Introduction: Needlefish (Belonidae family) comprises 44 known species distributed worldwide. These species are predominantly marine but include estuarine representatives and 12 freshwater species. Among the recognized species, 8 are endemic to South American rivers. Cytogenetic studies of Belonidae are scarce and mostly limited to describing the diploid chromosome number (2n) and karyotypic structure. Methods: We used classical and molecular cytogenetic markers to karyotypically characterize Pseudotylosurus microps to understand the evolutionary processes of Belonidae species in the Amazon basin. Results:P. microps exhibited different diploid numbers between males (2n = 47, 3m + 3sm + 41st/a FN = 53) and females (2n = 48, 4m + 4sm + 40st/a FN = 56). Our study revealed the first case of multiple sex chromosomes in the Belonidae family. Conclusion: These findings describe a multiple sex chromosome system of the type X1X1X2X2/X1X2Y. The C-banding pattern and 5S rDNA mapping suggest that this system likely resulted from a tandem fusion between a homolog of pair 1 and a homolog of pair 3, producing a large acrocentric Y chromosome. We propose that karyotypic changes due to internal chromosomal rearrangements, as observed in P. microps, can lead to species diversification and, in some cases, the emergence of a heteromorphic and multiple sex chromosome system.

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