Genome‐Wide Characterization of Sex‐Linked Regions in the Sangzhi Horned Toad ( Boulenophrys sangzhiensis ) Reveals Complex Sex Determination Mechanisms

Sex chromosomes in amphibians exhibit substantial variability and often remain largely homomorphic, providing a powerful system for studying sex determination. The Sangzhi horned toad (Boulenophrys sangzhiensis) is an ideal model, but limited genomic resources have hindered insights into sex determination mechanisms. Here, we assembled a 2.8 Gb chromosome-level genome of B. sangzhiensis generated using PacBio HiFi and Hi-C data, achieving a Contig N50 of 30 Mb and identifying 21,775 protein-coding genes. Genome-wide analyses of coverage, FST, SNP density, and linkage disequilibrium identified two putative sex-linked regions on chromosome 2 and 6, showing sex-specific coverage and strong genetic differentiation. Within these sex-linked regions, we identified a Y-specific sequence and several candidate sex-determining genes, including Hsd11b2, Nlrp14, and zinc finger genes. Our findings suggest that sex determination in B. sangzhiensis involves a complex, possibly polymorphic mechanism, with multiple Y haplotypes segregating within populations. Additionally, the sex-linked regions exhibit accumulation of repetitive sequences, multi-copy genes, and chromosomal rearrangements, which may contribute to the evolution of sex chromosomes. This chromosome-level genome provides a valuable resource for understanding the dynamic evolution of sex-determining mechanisms in amphibians.