Testing sex determining markers in hornbills (Bucerotidae) and ground-hornbills (Bucorvidae)

Abstract Hornbills (Bucerotidae) and ground-hornbills (Bucorvidae) are families of birds found in Asia and Africa. At least 29 species are considered Near Threatened or above by the IUCN, with hunting for trade being the main threat to these birds. As many species have a unique feature on the top of their beaks called a casque, they are most commonly legally or illegally traded for their skulls or, in the case of the Helmeted Hornbill ( Rhinoplax vigil ), ivory. Hornbill casques are sexually dimorphic, vary in size within species across age classes, juvenile individuals can look like females, and, further, can be convergent across species. As not all hornbill species are subject to CITES regulation, the use of morphological identification guides is essential for efficient and quick identification especially of often difficult to identify non-adult male individuals In this study, we evaluated the performance of multiple DNA sex primers for the ZW system in birds, focusing on the chromo-helicase-DNA binding protein gene (CHD-ZW-common/CHD-W-specific/P2; M5/P8; P2/P8; 2250 F/2718R; CHD1F/CHD1R). The amplification of these markers was performed using a polymerase chain reaction (PCR), followed by visualisation via capillary electrophoresis. The primer sets CHD1F/CHD1R and 2250 F/2718R were found to be the most effective, together successfully amplifying in 18 hornbill species from diverse genera. Both sets also exhibited functionality with diverse tissue types (muscle, liver, whole blood, dried blood, and bone) and varying DNA quantities (1.991–23.356 ng/µL). CHD1F/CHD1R and 2250 F/2718R further benefit from the trait that the female-determining allele is smaller. Consequently, the potential for a misidentification resulting from degraded samples can be ruled out. By using the CHD1F/CHD1R and 2250 F/2718R markers, the sex of hornbills can be reliably determined in morphologically referenced specimens, which provides documentation of the casque variability within a species from adult males, juvenile individuals, and females. Such a guide can account for interspecific and ontogenetic variation and support relevant authorities in the rapid identification of hornbill species in both legal and illegal trade.