Genetic parentage in the indigo bunting: a study using DNA fingerprinting

Parentage of nestlings in a North Carolina population of indigo buntings (Passerina cyanea) was analyzed using DNA fingerprinting. Three minisatellite DNA probes (wild type M13, Jeffreys' 33.15 and 33.6) were used to analyze nuclear DNA isolated from muscle biopsies of 63 nestlings, their parents, and other local adults. Each probe detected approximately 15 scorable fragments per individual, with 18%-39% overlap between probes. The proportion of bands shared (using all fragments over all three probes) between presumably unrelated adults averaged 0.23. Of the 63 offspring analyzed, 35 had at least one fragment not present in either putative parent. The distribution of offspring with novel fragments was distinctly bimodal. The lower mode (offspring with 0, 1, or 2 novel fragments, N=41) fit a Poisson distribution, a pattern expected if mutation (estimated rate per fragment= 0.01) were the source of the novel fragments. The remaining 22 offspring had more novel fragments than could be explained by mutation alone (minimum of four independent fragments across all three probes, {Mathematical expression}=8.2). A low band-sharing proportion with the resident male ( {Mathematical expression}=0.24) and high band-sharing with the resident female ( {Mathematical expression}=0.60) implicated extra-pair fertilizations as the source of all 22. Thus in this sample, 35% of all nestlings came from extra-pair fertilizations and none from intra-specific brood parasitism. Of 25 broods sampled, 12 (48%) had at least one excluded offspring. In 3 broods all of the offspring excluded the resident male. Band-sharing proportions between excluded nestlings within a brood could not distinguish between single and multiple extra-pair paternity. Although young males tended to be excluded less often than older males, wing length and weight were not associated with the frequency of exclusion. Weight and wing length of females also were not associated with involvement in EPCs. Six of the 22 excluded offspring (in 3 broods) shared a large proportion of bands and had fewer than four novel fragments when compared to the fingerprints of a neighboring territorial male, implicating those males as actual fathers. The parentage of the remaining 16 offspring (in 9 broods) could not be clearly assigned because (1) one or more neighbors were not sampled or (2) difficulties in scoring across gels prevented confident alignment of fingerprint bands, and insufficient DNA was obtained from muscle samples to allow reanalysis of potential actual fathers on the same gel as excluded nestlings.