Multiple laying females in nests of the Cattle Egret (Bubulcus ibis): behavioral and genetic evidence

Behavioral and genetic data were used to detect multiple females laying eggs in nests of the Cattle Egret (Bubulcus ibis). We used firstly a classic method of behavioral observations to select suspected nests in which the probability of detecting multiple laying females is greater than nests in general. Molecular genetics was then applied using seven microsatellite loci to compare genotypes of maternal DNA collected from eggs of the same clutch. We monitored 85 nests and collected swabs from eggshell from all clutches. A group of 18 nests with eggs that were laid in an interval of less than one day or more than four days was selected since they have eggs suspected origin from multiple laying females. After molecular sexing, 7.3% of the DNA swab samples were discarded due to contamination by DNA of a male origin, leading to the elimination of three clutches. Of the remaining 15 nests, DNA was extracted from all eggs (n = 43) and genotyped at seven microsatellite loci. Genotypes from maternal DNA found in eggs from same clutch were compared and genotypes with non-matching alleles at a minimum of two loci within the same clutch were classified as being the result of different laying females. Among 16 eggs classified by behavioral criterion as placed by a different female 14 eggs were confirmed by the genetic approach. Evidence of multiple females laying eggs in a same nest was discussed supposing the occurrence of conspecific brood parasitism or polygyny, previously reported for the Cattle Egret.