Evolutionary dynamics of conserved non-exonic elements and distinctive X-linked regulatory evolution in Felidae

Conserved non-exonic elements (CNEEs) are widely recognized as key cis-regulatory elements contributing to developmental regulation and phenotypic evolution in vertebrates. However, their chromosomal organization and evolutionary dynamics remain poorly understood, particularly in non-model animals. The family Felidae, characterized by rapid evolutionary radiation and a highly conserved karyotype, provides an ideal system for investigating these questions. Here, we leveraged long-read genome assemblies from 17 felids and two carnivoran outgroups to identify CNEEs and assess their chromosomal organization, lineage-specific loss, and accelerated evolution. We identified 420,225 feline CNEEs and further defined 490 high-density CNEE clusters, which are associated with developmental regulatory landscapes. The X chromosome exhibited distinct regulatory evolutionary features, with an enrichment of high-density CNEE clusters and higher putative CNEE loss rates relative to autosomes. In contrast, CNEE acceleration was not elevated on the X chromosome relative to autosomes. Additionally, we characterized lineage-specific accelerated CNEEs associated with genes involved in locomotion, sensory ecology, hypoxia adaptation, and musculoskeletal traits, including a Pantherinae-shared 5-bp deletion in a candidate regulatory element associated with LAMA2. Our study provides a genome-wide view of CNEE organization and evolution in felids, highlights distinct regulatory dynamics on the X chromosome, and offers new insights into the non-coding genomic mechanisms underlying felid diversification and adaptation.