Heritability and genetic correlation between the sexes in a songbird sexual ornament

two out of the three predictions supported Z-linkage and lack of significant mother–daughter resemblance could be due to small sample sizes caused by limited female trait expression. Therefore, the evidence was mixed as to the contribution of the Z chromosome and autosomal genes to trait size. The threshold heritability of trait expression in females was lower, around 0.3, supporting autosomal-based trait expression in females. Environmental (birth date) and parental effects on ornament size mediated by the mother’s condition after accounting for maternal and paternal genetic influences are also highlighted. The genetic correlation between the sexes did not differ from one, indicating that selection on the character on either sex entails a correlated response in the opposite sex

The amount of linkage to the sex chromosomes of secondary sex traits subjected to sexual selection in breeding systems with genetic sex determination (XX/ XY or ZZ/WZ; Rice, 1984; Lande, 1987; Fairbairn and Roff, 2006; Moore and Moore, 2006; Qvarnstro¨ m and Bailey, 2008) is a subject of certain controversy. This debate is important for at least two reasons. First, the genomic location can influence the correlation between sexually selected traits and preferences for those traits (Kirkpatrick and Hall, 2004; Sæther et al., 2007) and thus directly impinges on Fisher ’s runaway and the good- genes processes of sexual selection (Kirkpatrick and Ryan, 1991; Kirkpatrick and Hall, 2004; Mank et al., 2006). Second, resolution of the controversy also affects estimates on the rate of evolution, which on the basis of the hemizygosity and subsequent smaller effective population size, is predicted to be steeper for Y/Z chromosomes than for X/W chromosomes and auto- somes (Charlesworth et al., 1987; Rice, 1988; Rowe and Houle, 1996; Fitzpatrick, 2004; Kirkpatrick and Hall,

2004; Fairbairn and Roff, 2006; Mank et al., 2007, 2010). Empirical tests of these ideas have usually approached the issue from comparative perspectives or inferences from reciprocal crosses (Reinhold, 1998; Mank et al.,
Correspondence: Dr J Potti, Department of Evolutionary Ecology, Estacio^´n Biolo^´gica de Don˜ ana—CSIC, Av. Ame^´rico Vespucio s/n, Seville

41092, Spain.

E-mail: jpotti@ebd.csic.es
2006), genomic scans (Fitzpatrick, 2004; Ellegren et al.,

2007, Itoh et al., 2007) or, in birds, wherein males are the

homogametic (ZZ) and females the heterogametic (WZ)

sex, mainly in the context of gene flow and introgression in hybrid zones (Sætre et al., 2003; Sæther et al., 2007; Storchova^´ et al., 2009). However, empirical, molecular results have not settled the question whether sexually selected genes are or not preferentially sex linked (Reinhold, 1998; Fitzpatrick, 2004) and the evidence from natural scenarios is even more limited. Using single- nucleotide polymorphisms, Sætre et al. (2003) concluded that male plumage characteristics, including a white patch in the male forehead, were probably linked to the Z chromosome in the complex of hybridising European black-and-white flycatchers (pied flycatcher Ficedula hypoleuca and collared flycatcher F. albicollis). This is an important result obtained in species that have become favourite models in sexual selection studies, one that is customarily cited in the literature on speciation and evolution of sex traits as one of the few rendering evidence for Z(X)-linked sex traits (Kirkpatrick and Hall,

2004; Mank et al., 2007; Qvarnstro¨ m and Bailey, 2008; Ellegren, 2009; Mank and Ellegren, 2009). However, Sætre et al. (2003) also recognised that their coarse- grained linkage mapping and the co-linearity they noted between autosomal and Z-genotypes could have affected their results. Independent validation or refutation of sex linkage in other populations and biological contexts (for example, in non-hybridising settings) and arrived at through different approaches would thus help to shed light on the possibility of Z-linkage.

Despite the increasing availability of long-term studies

examining (co)variation of sex and life history traits

(Merila¨ and Sheldon, 2001; Poissant et al., 2009) the genetic correlation between the sexes in the expression of secondary sex traits in wild bird populations has attracted very few previous empirical efforts of field researchers, with some exceptions (for example, barn owls, Tyto alba; Roulin and Dijkstra, 2003; barn swallows, Hirundo rustica; MØller, 1993). In Iberian pied flycatcher (F. hypoleuca iberiae) populations a substantial number of breeding females express a white forehead patch (FP) (42% expressing it at least once in their lifetimes; N ¼ 851 individuals; J. Potti, unpublished data; Potti, 1993; Potti and Merino, 1996a; Morales et al., 2007) that is displayed by all breeding males. We capitalised on a relatively large data set obtained throughout long-term (420 years) measurements and experimental cross-fostering to ex- amine the genetic architecture of a secondary sex trait through both the male and female germ lines. We also aimed to test with field data if genetic transmission may occur through linkage to the Z chromosome, as suggested by the analyses of Sætre et al. (2003). To that end, we used familial data to try to discriminate among alternative modes of inheritance. As half of their genes in autosomes is inherited by birds from either parent, but the single Z chromosome of WZ females only from their father, heritability analyses made possible by the measurement of the trait in both sexes can test whether ornament size is inherited on autosomes or may be determined by linkage to the Z chromosome. Under the hypothesis of Z-linkage at least three testable predictions should hold (see also Trivers, 1985; Lynch and Walsh,

