Verena Matejas

Bernward Hinkes

Faisal Alkandari

Lihadh Al-Gazali

Ellen Annexstad

Mehmet B Aytac

Margaret Barrow

Kveta Blahova

Detlef Bockenhauer

Hae Il Cheong

Jens ; Koenig

Laurie Seaver

John A Burns

Mohnish ; Suri

Květa Bláhová

Iwona Maruniak-Chudek

Pierre Cochat

Jörg Dötsch

Priya Gajjar

Raoul C Hennekam

Françoise Janssen

Mikhail Kagan

Ariana Kariminejad

Markus J Kemper

Jillene Kogan

Hester Y Kroes

Eberhard Kuwertz-Bröking

Amy F Lewanda

Ana Medeira

Jutta Muscheites

Patrick Niaudet

Michel Pierson

Anand Saggar

Alexey Tsygin

Elke Wühl

Aleksandra Zurowska

Steffen Uebe

Friedhelm Hildebrandt

Corinne Antignac

M.D. Martin Zenker
email: martin.zenker@med.ovgu.de

Mutations in the Human Laminin β2 (LAMB2) Gene and the Associated Phenotypic Spectrum

Keywords: nephrotic syndrome, Pierson syndrome, laminin, autosomal recessive, podocyte, ocular malformation LAMB2, Pierson syndrome, nephrotic syndrome, autosomal recessive, podocyte, laminin, ocular malformation

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

BACKGROUND

Mutations of LAMB2 (MIM# 150325), the gene encoding laminin β2, were first detected in patients who suffered from congenital nephrotic syndrome (NS) histologically presenting as diffuse mesangial sclerosis, in combination with a complex ocular maldevelopment the most impressing clinical sign of which is extreme and fixed narrowing of the pupils (microcoria) (Zenker et al., 2004a). This unusual association was first described by [START_REF] Pierson | an Unusual Congenital and Familial Congenital Malformative Combination Involving the Eye and Kidney[END_REF][START_REF] Pierson | an Unusual Congenital and Familial Congenital Malformative Combination Involving the Eye and Kidney[END_REF], and therefore the term Pierson syndrome was coined for this disorder (MIM# 609049) (Zenker et al., 2004b). Microcoria-congenital nephrosis syndrome is a synonym. Patients with Pierson syndrome are also at risk for severe neurodevelopmental deficits including congenital muscular weakness / myasthenia and developmental retardation [START_REF] Maselli | Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome[END_REF][START_REF] Wuhl | Neurodevelopmental deficits in Pierson (microcoria-congenital nephrosis) syndrome[END_REF]. The clinical manifestations in Pierson syndrome correspond well to the defects observed in mice deficient of laminin β2, who display severe glomerular kidney disease, ocular and neurologic abnormalities [START_REF] Miner | Transgenic isolation of skeletal muscle and kidney defects in laminin beta2 mutant mice: implications for Pierson syndrome[END_REF]Noakes et al., 1995a;Noakes et al., 1995b).

Laminins represent a group of cross-shaped heterotrimeric proteins each consisting of α, β and γ subunits joined together through a coiled coil. Laminins are indispensable basement membranes constituents with important roles in cell adhesion, proliferation, differentiation and migration [START_REF] Miner | Laminin functions in tissue morphogenesis[END_REF][START_REF] Ryan | The functions of laminins: lessons from in vivo studies[END_REF][START_REF] Tunggal | Laminins: structure and genetic regulation[END_REF]. Laminin-521 (consisting of α5, β2, and γ1 subunits; formerly called laminin-11) is the most common laminin isoform that contains a β2-chain [START_REF] Miner | Laminin-11[END_REF]. This isoform is specifically expressed at distinct sites such as the glomerular basement membrane, various ocular structures, and the neuromuscular system, (Zenker et al., 2004a).

The human LAMB2 gene maps to chromosome band 3p21 and is composed of 32 densely packed exons spanning about 12 kb of genomic DNA (Fig. 1a). The gene encodes a protein of 1,798 amino acids, containing the typical laminin domains: the Nterminal globular laminin domain (LN) making interactions with neighboring laminins, multiple EGF-like repeats (LE) with an interjacent second globular domain (LF) whose function is currently unknown, and a coiled coil domain (LCC) (Fig. 1b). The most Nterminal 32 amino acids represent a cleavable signal peptide.

Although Pierson syndrome was not recognized as a separate entity before 2004, several reports on single cases and a few patient series have appeared in the years since then [START_REF] Bredrup | Ophthalmological aspects of Pierson syndrome[END_REF][START_REF] Choi | Variable phenotype of Pierson syndrome[END_REF][START_REF] Hasselbacher | Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders[END_REF]Kagan et al., 2008;[START_REF] Maselli | Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome[END_REF][START_REF] Matejas | A syndrome comprising childhoodonset glomerular kidney disease and ocular abnormalities with progressive loss of vision is caused by mutated LAMB2[END_REF][START_REF] Vandevoorde | Pierson syndrome: a novel cause of congenital nephrotic syndrome[END_REF][START_REF] Wuhl | Neurodevelopmental deficits in Pierson (microcoria-congenital nephrosis) syndrome[END_REF], indicating that this disease had likely been overlooked before. The current literature also includes some descriptions of milder variants of the disease as well as two observations of apparent isolated infantile NS caused by homozygous or compound heterozygous mutations of LAMB2 [START_REF] Choi | Variable phenotype of Pierson syndrome[END_REF][START_REF] Hasselbacher | Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders[END_REF][START_REF] Kagan | A milder variant of Pierson syndrome[END_REF][START_REF] Matejas | A syndrome comprising childhoodonset glomerular kidney disease and ocular abnormalities with progressive loss of vision is caused by mutated LAMB2[END_REF]. Based on the finding of missense mutations or small in frame deletions at least on one allele in these cases in contrast to the predominance of biallelic truncating mutations in the classic Pierson syndrome, it has been proposed that the level of residual laminin β2 function/expression is the main modifier of the phenotype [START_REF] Hasselbacher | Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders[END_REF][START_REF] Kagan | A milder variant of Pierson syndrome[END_REF]. Herein we review a total of 1, 3, and4. The genotype and phenotype features of patients with LAMB2 mutations are listed in Table 2.

Mutations

Sequence changes regarded as disease-causing mutations are presented in Table 1.

They comprise missense, nonsense, and splice site mutations, as well as small deletions and insertions, found either as homozygous or compound heterozygous sequence changes in patients affected by typical Pierson syndrome or its milder variants [START_REF] Choi | Variable phenotype of Pierson syndrome[END_REF][START_REF] Hinkes | Nephrotic syndrome in the first year of life: two thirds of cases are caused by mutations in 4 genes (NPHS1, NPHS2, WT1, and LAMB2)[END_REF][START_REF] Kagan | A milder variant of Pierson syndrome[END_REF][START_REF] Maselli | Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome[END_REF][START_REF] Matejas | A syndrome comprising childhoodonset glomerular kidney disease and ocular abnormalities with progressive loss of vision is caused by mutated LAMB2[END_REF][START_REF] Vandevoorde | Pierson syndrome: a novel cause of congenital nephrotic syndrome[END_REF][START_REF] Wuhl | Neurodevelopmental deficits in Pierson (microcoria-congenital nephrosis) syndrome[END_REF]Zenker et al., 2004a[START_REF] Zenker | Demonstration of two novel LAMB2 mutations in the original Pierson syndrome family reported 42 years ago[END_REF], as well as in two previously published siblings with isolated NS [START_REF] Hasselbacher | Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders[END_REF]. The 12 novel mutations were identified by automated sequencing of PCR products of genomic DNA as described previously (Zenker et al., 2004a). All coding exons and flanking intronic regions were analyzed in each patient. Sequence changes were classified as causative mutations, if they produce a premature translational stop codon, if they affect the conserved nucleotides at the splice acceptor and donor sites, respectively, or if they delete or substitute a conserved amino acid and were observed together with a mutation on the second allele.

The majority of mutations (35 out of 49) are predicted to lead to a premature translational stop codon. These mutations include 14 nonsense, 19 frameshift, and two c.3798-2A>C; c.3982+1G>T; c.4573+1G>A) has not been determined experimentally, because no appropriate material from the respective patients was available. However, in all of them the most likely consequence predicted by in silico analysis is either exon skipping or intron inclusion, shifting the reading frame. This is also true for the mutation c.1036+6_9delTGAG, the only identified splice site mutation that does not affect the invariant nucleotides +1 or +2 of the splice donor, but which is nevertheless predicted to lead to loss of splice donor function. Only eight missense changes and two in frame deletions leading to the loss of a single amino acid have been identified as likely causative mutations, to date. All of them affect highly conserved amino acid residues of the laminin β2 protein (Supp. Figure S1) and were found either in the homozygous state or in compound heterozygosity with another bona fide mutation on the second allele (Table 2). All missense mutations except for two (p.L139P, p.S80R) have been reported previously [START_REF] Choi | Variable phenotype of Pierson syndrome[END_REF][START_REF] Hasselbacher | Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders[END_REF][START_REF] Kagan | A milder variant of Pierson syndrome[END_REF]Matejas et al., 2006;Zenker et al., 2004a). None of these changes was found in over 200 controls.

Five mutations were recurrent (Table 2). Four of them (c.1405+1G>A, c.1477delT, c.3174_3175delTG, andc.4504delA) were found in two unrelated families each, while the mutation p.R246W was independently observed in 5 unrelated families. The remaining changes are "private mutations" observed only in single families.

Mutations creating premature stop codons are almost evenly distributed along the LAMB2 gene (Fig. 1a). They may either lead to nonsense mediated mRNA decay or result in truncated proteins. Notably, the mutation c.5258dupA, which is predicted to produce the premature translational stop located most closely to the 3' end of the mRNA and to delete only 39 amino acids from the protein, has been demonstrated to result in complete lack of protein expression (Zenker et al., 2004a). This may be due to the fact that the C-terminus of laminin β2 is important for the proper assembly of the laminin chains. Mutant laminin β2 chains that cannot be stably assembled into a trimeric laminin complex are probably degraded [START_REF] Utani | Laminin chain assembly. Specific sequences at the C terminus of the long arm are required for the formation of specific double-and triple-stranded coiled-coil structures[END_REF]. Consequently, all truncating mutations known to date likely represent functional null alleles.

