Sex identification in Gentoo (Pygoscelis papua) and Chinstrap (Pygoscelis antarctica) penguins: Can flow cytometry be used as a reliable identification method?

An important scientific question in ecology is how to differentiate males from females that have similar morphology. In penguins, due to their monomorphic plumage, it is difficult to determine gender. So far, most approaches to address this problem have focused on using sex based discriminant functions combined with DNA based tools as a validation method. As discriminant functions can be species and locality specific, in this study we explored the feasibility of using flow cytometry to determine the sex of two species of penguins, Pygoscelis antarctica and Pygoscelis papua. Our results for sex assessment determined by flow cytometry were compared with those obtained using morphological characters (bill length and depth), and DNA based methods (using the PL/PR primer pair) were used to validate both approaches. For both species, statistically significant differences were observed between males and females, with males presenting on average 2.1% more nuclear DNA than females. Flow cytometry enabled similar or better rates of correct sex assignment (86.4% and 80.0%) than sex discriminant functions (31.8% and 90.0%) for P. antarctica and P. papua, respectively, and thus may be considered as a promising alternative to the use of morphological data for sex identification. Nevertheless, some individuals with intermediate genome size values were observed, which increased the difficulty of categorically assigning their sex. Therefore, flow cytometry alone cannot be considered in all cases, but if used together with DNA-based methods in targeted samples, it can be used to efficiently estimate the gender of, at least, these two penguin species, with the potential to be used in other species.