1997; Iyengar et al., 2002): (1) females should resemble their fathers, not their mothers, as by being WZ females always receive their Z chromosome from their father (Lande, 1980). Hence, the female (that is, her father ’s) Z chromosome is lost in the female’s daughters but remains in the female’s male (ZZ) offspring and grand- offspring (Figure 1). Simple autosomal inheritance, on the contrary, does not predict sex biases in familial resemblance and thus both males and females should resemble their parents and grandparents of either sex in the size of their FP. (2) Under Z-linkage, male half-sibs sired by the same father should show lower resemblance than those sharing their mother. This is due to the former inheriting, on average, one of their Z chromosomes from each of their paternal grandparents while all mother- sharing sibs share one of their Z chromosomes with their maternal grandfather. (3) Similarly, males should show higher resemblance to their maternal than their paternal grandfather (Figure 1), as they always bear a Z chromosome from the former while the likelihood of sharing the other Z chromosome with their paternal grandfather is diluted by a half when assuming complete Z-linkage and no dominance or epistatic interactions (Lynch and Walsh, 1997).

Similar to male collared flycatchers in the Baltic islands

(summarised in Qvarnstro¨ m et al., 2006) there is apparent directional selection in at least some Iberian populations of pied flycatchers favouring large size of this demela- nised ornament in both males and females. Males with larger patches are apparently preferred by female pied flycatchers when choosing mates (Potti and Montalvo,

1991a) and females are mainly fertilised by males bearing ornaments larger than those displayed by both their


Figure 1 (a) Female (left) and male Iberian pied flycatchers displaying white FP. (b) Schematic relationships of the Z sex chromosome of a male individual (grandson) with those of his mother (left) and father, and maternal and paternal grandparents. Numbers besides dotted lines indicate the probability of sharing the same Z chromosome across generations.

social mates and neighbours in their extra pair relation- ships resulting in extra-pair young (D Canal, J Potti and JA Da^´ vila, manuscript under review). Unusually among European populations (Lundberg and Alatalo, 1992; Cramp and Perrins, 1993), some females express with an advancing age FPs as those all males display (Figure 1), although often smaller. Benefits of its expression (absence vs presence) and size in females are less understood, but there is evidence that the ornament signals their age (Potti, 1993; Morales et al.,

2007), condition, fecundity and resistance to both endoparasites (trypanosomes; Potti and Merino, 1996a)

and nest mite (Dermanyssus) ectoparasites (J Potti and D

Canal, manuscript under review). Thus, as suggested for a handful of species (Amundsen, 2000; Griggio et al.,

2009), both ornament expression (in females) and size (in both sexes) may act as quality indicators, that is, targets

for the establishment of mate preferences in both sexes

(Potti and Montalvo, 1991a; Potti and Merino, 1996a;

Osorno et al., 2006). The overall scenario of selection apparently favouring large expression of the ornament in

males and females is thus of interest in the context of hypotheses stating that the ‘vestigial’ nature of the

expression of sex traits in females has no adaptive utility in itself (Darwin, 1874). In nowadays terminology, this is

widely stated as being a by-product of the genetic

correlation between the sexes (Lande, 1980, 1987; Bonduriansky and Chenoveth, 2009). A large genetic

correlation may severely constrain the rate of the independent evolution of sexual dimorphism (Fisher,

1958; Lande, 1980, 1987; Bonduriansky and Chenoveth,

2009, Poissant et al., 2009) as, among others, has been put

in evidence for beak colouration in captive zebra finches

Taeniopygia guttata (Price and Burley, 1993, 1994; Price,

1996) and for a non-sexual trait (tarsus length) in collared

flycatchers in the wild (Merila¨ et al., 1998). Here, we first ask whether there is evidence for additive genetic

transmitted through both sexes. We examine patterns

in trait heritabilities in the wild and the genetic correlation between the sexes as revealed through

several familial comparisons across three generations in male and female pied flycatchers. Heritability of its

limited displaying in females is also estimated to gain insight into the trait genetic architecture. As a test of the

validity of field results for the male sex, wherein expression is universal in all populations, we use

experimental cross-fostering of individuals to assess whether genotype–environment correlation biases our

field-derived estimates (Merila¨ and Sheldon, 2001). We also examine environmental (hatching date) and mater- nal effects on ornament size and look for testable evidence discriminating on the most likely modes of genetic transmission, that is, Z-linked vs autosomal inheritance.