In contrast, missense mutations and small in frame deletions obviously cluster in the LN domain of laminin β2 (Fig. 1b). This protein domain is critical for interacting with the LN domains of α and γ chains of neighboring laminins to form the monolayer network which represents a scaffold for basement membrane assembly [START_REF] Colognato | Laminin polymerization induces a receptor-cytoskeleton network[END_REF][START_REF] Yurchenco | Self-assembly and calcium-binding sites in laminin. A three-arm interaction model[END_REF]. This suggests that changes of highly conserved amino acid residues in that domain might perturb laminin polymerization. However, it has also been shown that the mutation p.R246W leads to significant reduction in protein expression (Zenker et al., 2004a). This may be due to disturbances at various stages of protein processing. Consistently, studies on a mouse model expressing the laminin β2 mutant R246Q suggested that the impact of this mutant on glomerular function stems in part from impaired laminin secretion (Cheng et al., 2008). The missense mutation p.C321R affects one of the invariant cysteines in the first EGF-like domain, LEa1 (Fig. 1c). As these cysteine residues form disulfide bonds that stabilize the structure of the EGF-like domains, substitution by other amino acids probably result in alteration or destabilization of protein structure. The consequences of the missense change p.L1393F affecting the LCC domain remain elusive [START_REF] Hasselbacher | Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders[END_REF]. In our own study population, the causative sequence changes were identified on both alleles in 29 out of 30 unrelated patients with typical Pierson syndrome (as defined by the presence of congenital microcoria plus NS). In a single patient (case 3.1, Table 2) only one allele, a nonsense mutation (p.Q125X) was identified, while the second allele has remained undetected despite sequencing of all introns and the presumed promoter region as well as screening for larger genomic deletions (using long range PCR covering the entire gene). RT-PCR on mRNA from kidney tissue of this patient, however, showed severely reduced LAMB2 mRNA expression from the allele that was not affected by the nonsense mutation (data not shown). These findings strongly support the existence of a mutation on the second allele, which escaped the employed screening methods (maybe an inversion or translocation affecting the LAMB2 locus).

Together, these results provide clear evidence that Pierson syndrome is not heterogeneous and that LAMB2 mutation detection rate reaches 98-100% in typical cases.

Polymorphisms

Table 3 13 polymorphisms listed in the dbSNP database, while 18 variants are novel. 15 of the latter are either intronic with no obvious effect on splicing or do not cause changes on the protein level and, thus, are not supposed to affect the protein structure or function. A heterozygous variant in exon 24, predicting a substitution of glycine by arginine (p.G1243R) was identified in a patient with Pierson syndrome together with the mutation p.G1693VfsX20 on the same allele. Moreover, interspecies alignment revealed only poor conservation of the glycine at position 1243, together suggesting that this change likely represents a neutral polymorphism. Similarily, the sequence variant c.4140C>A in exon 26 leading to an exchange of asparagine by lysine (p.N1380K) was detected in a patient together with the mutation p.L1393F on the same allele. Based on evolutionary conservation and on the fact that p.N1380K but not p.L1393F was also found in one of 96 controls, the former was regarded as a probable polymorphism and the latter as the disease-causing mutation [START_REF] Hasselbacher | Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders[END_REF]. The c.5293G>A variant (predicting the change p.A1765T) was found repeatedly in both patients and healthy controls, and it was found in one family to be located on the same allele as a truncating mutation (p.Q1006NfsX144) [START_REF] Matejas | A syndrome comprising childhoodonset glomerular kidney disease and ocular abnormalities with progressive loss of vision is caused by mutated LAMB2[END_REF]. For evolutionary conservation of these missense variants see Supp. Figure S2.

Sequence Variants of LAMB2 with Unknown Phenotype Effect

For nine missense variations in the LAMB2 gene and three variations located 5' to the start codon in the potential promoter region, the pathogenetic significance could not definitively be determined. These changes are listed in Table 4. All but two of them have been found as heterozygous changes in patients with NS who lacked other typical The amino acids affected by those changes show various levels of evolutionary conservation (Supp. Figure S2). Three variations are located in the putative LAMB2 promoter region. Two of them were heterozygous (c.-1925G>C and c.-408_404delTAGTT) while the c.-165C>A substitution was found in a homozygous state in a patient with isolated NS. Their significance cannot be determined clearly, since the LAMB2 promoter is poorly characterized, so far. The heterozygous variant p.P37A affects the relatively conserved signal peptide cleavage site and is predicted to possibly favour aberrant cleavage 4 amino acids more downstream (SignalP 3.0 Server at http://www.cbs.dtu.dk/services/SignalP/), thus leading to a protein that is slightly shortened at its N-terminus. A heterozygous substitution p.H882Y was found in a healthy carrier for Pierson syndrome, who was heterozygous for the mutation p.C374X, but whether the variation p.H882Y was on the same allele could not be determined, because the affected children were not available for genetic testing [START_REF] Zenker | Demonstration of two novel LAMB2 mutations in the original Pierson syndrome family reported 42 years ago[END_REF]. Since His-882 is relatively conserved (Supp. Figure S2) and we cannot absolutely exclude the possibility of two clinically significant sequence changes on the same allele, we conservatively classified p.H882Y in this category.

Haplotype Analysis of Recurrent LAMB2 Mutations

In patients harbouring the five recurrent LAMB2 mutations we determined haplotypes by genotyping 13 microsatellite markers within a range of 15 Mb flanking the LAMB2 gene 15 as well as 5 to 6 common intragenic single nucleotide polymorphisms in the index patient and his/her parents, as far as parental DNAs were available (Supp. Figure S3). The mutation p.R246W, which has been observed in 5 unrelated families, was found on the same haplotype in three families of Portuguese origin (two from Portugal and one from Brazil), thus suggesting a founder effect in this population. However, the same mutation was also found in one family of Asian origin on a different haplotype and in a patient of African origin. Shared haplotypes were also found in two families with Slavic background (originating from Poland and the Czech Republic), who carried the mutation c.4504delA, two families of German/French ancestry (mutation: c.1477delT), and two families of Middle European origin (mutation: p.C1058X), respectively. In all cases the shared haplotypes encompassed about 5 Mb. However, the precise extent could not be determined because it is impossible to distinguish identity by descent from identity by state for individual markers.

GENOTYPE-PHENOTYPE CORRELATIONS

All individuals carrying homozygous or compound heterozygous LAMB2 mutations were affected by NS in the first decade, the vast majority in the first year of life. All but two patients (affected siblings from family 13) had ocular anomalies (Table 2). Although an ascertainment bias cannot be excluded, the current data suggest that kidney involvement is an invariant manifestation of genetic defects of the LAMB2 gene.

However, some patients may be recognized because of their eye findings, and the renal symptoms only arrive thereafter [START_REF] Bredrup | Ophthalmological aspects of Pierson syndrome[END_REF]. As mentioned above, there is strong evidence that truncating mutations, even those creating truncations of less than Comparison of the group of patients with biallelic truncating mutations versus those with non-truncating mutations on at least one allele reveals a somewhat earlier onset of nephrotic symptoms and delayed occurrence of end stage renal disease (ESRD) at least in some of them (Fig. 2). Patients with two putative functional null alleles typically develop ESRD within the first year of life. ESRD may already be present at birth and mask nephrotic symptoms (hypoproteinemia, edema) through the limitation of renal protein waste in the presence of oliguria. However, in two patients with biallelic truncating mutations and presumed complete loss of laminin β2 production, ESRD was delayed until childhood age (patients 24.1 and 29.1) (Choi et al., 2008 and unpublished observation). Regarding ocular manifestations, all patients harboring biallelic nonsense or frameshift mutations exhibited congenital microcoria in association with variable other eye abnormalities as reviewed previously [START_REF] Bredrup | Ophthalmological aspects of Pierson syndrome[END_REF], except for one unpublished case (patient 29.1). With this single exception, patients who lacked this typical ocular sign of Pierson syndrome, including the two reported families with initial presentation of isolated NS [START_REF] Choi | Variable phenotype of Pierson syndrome[END_REF][START_REF] Hasselbacher | Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders[END_REF], had at least one non-truncating allele or a splice site mutation whose effect on protein expression could not be definitely determined. This may suggest that only little residual function of laminin β2 is required for an apparently normal development and maintenance of the iris muscles. Notably, those patients who presented initially with only minor or without ocular changes appear to remain at high risk of developing serious ocular complications, e.g. retinal detachment, in later infancy or childhood [START_REF] Bredrup | Ophthalmological aspects of Pierson syndrome[END_REF][START_REF] Choi | Variable phenotype of Pierson syndrome[END_REF][START_REF] Matejas | A syndrome comprising childhoodonset glomerular kidney disease and ocular abnormalities with progressive loss of vision is caused by mutated LAMB2[END_REF]. Generally, isolated NS is rarely caused by [START_REF] Hinkes | Nephrotic syndrome in the first year of life: two thirds of cases are caused by mutations in 4 genes (NPHS1, NPHS2, WT1, and LAMB2)[END_REF]. In addition to what was published before [START_REF] Choi | Variable phenotype of Pierson syndrome[END_REF][START_REF] Hasselbacher | Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders[END_REF][START_REF] Hinkes | Nephrotic syndrome in the first year of life: two thirds of cases are caused by mutations in 4 genes (NPHS1, NPHS2, WT1, and LAMB2)[END_REF], we have meanwhile studied more than 70 further cases with isolated NS without detecting another LAMB2 mutationpositive individual in this cohort. The same is true for nonspecific syndromic NS cases lacking ocular abnormalities of the Pierson syndrome spectrum. Significant neurodevelopmental abnormalities (muscular weakness / myasthenia, global delay, presumed retinal blindness) have been described repeatedly in patients with presumed complete deficiency of laminin β2 [START_REF] Maselli | Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome[END_REF][START_REF] Wuhl | Neurodevelopmental deficits in Pierson (microcoria-congenital nephrosis) syndrome[END_REF] . However, the nature of these deficits is not well characterized, and only few patients with the classic Pierson syndrome phenotype survived beyond the age of 2. One long-term survivor has recently been described with a neurologic picture resembling congenital myasthenia [START_REF] Maselli | Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome[END_REF]. In contrast, we are aware of several patients (case 21.1, 24.1, 29.1, 31.1, 32.1, and 35.1; Table 2) with either truncating or splice site mutations on each allele, who had a normal neurologic and cognitive development up to the age of 4-21 years [START_REF] Bredrup | Ophthalmological aspects of Pierson syndrome[END_REF][START_REF] Choi | Variable phenotype of Pierson syndrome[END_REF][START_REF] Wuhl | Neurodevelopmental deficits in Pierson (microcoria-congenital nephrosis) syndrome[END_REF]. In one of them, possible residual function of one allele carrying a de novo splice site mutation (c.3798-2A>C) was discussed but could not be demonstrated [START_REF] Wuhl | Neurodevelopmental deficits in Pierson (microcoria-congenital nephrosis) syndrome[END_REF]. Most patients who showed a favourable neurodevelopmental outcome despite the presence of biallelic mutations predicting probable complete loss of function also had delayed onset of ESRD. This observation would be compatible with the existence of genetic modifiers that may to some extent compensate for the laminin β2 defect, thereby rescuing the neurologic deficits and ameliorating the renal phenotype. Admittedly, considering truncating mutations as functional null alleles may be an oversimplification and does not [START_REF] Kellermayer | Translational readthrough induction of pathogenic nonsense mutations[END_REF].

