Peculiar pigment pattern and population profile of a poisonous pufferfish

Pufferfish are well-known for their toxicity, yet they also exhibit a remarkable diversity of pigment patterns. Mushifugu ( Takifugu exascurus ) is a pufferfish endemic to Japan’s coastal waters and is characterized by conspicuous labyrinthine patterns. Despite being recorded along both the Sea of Japan and the Pacific coasts, it has a limited distribution and is rarely observed. Aside from its unique body pattern, mushifugu shows little to no morphological differences from other Takifugu species, often leading to speculation that it may be an interspecific hybrid. In addition, previous theoretical and empirical studies have shown that complex camouflage-like labyrinthine patterns can emerge through the ‘pattern blending’ caused by hybridization between spotted species, providing support for this possibility. Here, we investigate the phylogenetic origin of mushifugu and its distinctive pattern through population structure analysis and demographic inference in comparison with its closest spotted relative, komonfugu ( T. flavipterus ). Mitochondrial DNA (mtDNA) analysis revealed two regional haplogroups within mushifugu—one in the Sea of Japan (SJ) and the other in the Pacific Ocean (PO). In the haplotype network, the SJ haplogroup formed a distinct cluster, whereas the PO haplogroup appeared as its own cluster connected to the komonfugu haplogroup. By contrast, genome-wide single nucleotide polymorphism (SNP) analyses indicated limited structure between the SJ and PO mushifugu populations, while clearly separating mushifugu from komonfugu. Coalescent-based demographic inference suggested that the two species diverged following a bottleneck event in the early Pleistocene. These results confirm that mushifugu is a distinct species rather than a recent interspecific hybrid. Nevertheless, evidence of introgression was detected in both mitochondrial and nuclear genomes, suggesting multiple episodes of past hybridization between mushifugu and komonfugu, highlighting the potentially complex evolutionary processes shaping Takifugu species and their pigment patterns.