High error rates for avian molecular sex identification primer sets applied to molted feathers

Molted feathers are becoming increasingly important as a source of DNA for identifying the sex of individuals, and accurate methods for molecular sex identification are needed. Three molecular sex identification primer sets have been developed for use in nearly all nonratite birds, but performance of these primer sets has not been evaluated for molted feathers. For two species of birds, the Ring-necked Pheasant (Phasianus colchicus) and the Scarlet Macaw (Ara macao), we evaluated success and error rates among primer sets using DNA from molted feathers and assessed the percentage of times an incorrect sex would be assigned when analyses are completed in duplicate. Amplification success rates differed among the primer sets for both species, ranging from 67.5% to 89.2% (P = 0.0002 and 0.009), and error rates were high, ranging from 1.9% to 24.2%. Success rates and error rates were not consistent between species and among primer sets. To improve the accuracy of molecular sex identification tests when using molted feathers, we suggest determining acceptable confidence levels in the accuracy of sex assignment, conducting pilot tests to evaluate the performance of different primer sets, and using high-resolution electrophoresis systems to increase detection of errors.