Taken together, the current data provide evidence of significant genotype phenotype correlations. On the other hand, it is has become obvious that the genotype alone does not explain all the clinical variability among LAMB2-associated disorders. The neurological manifestations of Pierson syndrome remain the most enigmatic part of the disease spectrum and require further elucidation.

CLINICAL AND DIAGNOSTIC RELEVANCE

The diagnosis of Pierson syndrome is based on the recognition of the typical association of glomerular kidney disease and ocular abnormalities. In the typical cases with microcoria and early onset NS the diagnosis is obvious. Molecular testing of LAMB2 will very likely confirm the diagnosis in such patients. Mutational screening by sequencing of all coding exons and flanking intronic regions should particularly be attempted, if the parents wish to have prenatal testing in a further pregnancy. Although affected fetuses may also present with kidney abnormalities on prenatal ultrasound [START_REF] Mark | Prenatal findings in four consecutive pregnancies with fetal Pierson syndrome, a newly defined congenital nephrosis syndrome[END_REF], only molecular genetic testing allows an early and reliable prenatal diagnosis. Molecular analysis of the LAMB2 gene may also be indicated to identify a familial mutation in order to offer subsequent carrier testing in healthy family members, although the risk of having a child with Pierson syndrome in relatives of a patient is very small (excluding consanguinity of the partners), given a presumably low carrier frequency in the general population. In cases with congenital or infantile NS and less specific ocular symptoms, molecular analysis of the LAMB2 gene provides a clue in the (Hinkes et al., 2007 and unpublished results). Analysis of this gene may however still be justified in patients with congenital or infantile nephrosis, mesangial sclerosis on biopsy, and negative results upon testing for mutations in the genes NPHS1, NPHS2, WT1, and PLCE1. Early development of ESRD (from birth or in the first 3 months of life), which is unusual in other types of congenital NS, may be a further indication for testing for LAMB2 mutations. Considering the fact that microcoria may be the first presenting symptom of Pierson syndrome, LAMB2 testing may be of important diagnostic and prognostic value in any child with congenital microcoria.

Considering the clinical variability of LAMB2-associated disorders, predictions on the phenotypic expression on the basis of the genotype should be made with caution.

Unfortunately, this is particularly true with respect to the possibility of relevant neurologic involvement which is an important determinant of long-term prognosis.

Within the same family, however, our current experience suggests a rather high consistency of the phenotype.

Patients with Pierson syndrome are mainly taken care of by pediatric nephrologists and ophthalmologists. There is so far no specific treatment available. Nephrectomy for the treatment of severe renal protein waste may be considered similar to the management of patients with Finnish type nephrosis [START_REF] Holmberg | Management of congenital nephrotic syndrome of the Finnish type[END_REF], but it should be taken into account that ESRD, which usually occurs much earlier in patients with Pierson syndrome compared to Finnish type nephrosis, will spontaneously limit protein loss. Kidney transplantation is currently the only long-term renal treatment option. There is so far no evidence of recurrence of mesangial sclerosis in the transplant, but the number of successfully transplanted patients who could be monitored for a longer time is very small. Of note, patients with a proven laminin β2 defect should receive careful ophthalmological follow-up, as they are obviously at high risk of retinal detachment even in the absence of significant congenital ocular anomalies. High grade myopia seems to be another common feature developing in infancy and early childhood [START_REF] Bredrup | Ophthalmological aspects of Pierson syndrome[END_REF].

An important differential diagnosis to Pierson syndrome is the nephrosis-microcephaly syndrome (Galloway-Mowat syndrome; GMS; MIM# 251300). Microcephaly which is a key finding in GMS has also been described in some patients with Pierson syndrome [START_REF] Wuhl | Neurodevelopmental deficits in Pierson (microcoria-congenital nephrosis) syndrome[END_REF]. However, in the latter microcephaly is usually not congenital, but may develop during the first year of life. Moreover, other features, such as structural brain anomalies, epilepsy, and hiatus hernia usually allow distinguishing GMS from Pierson syndrome clinically. Notably, however, there have been a few reports on patients with GMS and ocular changes resembling the manifestations in Pierson syndrome [START_REF] Mildenberger | Diffuse mesangial sclerosis: association with unreported congenital anomalies and placental enlargement[END_REF][START_REF] Shapiro | Congenital microcephaly, hiatus hernia and nephrotic syndrome: an autosomal recessive syndrome[END_REF]. Despite the obvious clinical overlap, we have found no evidence that GMS and Pierson syndrome are allelic disorders [START_REF] Dietrich | Analysis of genes encoding laminin beta2 and related Page 24 of 46[END_REF].

There is so far no evidence for isolated ocular anomalies or ocular plus neurologic abnormalities without kidney involvement being caused by mutations in LAMB2.

Isolated microcoria was been reported as an autosomal dominant trait (MIM# 156600).

A provisional locus has been assigned to 13q31-q32 [START_REF] Rouillac | Mapping of a congenital microcoria locus to 13q31-q32[END_REF], thus ruling out LAMB2 as the causative gene for families linked to this locus. However, genetic heterogeneity is not excluded and studies of a possible significance of LAMB2

FUTURE PROSPECTS

Future research will be directed towards the identification of possible modifiers of the phenotype caused by laminin β2 defects. Considering that a lack of expression on the β2-chain probably leads to the expression of aberrant laminin isoforms in the basement membrane rather than the complete absence of laminin (Noakes et al., 1995b), it is tempting to speculate that laminin β1 (or other laminin β isoforms) might to some extent be able to compensate for the lack of β2. Treatment prospects might arise from the knowledge on modifying factors. As a significant proportion of LAMB2 mutations are nonsense mutations, aminoglycosides which are known to be able to induce translational stop codon readthrough [START_REF] Allamand | Drug-induced readthrough of premature stop codons leads to the stabilization of laminin alpha2 chain mRNA in CMD myotubes[END_REF][START_REF] Linde | Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin[END_REF], and which reach high concentrations particularly in the kidney, may be evaluated as a therapeutic target in the future.

A locus-specific mutation database is available at: http://www.med.unimagdeburg.de/LAMB2mutdb. 3 and4).

P37A

Homo

R575Q

Homo sapiens 560 FRPFLDHLIWEAEDT---------------RGQVLDVVERLV 586 Macaca mulatta 559 FRFPLDHLTWEAEDT---------------RGQVLDVVERLV 585

  pattern of organ involvement in Pierson syndrome

  LAMB2 including 12 novel ones, we summarize clinical findings and discuss genotype phenotype correlations. LAMB2 GENE All LAMB2 sequence changes published thus far, together with the novel variants characterized in our laboratories since 2004, are divided into three subgroups and listed in Tables

  whose consequences on splicing could be confirmed on the mRNA level. The precise effect of four additional splice site mutations (c.1036+6_9delTGAG;

  syndrome. None of these patients was found to have a diseasecausing change on the second allele. Nevertheless, we cannot exclude the possibility that the LAMB2 alteration might have contributed to the renal disorder in these patients.

  from the C-terminal part of the protein, represent functional null alleles.

  rescue mechanisms on the transcriptional level

  Among patients with isolated NS LAMB2 mutations are apparently rare

Figure 1 .

 1 Figure 1. LAMB2 gene, laminin β2 protein domains, and distribution of known

Figure 2 .

 2 Figure 2. Age of onset of gross proteinuria / nephrosis (a) and age of onset of end

  , +1 corresponds to the A of the ATG translation initiation codon in the reference sequence

Figure

  Figure 1 RGB colors, 300 dpi 160x119mm (300 x 300 DPI)

lists sequence variants that likely do not lead to development of Pierson

	F o
	r
	P
	e e r
	syndrome or NS. These changes were quoted as probable neutral polymorphisms R e v i e w
	because they have been found either in homozygous state in healthy controls, in
	compound heterozygosity together with a clearly disease causing mutation in healthy
	Pierson syndrome carriers, or together with two bona fide mutations in patients.
	Altogether, 26 polymorphisms have been detected by sequencing of more than 200
	individuals of various ethnic backgrounds (Table 3). Eight of them are known

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	Human Mutation
	LAMB2 mutations
	F o
	r
	P
	e e r
	R e v i e w
	17

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	Human Mutation
	mutations in sporadic cases with isolated microcoria or related iris symptoms as well as
	in families unlinked to 13q31-q32 are warranted.
	F o
	r
	P
	e e r
	R e v i e w
	21

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	Human Mutation
	F o
	r
	P
	e e r
	R e v i e w
	22

Table 1 . Mutations in the LAMB2 gene causing Pierson syndrome and milder disease variants*

 1 The numbering for the nucleotide changes are based on cDNA sequence in accordance with the GenBank entries NM_002292.3, NP_002283.3, and NT_022517.18 (Genome Reference Consortium Human Build 37). Novel mutations are printed in bold.

