Genomic DNA was isolated from blood or other tissues using modified LiCl method

Materials and methods Sample collection

We studied green-backed tits from 2002 to 2006 in the Ao-Wan-Da National Forest Recreation Area, Nautou County, Taiwan (23°56’ N, 121°10’ E). Up to 200 next-boxes were set up in a total area of 5.3ha. From February to July, all next-boxes were monitored continuously to determine the social pairs, their laying dates, clutch sizes, juvenile hatching dates, brood sizes, and fledging successes. Adults were caught with mist nets or inside next-boxes; nestlings were sampled about seven days after hatching. Yearly sample sizes are summarized in Table 1. All birds were individually ringed with an aluminium ring and two colored plastic rings, and approximately 15µL blood was sampled then added into 800µL of 100% ethanol for subsequent genetic analysis. Tissues of unhatched eggs and dead nestlings were collected and stored in 100% ethanol. The green-backed tits in our study population brooded one to three times per year. Individuals were categorized as ‘breeder’ if they were found to breed at the study site, or

‘non-breeder’ if they were only captured and observed before the onset of breeding season. Male breeders were further categorized into uncuckolded male (all offspring were sired by social father), and cuckolded male (at least one offspring were sired by males other than social father). Only broods for which all nestlings and putative parents were sampled were included in our genetic analysis.

Molecular techniques

Genomic DNA was isolated from blood or other tissues using modified LiCl method

(Gemmell & Akiyama, 1996). Molecular sexing (Fridolfsson & Ellegren, 1999) was

performed to confirm the sex of each individual. All adults were genotyped at 18

polymorphic microsatellite loci, in which 13 were isolated from Green-backed tits:

Titgata02, Titgata39, Titgata67, Titgata79, Titgata84, Titgata87, Titgata88, Titgata89, and Titgata94 (Wang et al., 2005), and Titgata06, Titgata11, Titgata28, and Titgata82 (newly isolated in this study, polymorphism content information is summarized in Table 3), and five were isolated from great tits (Parus major): PmaCAn1, PmaTGAn45, PmaTAGAn71, and PmaGAn27 (Saladin et al., 2003), and Pma69u (Kawano, 2003).

Nestlings were genotyped using the first nine Green-backed Tit specific microsatellites (Wang et al., 2005). Polymerase chain reaction (PCR) and genotyping of microsatellite loci were performed according to the protocol described in Wang et al. (2005) and conditions described in Table 2.

For MHC typing, the PBR and intron 3 region of MHC class I alpha chain were amplified using HEX labelled primer PBRuniF (F: GGG CCA CAA TTC CAT GGG TCT CTG T) and TAMAR labelled primer P.m.ex4 (R: GAC ACA TGG ACG TCA GGG ACC AC). PBRuniF and P.m.ex4 were designed at aligned intron 2 and exon 4 conservative regions of green-backed Tits (for more details, see Appendix). PCR was performed in a reaction volume of 12.5 µL, containing 50-200ng of genomic DNA, 0.25µM of each primer, 1X reaction buffer (TOYOBO), 2.0 mM MgCl

, 0.3U Blend Taq-Plus with 0.125µg anti-Taq DNA polymerase antibody (TOYOBO). PCR condition was set as the following: at 95°C for 2 min, then 40 cycles of 95 °C for 20 s, 55 °C for 30 s and 72 °C for 30 s, followed by a final extension at 72 °C for 2 min (iCycler Thermal Cycler, Bio-Rad). The PCR product was further analyzed by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP; (Lento et al., 2003; Bryja et al., 2005) performed on a MegaBACE

1000 DNA analysis System (Amersham

Biosciences), which was equipped with 96 coated capillaries (effective length: 40cm,

internal diameter: 75µm) using the following modified method: 6.25µL of PCR product was ethanol precipitated then resuspended in 5µL of loading buffer (Amersham

Biosciences) with 0.2µL ROX-labelled internal marker MegaBACE

ET-900 R size standard (Amersham Biosciences), and denatured for 5 min at 95 °C and snap-cooled on ice for 2-4 min before electrophoresis. The sample was injected (45 s, 3 kV) into the capillary containing 3% MegaBACE™ nondenaturing Long Read Matrix (LRM, Amersham Biosciences). MegaBACE™ Running Buffer (Amersham Biosciences) supplied with 10% glycerol was used as running buffer. In the majority of capillaries, 160 min runs at 27°C and 9 kV were sufficient to detect all peaks of internal marker.

