Analysis of the correlation between phenotypic color differences and molecular regulation of <em>Amphiprion percula</em> and <em>A. ocellaris</em>

Abstract

This study investigates phenotypic characterization with molecular analysis to elucidate the mechanisms underlying skin color divergence between Amphiprion percula and A. ocellaris. These two closely related clownfish species differ markedly in skin pigmentation and are compared here to understand how ecological adaptation and genetic divergence shape phenotypic traits. HSB（Hue, Saturation, Brightness）colorimetry revealed significant interspecific differences (A. ocellaris: L=-57.11±2.59, a=29.91±4.77, b=20.70±4.68; A. percula: L=-59.87±4.30, a=14.08±3.42, b=13.32±5.03), with A. ocellaris exhibiting brighter reddish-yellow tones (ΔE=67.85), contrasting with A. percula's darker greenish-blue hues (ΔE=63.26). The pigment content analysis showed that A. percula had higher melanin content (90 mg/g vs 60 mg/g, p <0.05) while A. ocellaris showed elevated carotenoids (30 μg/g vs <10 μg/g, p <0.05). Histological analysis identified distinct melanocyte architectures—A. percula displayed tightly packed, irregular melanocytes in band-like arrangements, whereas A. ocellaris had sparsely distributed uniform cells with punctate patterning. Transcriptomic analysis identified 1925 differentially expressed genes (641 upregulated, 1,284 downregulated), based on thresholds of |log₂ fold change| ≥ 1 and false discovery rate (FDR) < 0.05, with enrichment in pigment biosynthesis pathways including cytochrome P450-mediated metabolism. Critical regulatory genes (MC1R, tyrosinase, CYPs) exhibited species-specific expression patterns. Melanogenic genes (tyrosinase) were upregulated in A. percula (1.5-fold, p <0.01), correlating with melanocyte proliferation and dark skin, whereas carotenoid-associated CYPs were overexpressed in A. ocellaris (2.3-fold, p <0.01), driving its vibrant coloration. This research highlights the genetic and cellular mechanisms underlying color variation in clownfish and provides valuable insight for ornamental fish breeding and genetic improvement.