	Exon/Intron	DNA Variant	a	Predicted and/or	Family-ID	Previous Publications
	Exon 2	c.235_237delGTC	Demonstrated Effect p.V79del	1	Matejas et al. [2006]
	Exon 2	c.240T>G		p.S80R	2	This report
	Exon 3	c.373C>T		p.Q125X	3	Bredrup et al. [2008]
	Exon 4	c.416T>C		p.L139P	4	This report
	Exon 4	c.447_449delTAT	p.I149del	5	Bredrup et al. [2008]
	Exon 5	c.499G>T		p.D167Y	6	Kagan et al. [2008]
	Exon 5	c.536C>T		p.S179F	7	Choi et al. [2008]
	Exon 7	c.736C>T		p.R246W	8, 9, 10, 11,12	Zenker et al. [2004]
	Exon 7	c.737G>A		p.R246Q	13	Hasselbacher et al. [2006]
	Exon 7	c.825T>A		p.Y275X	14	This report
	Exon 8	c.961T>C		p.C321R	15	Hasselbacher et al. [2006]
	Intron 8	c.1036+6_9delTGAG	No mRNA studies	16	This report
	Exon 9	c.1122T>A		p.C374X	17	Zenker et al. [2005]
	Exon 10	c.1241_1242dupCC	p.M415PfsX83	18	Wühl et al. [2007]
	Exon 10	c.1252C>T		p.Q418X	18	Wühl et al. [2007]
	Intron 10	c.1405+1G>A	p.S409X	19, 20	Bredrup et al. [2008]
	Exon 11	c.1477delT		p.C493AfsX4	21, 22	Wühl et al. [2007]
	Exon 11	c.1478delG		p.C493SfsX4	23	Maselli et al. [2009]
	Exon 11	c.1503_1504delAT	p.C502X	24	Choi et al. [2008]
	Exon 13	c.1723C>T		p.R575X	9	Bredrup et al. [2008]
	Exon 14	c.1875_1879delGCGCT	p.L627AfsX5	25	Bredrup et al. [2008]
	Exon 16	c.2067C>G		p.Y689X	17	Zenker et al. [2005]
	Exon 17	c.2283_2286delCTCT	p.S762RfsX29	7	Choi et al. [2008]
	Exon 18	c.2422delG		p.V808WfsX343	26	Bredrup et al. [2008]
	Exon 19	c.2602C>T		p.Q868X	27	Bredrup et al. [2008]
	Exon 21	c.3015delG		p.Q1006NfsX145	28	Zenker et al. [2004]
	Exon 21	c.3015dupG	p.Q1006AfsX49	29	This report
	Exon 21	c.3094C>T		p.R1032X	30	Bredrup et al. [2008]
	Exon 22	c.3174_3175delTG	p.C1058X	27, 31	Bredrup et al. [2008]
	Intron 22	c.3327+2T>C	p.R1037LfsX18	21	Wühl et al. [2007]
	Exon 24	c.3440dupC	p.R1148SfsX27	29	This report
	Exon 24	c.3780_3781delGG	p.E1260DfsX8	19	Bredrup et al. [2008]
	Intron 24	c.3798-2A>C	No mRNA studies	31	Bredrup et al. [2008]
	Exon 25	c.3902delA		p.E1301GfsX58	22	Wühl et al. [2007]
	Intron 25	c.3982+1G>T	No mRNA studies	32	This report
	Exon 26	c.4177C>T		p.L1393F	15	Hasselbacher et al. [2006]
	Exon 27	c.4267delT		p.C1423VfsX29	24	Choi et al. [2008]
	Exon 27	c.4504delA		p.R1502GfsX18	33, 25	Zenker et al. [2004]
	Exon 27	c.4519C>T		p.Q1507X	33	Zenker et al. [2004]
	Exon 27	c.4534delC		p.L1512FfsX8	34	This report
	Intron 27	c.4573+1G>A	No mRNA studies	35	This report
	Exon 28	c.4684C>T		p.R1562X	36	Zenker et al. [2004]
	Exon 28	c.4780dupA	p.R1594KfsX5	37	This report
	Exon 29	c.4804delC		p.Q1602RfsX52	23	Maselli et al. [2009]
	Exon 29	c.4907_4908delAG	p.E1636AfsX22	38	This report
	Exon 30	c.5078delG		p.G1693VfsX21	5	Bredrup et al. [2008]
	Exon 31	c.5182C>T		p.Q1728X	1	Matejas et al. [2006]
	Exon 31	c.5197C>T		p.Q1733X	10	This report
	Exon 31	c.5258dupA		p.E1754GfsX7	39	Zenker et al. [2004]

*

Table 2 . Patients with LAMB2 mutations*: genotype and phenotypic features

 2

	Page 33 of 46					Human Mutation
	Family #	Patient	Genotype a	Total number of	affected individuals	Age of death ( ) /	last follow-up	Age at diagnosis	of nephrotic	proteinuria	Onset of ESRD / Age	at initiation of RRT	Kidney biopsy	result	Microcoria	Other major ocular	abnormalities	Microcephaly	Neurodevelopmental	deficits	Reference
	1	1.1	[p.V79del]+[p.Q1728X]	7	20 y	NA		4 y	NA	-	NA	-	-		Matejas et al.
		1.2				8 y ( )	NA		5 y	DMS	-	C, R, VI	-	-
		1.3				NA	4.5 y	16 y	FSGS	-	Mi, C, R, VI	-	-
	2	2.1	[p.S80R]+ [p.S80R]	1	12 y	6.5		-		atypical DMS	-	My, R	-	-		This report
	3	3.1	[p.Q125X] + ?	1	2 m ( )	1 w		2 w	DMS, MGN	+	L	+	H	Bredrup et al.[2008]
	4	4.1	[p.L139P] + [p.L139P]	3	12 m ( )	3 m	-		DMS?	+	L, VI	-	H, M, C This report
		4.2				1.5 w ( )	< 1 w	-		NA	-	VI	-	NA
		4.3				5 m ( )	< 1 w	-		NA	-	L, VI	-	H, M
	5	5.1	[p.I149del]+[p.G1693VfsX21]	1	2m ( )	1 w		< 1m	NA	+	VI	-	H	Bredrup et al.[2008]
	6	6.1	[p.D167Y]+ [p.D167Y]	2	3.7 y	1 m	19 m	MCD	-	My, R, VI	-	-		Kagan et al. [2008]
	7	7.1	[p.S179F]+[p.S762RfsX29]	1	1.5 y	2 w		1.5 y	FSGS	-	R, VI	-	-		Choi et al. [2008]
	8	8.1	[p.R246W]+[p.R246W]	2	8 m ( )	5 m	-		DMS	+	L	-	H, M	Zenker et al. [2004] 2
		8.2				8 m ( )	1 w		-		DMS	+	L	-	H. M
	9	9.1	[p.R246W]+[p.R575X]	2	16 m	1 w		5 m	NA	+	L, VI	+	H, M, C	Bredrup et al.[2008]
		9.2				1 y ( )	< 1 m	-		DMS	+	N, My	-	H, M, C
	10	10.1	[p.R246W]+[p.Q1733X]	1	2 m	1 m	-		NA	+	NA	-	NA	This report
	11	11.1	[p.R246W]+[p.R246W ]	1	19 m ( )	2 w		13 m	FSGS	+	L	-	M	Bredrup et al.[2008]
	12	12.1	[p.R246W]+[p.R246W]	5	3 m ( )	< 1 m	NA	NA	NA	N	-	NA	This report
							John Wiley & Sons, Inc.

a For cDNA numbering, +1 corresponds to the A of the ATG translation initiation codon in the reference sequence y, year(s); m, month(s); w, week(s); NA, not done / not available; MCD, minimal changes; DMS, diffuse mesangial sclerosis; FSGS, focal and segmental glomerulosclerosis; MGN, membranous glomerulonephritis; O, other; +, present; -, not present / none; Mi, microphthalmia; My, high myopia (> 5 diopters); N, nystagmus; L, abnormal lens (either lenticonus or cataract); R, retinal detachment; VI, severe visual impairment of any cause and despite correcting glasses; H, significant hypotonia / muscular weakness / myasthenia; M, significant motor delay; C, suspected or proven cognitive deficits, speech delay; S, seizures; MR, mental retardaion

Table 3 . Single nucleotide polymorphisms in the LAMB2 gene

 3

	Exon/Intron DNA variant a	Predicted and/or
		demonstrated effect
		on protein

Page 42 of 46 John Wiley & Sons, Inc.

  Alignment of human laminin β2 and laminin β2 orthologues from various species, as well as the human β-laminin paralogues showing the degree of conservation of the respective amino acid residues affected by variants. Conserved amino acids are shown with black background and similar amino acids with a grey background. Alignments were created according to outputs of BLAST (http://www.ncbi.nlm.nih.gov/BLAST) and the Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd).

		Human Mutation
	Matejas et al., Human Mutation	5
	Danio rerio	1416 GGLNCN-GAVAVADTALDRSKHAEKELDKAMGVVEELFK 1453
	Aedes aegypti	1400 GGLTCDKGALTRSEKALQYAKKTEQTIKEKEELADDILR 1438
	human LAMB1	1426 GGPGCG-GLVTVAHNAWQKAMDLDQDVLSALAEVEQLSK 1463
	human LAMB4	1404 RGPGCH-GSLTLSTNALQKAQEAKSIIRNLDKQVRGLKN 1441
	A1765T
	Homo sapiens	1748 KLQRLQELEGTYEENERALESKAAQLDGLE 1777
	Macaca mulatta	1747 KLQRLQELEGTYEENERALESKAAQLDGLE 1776
	Rattus norvegicus 1751 KLQRLQELEGTYEENERELEVKAAQLDGLE 1780
	Canis familiaris	1751 KLQRLQELEGTYEENERALEGKAAQLDGLE 1780
	Gallus gallus Danio rerio Aedes aegypti human LAMB1 human LAMB4	F o r F o r 1742 KLQKLRALEEAYERNERVLDAKVAQLDGLE 1771 1726 KLQRLAELEKDYEENQKVLEGKARQLDGLE 1755 F 1710 QLNKLHDLYKTYEQNQNELGSLETNIQGLT 1739 1736 KLQLLKDLERKYEDNQRYLEDKAQELARLE 1765 o r 1710 KIRRITDLERKIQDLNLSRQAKADQLRILE 1739
		P P P
		e e r e e r e e r
		R e v i e w R e v i e w R e v i e w

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGMENTS

We are indebted to the families of our patients for participation in our study. We are grateful to the members of the GPN (Gesellschaft für Pädiatrische Nephrologie) and Dr. Mike Urban for providing us with DNA samples from patients with syndromic forms of NS, and to Jeffrey H. Miner for helpful discussion. This work was supported by a grant from the German Research Foundation (DFG) to M.Z. (SFB 423, TP A19) John Wiley & Sons, Inc. Human Mutation

John Wiley & Sons, Inc. Human Mutation John Wiley & Sons, Inc.

