<i>Hox</i> Gene Variation Drives Morphological Specialization of Humpback Grouper <i>Cromileptes altivelis</i>

Morphological traits are central to traditional taxonomy, yet convergent and divergent evolution can lead to inconsistencies between morphological classification and molecular phylogenetics. The distinctive "sunken head and humpback" morphology of Cromileptes altivelis and its close phylogenetic relationship with Epinephelus make it an ideal model for evaluating the weighting of morphological traits in taxonomic classification and refining the classification system. We measured and analyzed the morphological specialization process of C. altivelis, identifying key developmental stages leading to its humpback phenotype. This trait develops through cranial remodeling, involving changes in the supraoccipital, frontal, and lateral occipital bones, with structural support from predorsal bones and the first neural arch and spine. Examining the Hox gene family, we found that C. altivelis possesses 49 highly conserved Hox genes, with no significant differences in gene copy number, arrangement, or exon count among groupers. However, unique amino acid variations were identified in the Hoxa7a, Hoxa10b, and Hoxc1a proteins of C. altivelis, which are otherwise highly conserved among other teleost fishes. Functional assays confirmed that mutations in these genes enhance gene transcription activity, promoting osteoblast proliferation and differentiation. qPCR analysis showed that the expression of hoxa7a and hoxa10b was significantly upregulated during the humpback stage, implicating their contribution to the morphological specialization of C. altivelis. hoxa10b remained elevated post-humpback, suggesting a role in bone strength and homeostasis, whereas hoxc1a exhibited consistently low expression, indicating limited involvement. Our findings provide insights into resolving taxonomic discrepancies in C. altivelis and offer a framework for understanding its adaptive evolution and speciation.