A novel molecular sexing method for greater one-horned rhinoceros (Rhinoceros unicornis) for reliable demographic assessments

Context Accurate estimation of demographic parameters, such as the sex ratio is critical for understanding patterns of population growth, reproduction, kin organisation, sex-biased dispersal, and differential mortality in threatened species. Such estimates are difficult to obtain, especially in elusive and sexually monomorphic species, where traditional field-based sex identification techniques often prove insufficient. The greater one-horned (GoH) rhinoceros (Rhinoceros unicornis), a sexually monomorphic species, lacks a validated molecular sexing protocol across various biological sample types. Aims We aimed to develop and validate a robust, reliable, and reproducible molecular sexing protocol for the GoH, applicable across a broad range of field-collected sample types. Methods We tested and optimised sex-linked markers through a comprehensive literature survey for multiplex polymerase chain reactions. The multiplex panel, comprising ZF, AMESC, and TSRY markers, was standardised using known-sex reference samples and validated on 286 individual rhino samples (blood, tissue, horn, and dung) collected from all existing wild populations in India. The panel’s performance was assessed through amplification success rates, allelic dropouts, consistency across replicates, and marker-specific reliability. Key results The three-marker multiplex panel achieved an overall sexing success rate of 97.9%, successfully identifying 209 males and 71 females. Sample-type specific success rates were high, being 100% for blood, horn and tissue, and 93.62% for dung, with low allelic dropout rates (5.3–8.5%). This approach validates a molecular sexing protocol with increased reliability and fewer amplification failures, facilitating large-scale demographic studies. Conclusions We have established a reliable and reproducible molecular sexing protocol for the GoH rhinos, suitable for various sample types and adaptable to large-scale demographic studies. Implications This validated protocol will support long-term monitoring, sex ratio estimation, and translocation planning for GoH rhinoceros populations. Its successful application to low-quality field samples highlights its utility for the conservation of this endangered megafauna with limited sexual dimorphism.