Human Mutation

Status

Protein domain


Elisa Lobato
email: elobato@mncn.csic.es

Juan Moreno

Santiago Merino

Judith Morales

Gustavo Tomás

Javier Martínez

Rodrigo A Vásquez

Alexandra Kuchar

Erich Möstl

José Luis Osorno

Gustavo Toma

Javier Martı ´nez

Rodrigo A Va ´squez •

Erich Mo

Jose ´luis Osorno

C G E Guglielmo

J Lobato

Á S Moreno

Á J Merino

Á G Morales

Toma

R A Va ´squez

Á E Mo

Arrival date and territorial behavior are associated with corticosterone metabolite levels in a migratory bird

Keywords: Arrival date, Glucocorticoid, Heat shock proteins, Forehead patch, Territorial behavior

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

Introduction

The expression of sexually selected traits may be limited by ecological and physiological constraints [START_REF] Andersson | Sexual selection[END_REF]. In order to unravel the physiological constraints involved in the evolution of sexually selected traits, the proximate mechanisms mediating the expression of these traits need to be understood. The endocrine system provides a mechanism by which the regulation of physiological and behavioral changes may take place [START_REF] Becker | Introduction to behavioral endocrinology[END_REF]. In particular, glucocorticoids are important mediators of allostasis, as they support readjustment of behavior and physiology [START_REF] Mcewen | The concept of allostasis in biology and biomedicine[END_REF]. At low baseline levels, glucocorticoids support daily life processes and, at elevated baseline levels, these hormones support predictable life history stages associated with high allostatic load or energy demand [START_REF] Landys | Actions of glucocorticoids at a seasonal baseline as compared to stressrelated levels in the regulation of periodic life processes[END_REF]). The stress-related levels of glucocorticoids are expected under life-threatening perturbations associated with allostatic overload or energy demands that exceed the requirement of the current life history stage [START_REF] Landys | Actions of glucocorticoids at a seasonal baseline as compared to stressrelated levels in the regulation of periodic life processes[END_REF]. Because of the role of CORT in energy mobilization and distribution and its involvement in cardiovascular, immune, or nervous diseases when secreted at high levels over long periods [START_REF] Sapolsky | How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions[END_REF][START_REF] Sapolsky | Stress hormones: good and bad[END_REF], it is possible that this hormone may also be involved in the expression of costly behaviors and characters and may provide a mechanistic link between the expression of these traits and diverse selective processes [START_REF] Husak | Stress hormones and mate choice[END_REF]. In fact, glucocorticoids have been suggested to be under the direct or indirect influence of sexual selection (reviewed in [START_REF] Husak | Stress hormones and mate choice[END_REF].

In migratory species, the pattern of arrival to the breeding grounds after migration and the defense of territories may be under the influence of sexual selection. The study of the variation of glucocorticoid levels in association with these patterns could provide important information about the involvement of these hormones in the expression of sexually selected traits. Migration constitutes a predictable life-history stage that imposes high energetic demands on birds and may be associated with elevated baseline levels of glucocorticoids [START_REF] Landys-Ciannelli | Baseline and stress-induced plasma corticosterone during long-distance migration in the bartailed godwit, Limosa lapponica[END_REF]. In several species, migration is associated with elevated levels of corticosterone (CORT), the predominant glucocorticoid in birds [START_REF] Holberton | Modulation of the adrenocortical stress response in neotropical migrans during autumn migration[END_REF][START_REF] Romero | Season and migration alters the corticosterone response to capture and handling in an artic migrant, the white-crowned sparrow (Zonotrichia leucophrys gambelli)[END_REF][START_REF] Holberton | Changes in patterns of corticosterone secretion concurrent with migratory fattening in a Neotropical migratory bird[END_REF][START_REF] Piersma | Baseline corticosterone peaks in shorebirds with maximal energy stores for migration: a general preparatory mechanism for rapid behavioral and metabolic transitions?[END_REF], and this association could be indicative of a role of this hormone in promoting energy acquisition at this stage. Moreover, CORT is suggested to play a regulatory role specifically associated with migratory flight [START_REF] Landys-Ciannelli | Baseline and stress-induced plasma corticosterone during long-distance migration in the bartailed godwit, Limosa lapponica[END_REF][START_REF] Landys | Plasma corticosterone increases during migratory restlessness in the captive white-crowned sparrow Zonotrichia leucophrys gambelli[END_REF]). In addition, hard environmental conditions experienced during migration could elicit high energy requirements. Also, the pattern of arrival on the breeding sites after migration may be related to differences in the exposure to hard environmental conditions. Particularly, early-arriving individuals may be subjected to harsher weather conditions or food scarcity [START_REF] Møller | Phenotype-dependent arrival time and its cosequences in migratory birds[END_REF][START_REF] Brown | Weather-mediated natural selection on arrival time in cliff swallows (Petrochelidon pyrrhonota)[END_REF][START_REF] Newton | Weather-related mass-mortality events in migrants[END_REF]. At the same time, early arrival may be advantageous for acquiring high quality territories and may increase the probability of mating (e.g., Potti and Montalvo 1991a;[START_REF] Kokko | Competition for early arrival in migratory birds[END_REF][START_REF] Smith | Arrival timing and seasonal reproductive performance in longdistace migratory landbird[END_REF]. Therefore, an early arrival after migration is presumably a character positively influenced by sexual selection that entails costs derived from severe environmental conditions [START_REF] Darwin | The descent of man, and selection in relation to sex. Murray, London Davies NB[END_REF][START_REF] Fisher | The genetical theory of natural selection[END_REF][START_REF] Møller | Phenotype-dependent arrival time and its cosequences in migratory birds[END_REF][START_REF] Kokko | Competition for early arrival in migratory birds[END_REF]). The study of the levels of CORT metabolites just after migration in relation to arrival date may give us insight into the physiological mechanisms that support the elevated energy demand associated with early arrival.

Territorial defense is also a costly activity closely related to sexual selection [START_REF] Andersson | Sexual selection[END_REF]. The defense of territories may be carried out by means of visual and auditory signals such as flight displays, songs, or calls, and may even involve chasing or attacking. Territorial defense may be costly due to elevated energy expenditure (i.e., Davies 1980) and to other costs associated with this behavior such as less time available for foraging and higher predation or injury risk [START_REF] Krebs | Optimal foraging, predation risk and territory defence[END_REF]. Recent studies have suggested that the secretion of CORT may facilitate energy mobilization during continuous aggressive or defensive behaviors [START_REF] Moore | Stress, reproduction and adrenocortical modulation in amphibians and reptiles[END_REF][START_REF] Goymann | Allostatic load, social status and stress hormones: the cost of social status matter[END_REF][START_REF] Berger | Corticosterone suppresses immune activity in territorial Gala ´pagos marine iguanas during reproduction[END_REF]. It has also been reported that territorial aggression in birds causes a rapid increase in CORT levels, suggesting that this hormone may play a specific role in the regulation of territorial behavior [START_REF] Landys | Hormonal responses to male-male social challenge in the blue tit Cyanistes caeruleus: single-broodedness as an explanatory variable[END_REF]. In fact, studies on rodents indicate that glucocorticoids may act directly on the facilitation of aggressive behaviors [START_REF] Hayden-Hixson | Cortisol exerts site-, contextand dose-dependent effects on agonistic responding in hamsters[END_REF][START_REF] Haller | Acute effects of corticosterone lack specificity but showed marked context dependency[END_REF].

Other secondary sexually selected traits such as plumage characteristics may be condition-dependent and costly to produce or maintain (e.g., [START_REF] Gustafsson | Trade-offs between life-history traits and a secondary sexual character in male collared flycatchers[END_REF]. One of the physiological costs that the expression of secondary sexual characters may impose is that derived from the impairment of the functioning of the immune system [START_REF] Saino | Sexual ornamentation and immunocompetence in the barn swallow[END_REF][START_REF] Kilpimaa | Trade-offs between sexual advertisement and immune function in the pied flycatcher (Ficedula hypoleuca)[END_REF]). It has been suggested that this cost may arise due to the immunosuppressive effects of sex steroids that facilitate the expression of secondary sexual traits [START_REF] Folstad | Parasites, brigth males and the immunocompetence handicap[END_REF] and also as a result of the allocation of resources to different costly traits [START_REF] Ra ˚berg | On the adaptive significance of stress-induced immunosupression[END_REF]. It is also reported that dominance may explain variation in glucocorticoid levels, as some studies have found that subordinates show high baseline glucocorticoid levels (i.e., [START_REF] Schwabl | Social status, circulating levels of hormones, and competition for food in winter flocks of the whitethroated sparrow[END_REF][START_REF] Belthoff | Plumage variation, plasma steroids and social dominance in male house finches[END_REF]; see also [START_REF] Creel | Social dominance and stress hormones[END_REF] for noncooperative breeders), while other studies did not find an association between social rank and baseline hormone levels (i.e., [START_REF] Holberton | Status signaling in dark-eyed juncos, Junco hyemalis: plumage manipulations and hormonal correlates of dominance[END_REF][START_REF] Pravosudov | The relationship between dominance, corticosterone, memory, and food caching in mountain chickadees (Poecile gambeli)[END_REF]. In general, as reviewed by [START_REF] Goymann | Allostatic load, social status and stress hormones: the cost of social status matter[END_REF], social conflicts may increase the energetic demands which is reflected in increased concentrations of glucocorticoids, and it is the relative allostatic load of social status that may determine the glucocorticoid levels of dominants and subordinate individuals. As social dominance may be related to early arrival and territorial behavior, it is necessary to control for social dominance in any analysis testing to see if early arrival and territorial aggression are associated with elevated CORT levels.

