial distribution variable, logit link function). The "Variable type" column indicates whether a variable was regarded as a covariable (C), fixed factor (F) or random factor (R). F values are given for covariables and fixed factors and Z values for random factors. The sign of the estimator indicates a positive or negative relation relation between covariables and the dependent variable.

Figure 1 .

 1 Figure 1. The difference in (a) spots darkness (b) spots spread (c) % white eggs

Table 1 Explanatory variables Variable type

 1

	Estimate	df	F/Z	p

Table 2 .

 2 GLIMMIX model which best explained variation in egg ground colour

Table 2 Explanatory variables Variable type Estimate df F/Z P

 2

	Ground colour	Female	R			2.65	0.004
		Year	R			1.03	0.151
		laying order	C	-0.23	1,515 10.24	0.001
		Clutch number	C	-0.28	1,535 11.64	< 0.001
		Clutch size	C	0.29	1,536	7.49	0.006
		Age	F		2,36.4 5.96	0.018

Acknowledgements

We are grateful to Manuel Soler for his extensive support and valuable comments to this study. Juan J. Soler and Manuel Martín-Vivaldi revised the manuscript and made useful comments to improve it. Francisco José Esteban, Juan Diego Ibáñez, David Martín, Javier Molina and Rubén Rabaneda helped during the capture and/or care of birds. The study was financed by the Junta de Andalucía through its support to the Comportamiento y Ecología Animal research group (RNM339). M.D.G. López de Hierro was financed by a PhD grant and L. De Neve by a post-doctoral fellowship (both financed by the Spanish Ministry of Education and Science). Comments by anonymous referees greatly improved the manuscript.