Materials and methods
Field methods

We studied a population of pied flycatchers breeding in nest boxes in La Hiruela, about 100 km northeast of

Madrid, central Spain, in the breeding seasons from 1987 to 2009 (for example, Potti, 2008). Breeding adults were

captured, measured for tarsus length (‘size’, hereafter;

with callipers, to the nearest 0.01 mm), weighed (preci-

sion 0.1 g), marked for individual identification with numbered and colour rings and released. The height and

width of the male FP were measured with callipers (to the nearest 0.1 mm) and the area calculated as FP

height x FP width. Data on FP dimensions could only be taken in a fraction of females (see above). When

present, FP length and breadth were measured and the area calculated as for males. Measurements of female FP

size were only available from 1993 onward. Many of the breeding adults were born in our nestboxes, wherein

they had been marked with a metal ring, and measured

and weighed (as for adults) at day 13 of nestling age

(Potti and Merino, 1994).
Heritability estimates of character size and expression Narrow-sense heritability (h²) estimates of ornament size assuming autosomal inheritance are presented as a null model to compare with the fulfilment of assumptions should Z-linked transmission be occurring (Falconer and Mackay, 1996; Lynch and Walsh, 1997). Heritabilities were calculated separately through the mother and father for male and female offspring from same-sex offspring means of FP size regressed on one parent (h² ¼ 2b, where b is the regression coefficient). As means and variances of FP differed between the sexes (below) it was first necessary to transform data, which we made by standardising FP values within each sex (zero mean, unit variance; see below).

Analyses of phenotypic resemblance with the grand- parent generation (h² ¼ 4b, when assuming autosomal inheritance) could only be made with males because of small sample sizes of females expressing FP because of age dependency in its first appearance (Potti 1993). We controlled for the assortative mating with respect to FP size that was earlier reported (Potti and Merino, 1996a) and can bias single-parent, full-sib and offspring–grand-

1996). Although the within-pair correlation was not

strong in the larger sample here analysed, it was also significant (R ¼ 0.16, N ¼ 145, P ¼ 0.048). We therefore

corrected those estimates with the expressions provided by Falconer and MacKay (1996) and Nagylaki (1978). The

standard errors (s.e.) of parent–offspring and grand- parent–grandoffspring heritability estimates were ob-

tained after doubling or quadrupling, respectively, the s.e. obtained by bootstrapping the samples 10 000 times.

Further estimates of heritability of FP size were made by analysing its variance components in male full-

brothers, as well as on paternal and maternal half- brothers (Becker, 1984; Falconer and MacKay, 1996;

Lynch and Walsh, 1997). This only could be made for males as we had data for only six female full-sisters in

three families and half-sister data were even sparser. The s.e. for these estimates were also found by bootstrapping

the samples 10 000 times. Variance components were also used to estimate repeatabilities of FP size between

individuals across years (Lynch and Walsh, 1997). Ornament expression is not universal in females (Potti,

1993) and thus may be treated as a threshold character by assuming its expression depends on an underlying

normally distributed continuous variable (Falconer and

MacKay, 1996; Lynch and Walsh, 1997). As described by

Falconer and MacKay (1996: pp. 300–305), we used the binary data on proportions of females expressing (yes/

no) the ornament across lifetime in the population at large (360/491) and in a sample of mothers and

daughters (26/44) to find out the heritability on the underlying scale and its s.e. by considering female

expression trait with two classes and one threshold, using the expression accounting for changes in variances

across generations (Falconer and Mackay, 1996, pp. 302;

Lynch and Walsh, 1997, pp. 733).

Cross-fostering experiment

To control for the possibility of genotype–environment correlation influencing familial resemblance in ornament

size, we used a sample of males recruiting from a limited data set of cross-fostered nests used for other experi-

ments (Potti et al., 2007). In short, we exchanged in 2006 all eggs in the second day of incubation between

matched pairs of nests (distant at least 1 km apart) of the same (^±1 day) breeding date and clutch size. As a result, all pairs of exchanged nests contained broods reared by unrelated adult birds (which was confirmed by DNA fingerprinting; D Canal et al., unpublished data). Twenty-nine males from cross-fostered nests recruited in the 2007 to 2009 breeding seasons, making possible comparisons of their average ornament size to those of their foster and genetic male parents. Familial data on cross-fostered females and their female parents were too limited to be of any use because of age dependence in female ornament expression ( J Potti and D Canal,

manuscript under review).

Genetic and phenotypic correlation between the sexes The intersexual genetic correlation r_MF was estimated with the expression Oh² _FD h² _MS/h² _MD h² _FS, where h² are the bootstrapped heritabilities (10 000 boot- strap iterations) calculated from father–daughter (FD),

120

100

₈₀

Table 1 Heritabilities (h²) with bootstrapped standard errors (s.e.) and associated probabilities (P) of forehead patch size in Iberian pied flycatchers

z
Familial comparison h² s.e. P h² (coef.) N