The elevated secretion of CORT in high demanding situations may be related to other physiological parameters that support processes associated with elevated energy requirements. For example, the cellular levels of heat shock proteins (HSPs) are good indicators of the exposition to different stressors in several species [START_REF] Merino | Increase in a heat shock protein from blood cells in response to parasitism of nestling house martins (Delichon urbica): an experimental approach[END_REF][START_REF] Merino | Blood stress protein levels in relation to sex and parasitism of barn swallows (Hirundo rustica)[END_REF][START_REF] Feder | Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology[END_REF][START_REF] Martı ´nez | Stress response to cold in Trichinella species[END_REF][START_REF] Moreno | Heterophil/lymphocyte ratios and the heat-shock protein levels are related to growth in nestling birds[END_REF][START_REF] Toma ´s | Stress protein levels and blood parasite infection in blue tits (Parus caeruleus): a medication field experiment[END_REF]; for a review, see [START_REF] Sørensen | The evolutionary and ecological role of heat shock proteins[END_REF]. HSPs are molecular chaperones that protect proteins from damage by means of their role in the maintenance of the functional conformation of protein structures and degradation of damaged proteins [START_REF] Schlesinger | Heat shock proteins[END_REF][START_REF] Sørensen | The evolutionary and ecological role of heat shock proteins[END_REF]. They are constitutively expressed to assist daily metabolic processes involving synthesis and degradation of proteins [START_REF] Feige | HSP70-a multigene, multistructure, multifunction with potential clinical applications[END_REF]. The inducible transcription of heat shock genes is associated with the exposure to situations that destabilize cell homeostasis and produce alterations in protein folding [START_REF] Morimoto | Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators[END_REF]. The increase in HSPs after an environmental challenge may last longer than other physiological responses, such as changes in glucocorticoid levels [START_REF] Herring | The role of stress proteins in the study of allostatic overload in birds: use and applicability to current studies in avian ecology[END_REF]. Moreover, HSP measure is independent of handling stress [START_REF] Washburn | Effects of handling on heat shock protein expression in rainbow trout (Oncorhynchus mykiss)[END_REF] and appropriate to measure long-term stress [START_REF] Herring | The role of stress proteins in the study of allostatic overload in birds: use and applicability to current studies in avian ecology[END_REF]. Similarly, in order to avoid problems induced by a rapid increase of CORT after handling of birds [START_REF] Romero | Corticosterone responses in wild birds: the importance of rapid initial sampling[END_REF], we used non-invasive measures of CORT metabolites in excreta of male Pied Flycatchers (Ficedula hypoleuca) that provide a hormonal measure independent of capture stress. Steroid metabolites concentrations in excreta constitute an integrated parameter of steroid levels over a long period of time [START_REF] Goymann | Noninvasive monitoring of hormones in bird droppings: physiological validation, sampling, extraction, sex differences and the influence of diet on hormone metabolite levels[END_REF] and may provide an accurate measure of long-term glucocorticoid levels (see [START_REF] Millspaugh | Use of fecal glucocorticoid metabolite measures in conservation biology research: considerations for application and interpretation[END_REF]. Therefore, the study of the association between HSPs and CORT metabolite levels may provide information about the general physiological state associated with the expression of high levels of glucocorticoids in a given situation.

In the present study, we tested the hypotheses that early arrival to the breeding grounds and territorial male-male competition are associated with elevated baseline levels of CORT metabolites in a tropical migrant, the Pied Flycatcher. We analyzed the association of the levels of CORT metabolites of male Pied Flycatchers just after arriving on the breeding area with timing of arrival. This test is controlled for the associations of social dominance with CORT metabolite levels by introducing two plumage traits that have been linked to male dominance in Pied Flycatchers, the white forehead patch size (Pa ¨rt and Qvarnstro ¨m 1997) and the amount of black plumage coloration [START_REF] Ja ¨rvi | Evolution of variation in male secondary sexual characteristics. A test of eight hypotheses applied to pied flycatchers[END_REF]. We also considered the HSP levels in the analyses as a measure of physiological response that may assist processes of protein synthesis and degradation during situations of elevated energy requirement. We also evaluated the associations of the levels of CORT metabolites of male Pied Flycatchers with two measures of territorial behavior, approaching and singing to an intruder. We considered the size of the forehead patch in the statistical analyses, as males with experimentally enlarged patches have been shown to express more intense territorial behavior [START_REF] Osorno | Evidence for differential maternal allocation to eggs in relation to manipulated male attractiveness in the pied flycatcher (Ficedula hypoleuca)[END_REF].

Methods

Capture and handling

The study was conducted in 2003 in a deciduous forest of Pyrennean oak Quercus pyrenaica at 1,200 m a.s.l. in the vicinity of La Granja, Segovia province, central Spain (40°53 0 N, 4°01 0 W.). The Pied Flycatcher is a small (12-13 g) hole-nesting migratory passerine of European woodlands [START_REF] Lundberg | The pied flycatcher[END_REF]. The population of Pied Flycatchers in the study area has been monitored since 1991 (see [START_REF] Sanz | Climate change and fitness components of a migratory bird breeding in the Mediterranean region[END_REF]. From mid-April, the study area was exhaustively searched for newly arrived males. The Pied Flycatcher is a tropical migrant that has a short time window available for reproduction. Males sing immediately upon arrival on the breeding grounds and get involved in territorial competition, in order to achieve and establish their territory, and start breeding as soon as possible [START_REF] Lundberg | The pied flycatcher[END_REF]; own observations). The song of Pied Flycatchers is unmistakable and their black and white plumage is very conspicuous. This makes their detection easy to an experienced observer, particularly at the beginning of the reproductive season, when the leaves of the oaks in the forest have not yet sprouted and the visibility is very good. Also, given that the territory defended by these males is restricted to the nestbox [START_REF] Lundberg | The pied flycatcher[END_REF], and the fact that they sing close to these boxes, makes their detection even easier. In our study, six expert ornithologists combed the whole study area before any male had arrived in a daily, systematic and exhaustive search for newly arrived Pied Flycatchers. We thus assumed that the first day a male was observed singing was the day of his arrival. Pied Flycatcher males were captured with nestbox traps on their first day of observation in the study area. They were identified or ringed in cases when they were not previously ringed, weighed with a Pesola spring balance (accuracy of 0.05 g), and their tarsus length was measured with a digital calliper to the nearest 0.01 mm (apparatus precision). As a measure of body condition, we used the ratio body mass/cube of tarsus length. We measured the white forehead patch of rectangular shape that Ficedula males exhibit [START_REF] Osorno | Evidence for differential maternal allocation to eggs in relation to manipulated male attractiveness in the pied flycatcher (Ficedula hypoleuca)[END_REF]). The patch size was estimated by multiplying the width and height as measured to the nearest 0.01 mm with a digital calliper. The percentage of black feathers in the head and mantle was scored as 'blackness', which is the exact inverse of the brownness score used by Potti and Montalvo (1991b). This score is highly correlated (Potti and Montalvo 1991b) with [START_REF] Drost | U ¨ber das Brutkleid ma ¨nnlicher Trauerfliegenschna ¨pper Muscicapa hypoleuca[END_REF] scale from I (darkest) to VII (lightest). Blackness scores were converted to 10-point intervals, with values ranging from 0 (0-10%) to 9 (90-100%). Male age was determined according to plumage characteristics following [START_REF] Svensson | Identification guide to European passerines[END_REF] or by previous ringing. All males were individually marked with color rings to allow identification in behavioral observations of territorial response on the following days (see below). At the time of capture, males were subjected to a forehead patch manipulation with the aid of black or white permanent paint markers in the context of a previously published study on the role of forehead patch size on differential maternal allocation of resources to eggs [START_REF] Osorno | Evidence for differential maternal allocation to eggs in relation to manipulated male attractiveness in the pied flycatcher (Ficedula hypoleuca)[END_REF]. Males were randomly assigned to one of three experimental groups: enlarged-patch, control, and reducedpatch. The modified patch size was measured in all cases. This modified patch size has only been considered in analyses explaining territorial response (see below), as these behavioral measures were taken after the treatment was performed, and they could be affected by the manipulation. The original patch size and not the modified patch size was considered in analyses explaining the variation in CORT metabolites as all explanatory variables considered had been taken before the treatment was performed.

In order to measure the concentration of excreted CORT metabolites, fresh excreta of male Pied Flycatchers were collected in 1.5-ml plastic vials, stored in cool bags with cold-blocks (not exceeding 15°C) and frozen at -20°C (1-5 h later) until laboratory analyses. Droppings were obtained by spontaneous defecation during the process of ringing. In order to increase sample size, when they did not defecate during the ringing process, they were introduced for some minutes into a dark wooden box to collect faeces from aluminium foil placed on the bottom of the box. To measure the levels of HSPs, a blood sample of approximately 80 ll was obtained by brachial venipuncture. Blood samples were centrifuged (2,000g, 5 min) in a field centrifuge (MiniCentrifuge, Cat. No 1201-220V; Labnet, Woodbridge, NJ, USA).The cellular and plasma fractions were separated and cold-stored until frozen at the end of the day not exceeding 8 h from collection which allows a reliable measure of stress proteins as shown by Toma ´s et al. (2004). The blood cellular fraction was used for further analyses.

Territorial response observations

A few days after capture (5.5 ± 0.8, mean ± SE, n = 36) and before initiation of nest building in their nestbox by females, the territorial response of each male was measured by observations during 10 min. A digital commercial recorder (Mundi Sound PQ10; Great Britain) playing Pied Flycatcher song was placed on the ground below the nestbox defended by a certain color-banded male (for details of territorial response observations, see [START_REF] Osorno | Evidence for differential maternal allocation to eggs in relation to manipulated male attractiveness in the pied flycatcher (Ficedula hypoleuca)[END_REF]). We used song from a single bird in all cases, so the responses to it have to be interpreted cautiously [START_REF] Kroodsma | Pseudoreplication in playback experiments, revisited a decade later[END_REF]. The recorder played a sequence of several vocalizations of Pied Flycatcher song of one individual from an unspecified Pied Flycatcher population. It is highly unlikely that the song recorded would be individually recognizable. Song rate was not atypical (about 40 strophes per 5 min) and falls within the natural range of an adult Pied Flycatcher male (e.g., average of 37.9 strophes per 5 min; [START_REF] Gottlander | Variation in the song rate of the male pied flycatcher Ficedula hypoleuca: causes and consequences[END_REF]. We assume that responses to this specific song reflect responses of males to circumstances when territorial intrusions may be associated to CORT metabolite levels, an assumption which may be unwarranted. A single song bout was used only once on each subject and gave rise to a unique data point. It is not likely that neighbors of subjects could hear the recording as the digital recorder was played at a volume that could presumably only be heard at a short distance from the subject. The volume of the loudspeaker was adjusted so that it could only be heard at a short distance from the nestbox of the subject male (at 50 m, the average distance between nestboxes in the study area, the playback song could not be heard by us). Each male's response was registered by an observer (always the same) hidden at a distance of at least 15 m. The approximation (whether a male approached to the playback recorder until almost touching it or not) and singing (whether males sang or not) behavior was registered during the 10-min playback observations. A second observation of territorial response was measured 13.2 (±1.0, n = 34) days after capture (see [START_REF] Osorno | Evidence for differential maternal allocation to eggs in relation to manipulated male attractiveness in the pied flycatcher (Ficedula hypoleuca)[END_REF], but for the study of the association of CORT metabolite levels and territorial response we only used the first observation, as this avoids problems of habituation, and it was temporally closer to the CORT metabolites measure.

