Economical genotyping of little blue penguin (Eudyptula minor) clades from feather-based DNA

Determination of clade membership is a crucial requirement for many research questions addressing phylogeography, population structure, mating patterns, speciation, and hybridisation. The little blue penguin (Eudyptula minor) can be separated into two deeply divergent clades. However, assigning clade membership in little blue penguins requires molecular methods. Genetic sequencing can be used to identify clade membership but is expensive. Here, we present an economical alternative to the use of sequencing to determine little blue penguin clade membership. We extracted DNA from feathers using a method that produced reasonable quantities of DNA. We then amplified the D-loop section of the mitochondrial control region from total genomic DNA extracts, using the primers ‘C L-tRNAglu’ and ‘D H-Dbox’ followed by digestion with the restriction enzyme AluI. When visualised on a gel, distinctive banding patterns clearly indicated clade membership. We sequenced a subset of our samples and verified the accuracy of this method. The methods we present should facilitate little blue penguin research through a cost-effective approach to clade analysis as well as a successful technique to extract DNA from feathers when blood or tissue samples are not available.