Peer Review #3 of "Sexual dimorphism in adult Little Stints (Calidris minuta) revealed by DNA sexing and discriminant analysis (v0.1)"

Abstract

Background. The sex of an individual organism plays such an important role in its life cycle that researchers must know a bird's sex to interpret key aspects of its biology. Sexes of dimorphic species can be easily distinguished, but sexing monomorphic bird species often requires expensive and timeconsuming molecular methods. The Little Stint (Calidris minuta) is a numerous species, monomorphic in plumage but showing a small degree of reversed sexual size dimorphism. Females are larger than males but the ranges of their measurements overlap, making Little Stints difficult to sex in the field. Our aim was to develop reliable sexing criteria for Little Stints in different stages of primary moult during their stay on the non-breeding grounds in South Africa using DNA-sexed individuals and discriminant function analysis. Methods. We caught 348 adult Little Stints in 2008-2016 on their non-breeding grounds at Barberspan Bird Sanctuary. To molecularly identify the birds' sex we used P2/P8 primers and DNA isolated from blood samples collected in the field. We used Storer's dimorphism index to assess the degree of sexual size dimorphism. Then we divided our sample into two groups: before or during and after primary moult. For each group we developed two functions: one using wing length only and the other a combination of morphometric features including wing, tarsus and total head length. Then we used a stepwise procedure to check which combination of measurements best discriminated sexes. To validate our result we used a jack-knife cross-validation procedure and Cohen-kappa statistics. Results. All the morphometric features we measured were bigger in DNA-sexed females than in males. Birds with fresh primaries had on average 2.3 mm longer wings than those with worn primaries. A discriminant function using wing length (D 1) correctly sexed 78.8% of individuals before moult, and a stepwise analysis showed that a combination of wing length and tarsus (D 2) correctly identified the sex of 82.7% of these birds. For birds with freshly moulted primaries a function using wing length (D 3) correctly classified 83.4% of the individuals, and a stepwise analysis revealed that wing and total head length (D 4) classified 84.7%. Discussion. Sexual size differences in Little Stints might be linked to their phylogenetics and breeding biology. Females are bigger, which increases their fecundity; males are smaller, which increases their manoeuverability during display flights and hence their mating success. Little Stints show an extreme lack of breeding site fidelity so we did not expect a geographical cline in their biometrics. Sexing criteria available for Little Stints in the literature were developed using museum specimens, which often shrink, leading to misclassification of live birds. The sexing criteria we developed can be used for studies on Little Stints at their non-breeding grounds and on past data, but should be applied cautiously because of the overlapping ranges.