Measurement of excreted CORT metabolites

In small birds, the study of droppings as a source of information of hormonal status is particularly useful due to the small volume of blood that can be obtained and the analytical disadvantages of small volumes of plasma. Steroid metabolites concentrations in excreta represent an integral measure of hormone metabolites [START_REF] Goymann | Noninvasive monitoring of hormones in bird droppings: physiological validation, sampling, extraction, sex differences and the influence of diet on hormone metabolite levels[END_REF]. Radiometabolism studies have determined that the length of the integral may range a few hours in birds (reviewed in [START_REF] Palme | Stress hormones in mammals and birds. Comparative aspects regarding metabolism excretion and noninvasive measurement in fecal samples[END_REF]. Thus, in our study, the integrated measure of CORT metabolites from samples obtained of birds on the day of their arrival refers to the CORT metabolite levels at the last stage of migration and the onset of territory acquisition. Excreta were homogenized, and steroids were extracted as described in [START_REF] Lobato | Corticosterone metabolites in blue tits and pied flycatcher droppings: effects of brood size, ectoraparasites and temperature[END_REF]. The concentration (ng/g) of excreted immunoreactive CORT metabolites was measured by means of the cortisone enzyme immunoassay described by [START_REF] Rettenbacher | Measurement of corticosterone metabolites in chicken droppings[END_REF], validated for Pied Flycatchers by [START_REF] Lobato | Corticosterone metabolites in blue tits and pied flycatcher droppings: effects of brood size, ectoraparasites and temperature[END_REF]. Enzyme immunoassays were performed on antirabbit-IgG-coated microtitre plates using the double antibody technique and biotinylated steroids as labels. Details of the procedure of the assay are described in Mo ¨stl et al. (2002).

Determination of heat shock proteins

The levels of HSP60 were determined from the blood cellular fraction by means of western blotting following the methodology described in Toma ´s et al. (2004). Protein bands were quantified using image analysis software for Windows (Scion Corporation) and immunoreactivities (arbitrary units) were obtained using the formula: Immunoreactivity = area 9 mean intensity of the band.

Statistics

We first evaluated the independent variables that significantly predicted the levels of CORT metabolites of male Pied Flycatchers just after arrival from migration. The variables considered in the analyses were: arrival date (1 = 1 April), body condition, HSP60 level, forehead patch size, plumage blackness, and male age. All these variables were introduced simultaneously as independent variables, and corticosterone metabolites as dependent variable, in a GLM (generalized linear models) analysis, using STATISTICA software. No interaction terms were considered. We systematically removed the variable with the lowest F value and ran the model again until we reached a model with all covariates significantly associated with CORT metabolite levels at P value \ 0.05.

We secondly analyzed if CORT metabolite levels predicted territorial behavior measured 5.5 ± 0.8 days after the sampling of droppings. We included in the analyses the arrival date, the original patch size, the modified forehead patch, and the levels of CORT metabolites as potential explanatory variables in GLM models with binomial distribution, with singing behavior (singing or not) and approaching behavior (approaching to playback recorder or not) as dependent variables in respective analyses. We systematically removed the covariates with lower associated Wald statistic until all covariates were significantly associated (P value \ 0.05) with the respective dependent variable.

Results

The final model explaining the variance in the concentration of excreted CORT metabolites after arriving from migration included arrival date, male HPS60 level, and the original forehead patch size as significant explanatory variables (n = 18, HSP60 level: F 1,14 = 6.53, P = 0.023, b = 0.54; arrival date: F 1,14 = 5.48, P = 0.035, b = -0.51; forehead patch: F 1,14 = 5.09, P = 0.041, b = -0.46). The adjusted r 2 of the model was 0.34. The residuals of the model were normally distributed. The HSP60 level was positively associated with the level of excreted CORT metabolites (Fig. 1a). Early-arriving individuals showed higher concentrations of excreted CORT metabolites (Fig. 1b). Larger forehead patches were associated with lower concentrations of CORT metabolites (Fig. 1c).

The only variable that significantly explained singing behavior was CORT metabolite levels (Wald = 4.77, P = 0.029). Males that sang during the 10-min observation period when playback was played showed higher levels of CORT metabolites at the moment of arrival from migration than males that did not sing during this period (Fig. 2a). Similarly, the only variable that significantly predicted approaching behavior was CORT metabolite levels (Wald = 4.55, P = 0.033). Males that approached the playback recorder during the playback showed higher levels of CORT metabolites than males that did not approach the recorder (Fig. 2b).

Discussion

Our data show that elevated CORT metabolite concentrations are positively associated with heat shock protein levels (HSP60) in Pied Flycatcher males just after arriving on their breeding area. Heat shock and glucocorticoid responses may be elicited under different environmental challenges, although the type or level of stimulus needed to induce each response may differ [START_REF] Herring | The role of stress proteins in the study of allostatic overload in birds: use and applicability to current studies in avian ecology[END_REF]. Also, these compounds may be synthesized to support daily metabolic processes and predictable lifehistory stages associated with high energy demand (i.e., [START_REF] Feige | HSP70-a multigene, multistructure, multifunction with potential clinical applications[END_REF][START_REF] Landys | Actions of glucocorticoids at a seasonal baseline as compared to stressrelated levels in the regulation of periodic life processes[END_REF]. The positive association between CORT metabolites and HSP60 levels found in our study may arise because migration itself or the differential conditions experienced during migratory flight trigger both physiological responses that therefore appeared correlated just when males arrived on the breeding grounds. It has been suggested that elevated glucocorticoid levels may regulate the synthesis of some HSPs (HSP27: [START_REF] Barr | Glucocorticoids regulate the synthesis of HSP27 in rat brain slices[END_REF]and HSP72: Fleshner et al. 2004). Also, it is known that high demanding activities such as physical exercise up-regulate proteins involved in energy metabolism and also specific proteins such as molecular chaperones, particularly HSP60 [START_REF] Ding | Exercise affects energy metabolism and neural plasticity-related proteins in the hippocampus as revealed by proteomic analyses[END_REF][START_REF] Thambirajah | Differential heat shock protein responses to strenuous standardized exercise in chronic fatigue syndrome patients and matched healthy controls[END_REF]. This HSP may provide cellular protection during changes in protein metabolism and assist protein formation and degradation in anabolic and catabolic processes [START_REF] Scha ¨fler | Overexpression of heat shock protein 60/10 in myocardium of patiens with chronic atrial fibrillation[END_REF]. In an avian model, adrenocorticotropic (ACTH) hormone administration increased corticosterone secretion and also other parameters such as haematocrit or the blood concentration of haemoglobin [START_REF] Olanrewaju | Stress and acid-base balance in chickens[END_REF]. This suggests that birds increased erythropoiesis to satisfy the need for O 2 to support energy production after ACTH infusion [START_REF] Olanrewaju | Stress and acid-base balance in chickens[END_REF]. Considering that HSP60 was detected in the cellular fraction of blood in our study, our HSP measure indicates, for the most part, the level of these proteins in erythrocytes. Therefore, the positive association of CORT and HSP found here may reflect the elevated need of molecular chaperones in processes involving elevated haemoglobin synthesis during increased need for O 2 to supply energy production in high demanding situations requiring CORT secretion. In wild, incubating Common Eiders (Somateria mollisima), HSP levels (HSP60 and HSP70) and CORT levels tended to vary in opposite directions with incubation stage, although a nonsignificant association was found between CORT and HSPs (HSP70 and HSP60; [START_REF] Bourgeon | Fasting-induced changes of immunological and stress indicators in breeding female eiders[END_REF], which could suggest that the potential association of these physiological parameters may be dependent on the specific stage of the reproductive cycle and the ecological context. Given the role of glucocorticoids in the increase of energy mobilization, high hormone secretion may drive a decrease in body mass. In fact, a negative relationship between CORT levels and body condition has been observed in many studies, frequently in association with adverse conditions (i.e., [START_REF] Cherel | Fasting in king penguin. I. Hormonal and metabolic changes during breeding[END_REF][START_REF] Marra | Corticosterone levels as indicators of habitat quality: effects of habitat segregation in a migratory bird during the non-breeding season[END_REF][START_REF] Kitaysky | Dynamics of food availability, body condition and physiological stress response in breeding black-legged kittiwakes[END_REF][START_REF] Breuner | Integrating stress physiology, environmental change, and behavior in free-living sparrows[END_REF], while other studies have not found an association between CORT levels and body condition [START_REF] Lorme ´e | Sex-specific patterns in baseline corticosterone and body condition changes in breeding red-footed boobies Sula sula[END_REF][START_REF] Poisbleau | Linear social dominance hierarchy and corticosterone responses in male mallard and pintails[END_REF]. In general, a negative association with body condition is expected at stress-related levels of glucocorticoids and not at seasonal glucocorticoid levels (reviewed at [START_REF] Landys | Actions of glucocorticoids at a seasonal baseline as compared to stressrelated levels in the regulation of periodic life processes[END_REF]. Our data show no relationship between CORT metabolites and body condition, suggesting that CORT metabolites are measured at a baseline level in our study. Moreover, male Pied Flycatchers were sampled at the last stage of migration, and it has been reported in other species of migrating birds that CORT levels circulate at elevated baseline levels during migration and not at stressed-related levels [START_REF] Landys-Ciannelli | Baseline and stress-induced plasma corticosterone during long-distance migration in the bartailed godwit, Limosa lapponica[END_REF].

As glucocorticoids promote the mobilization of energy stores, their secretion may facilitate the expression of energetically expensive reproductive functions. Migration and territorial competition in males are energetically demanding activities influenced by sexual selection that may be associated with the secretion of glucocorticoid hormones. Our study shows that an early arrival from migration in the Pied Flycatcher may require a high mobilization of energy in this tropical migrant, as indicated by the elevated baseline levels of CORT metabolites observed in early individuals. Arriving early on the breeding area provides benefits in terms of sexual selection, as early individuals show higher probabilities of acquiring the best mates (Potti and Montalvo 1991a). However, an early arrival may not be exempt from costs as it may be associated with hard weather conditions on migration or the onset of reproduction [START_REF] Kokko | Competition for early arrival in migratory birds[END_REF][START_REF] Brown | Weather-mediated natural selection on arrival time in cliff swallows (Petrochelidon pyrrhonota)[END_REF]. Thus, the variation in the exposure to harsh conditions during migration could translate into individual variation in physiological variables directed at surmounting high energy demanding situations. In fact, our study shows that early arriving individuals have higher baseline CORT metabolite levels, which could indicate that an early arrival may be associated with an increased allostatic load. We may speculate that, if the advance of arrival was notable, it could be that the allostatic load of early individuals increased and the CORT responses escalated until stressrelated levels were reached. In this case, the stress-related hormone levels could lead to immunosuppressive and other negative effects on the organism [START_REF] Sapolsky | How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions[END_REF][START_REF] Sapolsky | Stress hormones: good and bad[END_REF]) that would impose physiological costs on too-early individuals [START_REF] Kokko | Competition for early arrival in migratory birds[END_REF][START_REF] Moreno | Sex differences in cell-mediated immunity in the magellanic penguin Spheniscus magellanicus[END_REF].

