Summary
Sex ratio biases in avian species remain controversial, although several studies have documented apparent facultative adjustment of offspring sex ratios. While hybridizing pied and collared flycatchers have exhibited sex ratio skews that may be a response to sex‐based costs associated with hybridization, this appears not to be true of a hybridized population of blue‐winged Vermivora pinus and golden‐winged V. chrysopterawarblers. We examined the primary sex ratio of nestlings in a population of hybrid and introgressed golden‐winged warblers. The sex ratio of 298 nestlings from 81 nests in the population was approximately 50:50. We conducted paternity assignments and analyzed groups of nestlings with shared genetic parents (“genetic broods”) and found no difference from the expected binomial distribution, and no statistically significant relationship between parental species phenotype and nestling sex ratio. We saw no evidence of preferential production of male or female nestlings, and female hybrids were found to mate and breed in the population. This suggests that heterogametic (female) hybrids are both viable and fertile, and thus that Haldane's Rule does not apply to this system. While populations of hybridizing golden‐winged warblers should be monitored for evidence of costs of heterospecific pairings, it is unlikely that adjustment of sex ratios would be the form of compensation for sub‐optimal mating conditions. Our results provide support for the emerging hypothesis that hybrids suffer no disadvantage relative to golden‐winged and blue‐winged warblers.
Methodology
Field methods
We conducted fieldwork in Eastern Ontario, Canada at 15 study sites throughout the Queen's University Biological Station property (44° 34'N, 76° 19'W), from late April until mid-July of 2001 through 2005. Nests were located, and then were checked every two days to determine social pairs, laying and hatching dates of clutches, and outcomes of nests (abandoned, depredated, or fledged). Approximate male territories were determined by observation of singing behavior in the early part of the season, and social parents were determined through observation of nests and territories; males were considered social fathers if the nests were on their territory, and they were observed at the nest on multiple occasions. Targeted mist-netting, involving audio playback and painted models, was used to catch adult males. We caught adult females by placing a mist-net near the ground and flushing her from the nest on day six of incubation; none of the females abandoned a nest as a result of this disturbance.
Adults were fitted with a single aluminium Canadian Wildlife Services (CWS) leg band, as well as with unique combinations of plastic colour bands to allow for later identification. Each individual was categorized as pure golden-winged warbler or hybrid based on their plumage characteristics. Any individuals that did not exhibit classic golden-winged warbler plumage were categorized as hy- brids. This included introgressed individuals that did not conform to parental or hybrid phenotypes, as well as those with classic "Brewster's" or "Lawrence's" warbler phenotypes (Parkes 1951).
We drew 25µl blood samples using brachial venipuncture from all adults (Clarke et al. 2002). We banded nestlings with a single CWS band four days after hatching, and obtained 15µl of blood following tarsal venipuncture (Clarke et al. 2002).
DNA analyses: sex ratios and parentage
DNA was extracted from the 2001 and 2002 blood samples using a standard phenol chloroform extraction method, followed by ethanol precipitation (Sambrook and Russell 2001), and dried DNA was re-suspended in 50pl ddH20. Preparations of DNA from 2003 to 2005 were carried out using an Eppendorf Perfect gDNA Blood Mini Isolation Kit, with a final elution volume of 200µl 10mM Tris-C1, pH 9.0.
Polymerase chain reaction (PCR) was used to amplify genes found exclusively on the sex chromosomes, using primers P2 (5'-TCT GCA TCG CTA AAT CCTT T-3') and P8 (5'-CTC CCA AGG ATG AGR AAY TG-3'), following Griffiths et al. (1998) to determine the sex of each nestling. A known male sample, known female sample, and a negative control were run for each set of nestling samples.
Details of paternity analyses and assignment methodol- ogies can be found in Vallender et al. (2007a) and Reed et al. (2007). Briefly, samples from 2001 to 2003 were analyzed using three microsatellite primers isolated from the Swainson's warbler Limnothlypis swainsonii genome (Winker et al. 1999), and 2004 and 2005 samples were analyzed using four primers isolated from golden-winged warblers (Stenzler et al. 2004).