F

P

v.1.0 (Amersham Biosciences) was used to score alleles.

Two unrelated individuals were selected for further sequencing to check for the genuineness of MHC alleles and the occurrences of pseudogenes. The PCR products of the PBR and intron 3 of MHC class I alpha chain were cloned by using TOPO TA Cloning® Kits (Invitrogen). Subsequently, 50 clones for each individual were sequenced using MegaBACE™ 1000 DYEnamic ETDYE Terminator Cycle Sequencing

(Amersham Biosciences) using M13 primers. The sequences were then proofread and aligned in Sequencher v.4.2 (Life Codes, Inc.).

Genetic analyses

Polymorphism, kinship and parentage assignment

Genepop v.3.4 (Raymond & Rousset, 1995) was used to check for departure from

Hardy-Weinberg equilibrium and genetic disequilibrium between pairs of loci, only

adults were included in this analysis. Kinship analysis was performed by the KINGROUP

software (Dmitry et al., 2004), which was developed using the algorithm of Goodnight &

Queller (Goodnight & Queller, 1999) for estimating pedigree relationships between pairs of individuals and reports the likelihood ratio for a primary hypothesis vs. a null

hypothesis (e.g. the likelihood of being full-sibs vs. unrelated individuals). Paternity analysis was carried out using the likelihood approach implemented in C

.2.0 (Slate et al., 2000). Females captured inside or observed to attend the next-boxes were assumed to be the genetic mother of the clutch therein; genetic fathers were assigned only when their genotypes matched with offspring genotype at all nine microsatellite loci. In cases where none of the candidate male genotypes matched with offspring genotype, that offspring was considered to have been sired by an unsampled individual.

Individual heterozygosity and genetic similarity

We used standardized heterozygosity (SH) (Amos et al., 2001) for quantifying internal

genetic diversity (heterozygosity) of an individual. The genetic similarity (relatedness)

between adult tits was estimated as Queller and Goodnight’s relatedness coefficient (R

;

(Queller & Goodnight, 1989) based on microsatellite genotypes. F

test performed by

software Arlequin V2.0 2000 (Schneider et al., 2002) was used to examine the yearly

differentiation of microsatellite allele frequency (Table 3). Because F

among years was

not significantly different from zero, I pooled adults individuals captured in different

years into one population, and the average genetic distance was set to be zero for the

calculation of relatedness coefficient. MHC similarity between females and males was

calculated as allele-sharing coefficient; the proportion of allele-sharing of a pair is twice

the number of the allele shared by two individuals (F

) divided by the total number of

bands of each individual (F

and F

, respectively) [D=2F

/(F

+F

)]. (Wetton et al.,

1987)

Statistical analyses

Individuals or pairs observed in different years were counted as independent samples (weighted) or just used once (unweighted) for the following statistic tests. All parameters of heterozygosity and genetic similarity were analyzed using Monte Carlo simulations (Manly, 1997). To test the heterozygosity advantage, the average observed difference in standardized heterozygosity between breeders and non-breeders was compared with the 95% confidence interval of the average differences of 10,000 randomized sample pairs from the pool of breeders and non-breeders. To test the genetic compatibility hypothesis, non-breeders were first excluded (because they might have no chance to gain a territory and therefore be chosen by females), and the observed average value of relatedness and allele-sharing of MHC class I genes in breeding pairs was compared with the 95%

confidence intervals of that obtained from 10,000 randomized samples of potential dyads

among breeder. The Levene test based on randomization was conducted to compare the

variation of MHC allele number between breeders and non-breeders, whereas one-way

ANOVA based on randomization was used to compare the variance of MHC allele

number between random dyads from true pairs. All analyses were performed with the

Microsoft Excel ad-in PopTools (Hood, 2005). Pairwise correlation between parameters

was analyzed using JMP5.0 (SAS).