Dominant high-quality individuals may require lower CORT levels than subdominants for the same level of migratory exertion [START_REF] Creel | Social dominance and stress hormones[END_REF]. The white forehead patch in Ficedula males has been associated with increased mating advantage (Potti and Montalvo 1991a;[START_REF] Qvarnstro ¨m | Adaptive plasticity in mate preference linked to differences in reproductive effort[END_REF]; but see [START_REF] Dale | Population divergence in sexual ornaments: the white forehead patch of Norwegian pied flycatchers is small and unsexy[END_REF]. The size of this patch is a condition-dependent character that responds to past reproductive effort in the related Collared Flycatcher (Ficedula albicollis) [START_REF] Gustafsson | Trade-offs between life-history traits and a secondary sexual character in male collared flycatchers[END_REF]. Also, male behavior has been reported to be affected by the forehead patch size, as individuals with experimentally enlarged patches have been shown to display a more intense territorial behavior [START_REF] Osorno | Evidence for differential maternal allocation to eggs in relation to manipulated male attractiveness in the pied flycatcher (Ficedula hypoleuca)[END_REF]). The negative association of baseline CORT metabolite levels and forehead patch size could arise due to the role of forehead patches in male contest. It has been reported that male Collared Flycatchers with larger patches are more successful in gaining territories (Pa ¨rt and Qvarnstro ¨m 1997). Black male plumage has also been related to mating advantage [START_REF] Ja ¨rvi | Evolution of variation in male secondary sexual characteristics. A test of eight hypotheses applied to pied flycatchers[END_REF][START_REF] Saetre | Female pied flycatchers prefer brightly coloured males[END_REF]; but see [START_REF] Alatalo | Can female preference explain sexual dichromatism in the pied flycatcher Ficedula hypoleuca?[END_REF]). However, CORT metabolites were not associated with this ornamental trait. On the contrary, our data show that forehead patch size is inversely related to CORT metabolites, which suggests that dominance as expressed by bigger forehead patches is associated to low baseline CORT metabolites.

Territorial defense comprises a variety of aggressive and advertisement behaviors that may be energetically demanding. One of the aspects associated with territorial defense is singing, and different song characteristics have been shown to be limited by ecological and physiological constraints (i.e., [START_REF] Saino | Song and immunological condition in male barn swallows (Hirundo rustica)[END_REF][START_REF] Gil | The honesty of bird song: multiple constraints for multiple traits[END_REF][START_REF] Garamszegi | Indicators of physiological stress and the elaboration of sexual traits in the collared flycatcher[END_REF]. Elevated levels of CORT have been observed in territorial males of different species, probably as a reflection of the high energetic requirements of intense aggressive behaviors [START_REF] Moore | Stress, reproduction and adrenocortical modulation in amphibians and reptiles[END_REF][START_REF] Goymann | Allostatic load, social status and stress hormones: the cost of social status matter[END_REF][START_REF] Berger | Corticosterone suppresses immune activity in territorial Gala ´pagos marine iguanas during reproduction[END_REF]. It has also been reported that territorial challenges may affect CORT levels [START_REF] Sorenson | Hormonal dynamics during mate choice in the northern pintail: a test of the challenge hypothesis[END_REF][START_REF] Van Duyse | Opposite changes in plasma testosterone and corticosterone levels following a simulated territorial challenge in male great tits[END_REF][START_REF] Landys | Hormonal responses to male-male social challenge in the blue tit Cyanistes caeruleus: single-broodedness as an explanatory variable[END_REF]. Our data indicate that approaching a playback recorder and singing as a response to a playback of an intruder were positively associated with high CORT metabolite levels of Pied Flycatchers. The association between CORT metabolites and behavior may have also arisen through mechanisms not necessarily associated with the role of CORT in facilitating energy mobilization to support territorial behavior. It could be that the association of the history of CORT secretion as measured through CORT metabolites with territorial behavior registered 5 days later may have arisen due to a direct role of CORT on the promotion of aggressive behavior. In fact, studies in mammals have shown that glucocorticoids may have a direct regulatory role on the promotion of aggressive behaviors [START_REF] Hayden-Hixson | Cortisol exerts site-, contextand dose-dependent effects on agonistic responding in hamsters[END_REF][START_REF] Haller | Acute effects of corticosterone lack specificity but showed marked context dependency[END_REF][START_REF] Mikics | Genomic and non-genomic effects of glucocorticoids on aggressive behavior in male rats[END_REF][START_REF] Mikics | The effect glucocorticoids on aggressiveness in established colonies of rats[END_REF].

Overall, our study suggests that early arrival in males is associated with elevated CORT metabolite levels and implies a high allostatic load. Also, dominance in males as expressed by a larger white forehead patch is associated with lower CORT metabolite levels. Our data suggest that high levels of CORT metabolites may facilitate an intense territorial competition in males. Recently, [START_REF] Husak | Stress hormones and mate choice[END_REF] have reviewed the evidence showing that glucocorticoids may be under the direct or indirect influence of sexual selection. We show here that sexual selection for early arrival and territory acquisition are associated with baseline CORT metabolite levels in males. Thus, male-male competition may be a factor affecting observed baseline glucocorticoid levels in migratory birds.

Zusammenfassung

Fig. 1 a

 1 Fig. 1 a Association between HSP60 levels and the residuals of the regression of CORT metabolites on arrival date and forehead patch size. b Association between arrival date and the residuals of the

Fig. 2

 2 Fig.2Mean ± SE CORT metabolites concentration of males that expressed singing behavior (a) or approximation (b) behavior when exposed to a playback of a conspecific male under their nestbox

  Beim ma ¨nnlichen Trauerschna ¨pper (Ficedula hypoleuca) stehen Ankunftsdatum und Territorealverhalten mit den Corticosteroidmetabolitenwerten in Zusammenhang Glucocorticoide fo ¨rdern die Mobilisation von Energiereserven und ko ¨nnten die Expression energetisch kostspieliger Funktionen erleichtern. Bei Zugvo ¨geln sind eine fru ¨he Ankunft im Brutgebiet und die Konkurrenz um Territorien energetisch anspruchsvolle Aktivita ¨ten, die durch erho ¨hte Baseline-Glucocorticoidwerte gefo ¨rdert werden ko ¨nnten. Hier haben wir die Zusammenha ¨nge zwischen der Ankunftszeit ma ¨nnlicher Trauerschna ¨pper (Ficedula hypoleuca) und den Baseline-Werten ausgeschiedener Corticosteron(CORT)metabolite direkt nach der Ankunft im Brutgebiet ausgewertet, unter Beru ¨cksichtigung von Ornamenten, die den sozialen Status anzeigen (z.B. Gro ¨ße des Stirnflecks, schwarze Gefiederfa ¨rbung), sowie Hitzeschockproteinwerten (HSP60). Wir haben einen positiven Zusammenhang zwischen CORT-Metaboliten und den Werten von HSP60-Proteine, die unter schwierigen, die Zellhomo ¨ostase beeintra ¨chtigenden Umweltbedingungen synthetisiert werden, festgestellt. Unsere Daten zeigten einen negativen Zusammenhang zwischen dem Ankunftsdatum und den CORT-Metabolitwerten auf, mo ¨glicherweise als Ergebnis eines ho ¨heren Energiebedarfs infolge der harten Umweltbedingungen, welche die Zugvo ¨gel bei einer fru ¨hen Ankunft erfahren. Wir haben einen negativen Zusammenhang zwischen der Gro ¨ße des Stirnflecks und den CORT-Metabolitwerten beobachtet, was darauf hindeutet, dass soziale Dominanz mit niedrigen Baseline-CORT-Metaboliten zusammenha ¨ngt. Außerdem hatten Ma ¨nnchen, die sta ¨rkeres Territorialverhalten zeigten, wenn sie dem Playback-Gesang eines Artgenossen an ihrem Nistkasten ausgesetzt waren, ho ¨here CORT-Metabolite bei ihrer Ankunft als Ma ¨nnchen, die weniger ausgepra ¨gtes Territorialverhalten zeigten. Dies ko ¨nnte darauf hindeuten, dass erho ¨hte Baseline-CORT-Metabolitwerte bei Ma ¨nnchen starke Territorialkonkurrenz fo ¨rdern ko ¨nnten. Wir vermuten, dass die Konkurrenz zwischen Ma ¨nnchen ein Faktor ist, der die bei Zugvo ¨geln gemessenen Baseline-Glucocorticoidwerte beeinflusst.

J Ornithol (2010) 151:587-597

Acknowledgments This paper is dedicated to the memory of Jose Ĺuis Osorno who planned this study during a sabbatical leave in Madrid in [2002][2003]. The study has received financial support from projects CGL2004-00787/BOS and CGL2007-61251 to J. Moreno and BOS2003-05724 and CGL2006-14129-C02-01/BOS to S.M. (DGI-Ministerio de Ciencia e Innovacio ´n). All procedures conform to the requirements of animal welfare and conservation of Spanish laws. J. Morales and G.T. were supported by grants from MEC and Comunidad de Madrid respectively. E.L. was supported by a FPU grant from MEC. R.A.V. acknowledges support from a grant CSIC-Universidad de Chile 2003-04-09, ICM,-P05-002, and PFB-23-CONI-CYT-Chile. Consuelo Corral and Tonantzin Calvo helped in the field. We thank Chris Guglielmo and an anonymous reviewer for their constructive comments on a previous version of this manuscript. We were authorized by Javier Done ´s, Director of the Montes de Valsaı ´n reserve, to work in the study area. Direccio ´n General del Medio Natural-Junta de Castilla y Leo ´n authorized the capture, ringing, and blood sampling of birds. This paper is a contribution from the El Ventorrillo field station.