New primers for sexing juvenile buntings and to assist ex-situ conservation of endangered eastern iberian reed bunting emberiza schoeniclus witherbyi populations

accurate sex determination of an individual is essential for understanding the ecology of a species and to implement effective conservation measures. Molecular sexing is proving to be an effective tool to determine the sex ratio of difficult-to-sex species as well as for establishing breeding pairs in ex situ conservation programmes. In the last few decades, a putatively universal set of primers, based on the so-called intron a of the CHD1 gene, has been used to sex birds through PCR. Nevertheless, several issues have been reported in several taxa for which more specific alternative protocols are needed. In this paper we present a new pair of markers based on the so-called intron E of the CHD1 gene and designed for sexing difficult-to-sex Old World bunting Emberiza spp. individuals. This method was then used for supporting an ongoing Eastern Iberian Reed Bunting Emberiza schoeniclus witherbyi breeding programme and to estimate, for the first time, the sex ratio of this critically endangered passerine. Newly designed primers allowed for more rapid and accurate sex determination compared to other previously developed markers tested here, thanks to larger size differences between CHD1W and CHD1Z PCR products and lower allelic drop-out frequency. Our results suggest no significant sex bias in the Eastern Iberian Reed Bunting resident population (male:female ratio 0.8: 1), as opposed to strongly female-biased estimates (up to male:female ratio 0.33: 1) previously recorded in Spain for wintering populations. Differences in sex ratio estimates between resident and wintering populations could arise from a latitudinal sex segregation in Reed Buntings due to differential migration, and highlight the importance of using resident populations to obtain key demographic parameters. future work should focus on sampling resident populations to better characterise local variations in sex ratio, to obtain relevant data for the management of threatened subspecies.