The genus Belostoma, known colloquially as “giant water bugs,” presents striking cytogenetic diversity and extensive chromosome variability. Notwithstanding, its karyotype evolution is not well understood. We analyzed 8 species of Belostoma (77 samples). The meiotic analysis revealed 2n = 14 + XY for Belostoma horvathi and Belostoma candidulum; 2n = 22 + XY for Belostoma cummings; 2n = 26 + X1X2Y for Belostoma dentatum, Belostoma elongatum, and Belostoma discretum; and 2n = 26 + X1X2X3Y for Belostoma testacopallidum and Belostoma dilatatum. All species showed holokinetic chromosomes. Based on heterochromatin distribution patterns and 18S rDNA, the species of the genus Belostoma were separated into four groups. The analysis of C0t-1 DNA showed that the repetitive DNA, partly composed of microsatellite DNA, was absent on the Y chromosome. Fluorescent in situ hybridization (FISH) using a microdissected X chromosome in species with simple sex system presents uniform hybridization in the nuclear region corresponding to the X chromosome. Species with multiple systems revealed discrete markings. The present data in conjunction with the existing literature led us to propose a new evolutionary hypothesis for the group, with an ancestral karyotype with a low diploid number, simple sex determination system, and nucleolus organizer regions (NORs) on the sex chromosomes. That karyotype would have originated other karyotypes through agmatoploidy, simploidy, heterochromatinization, and movement of the 18S rDNA.

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