Chromosomes were prepared according to the Kligerman and Bloom method (1974). The chromosome preparations were obtained from head kidney tissue. Before preparation fish were treated intraperitoneally with 0.1% colchicine for 3–4 hours. The hypotonisation lasted 20–30 min at room temperature in 0.075 M KCl. Then tissue samples were fixed in 3:1 methanol : acetic acid for 24 hours. Six specimens of N. brieni (three males and three females) and three specimens of Nothobranchius sp. ‘Kasenga’ (one male and three females) were karyotyped with this method. Meiotic chromosome preparations of N. brieni were acquired from testes by the same technique.

Slides were dried by air and stained with 2% Giemsa solution in phosphate buffer at pH 6.8 for 10 min. Karyotypes were analysed under microscope “AxioImager” Karl Zeiss (Germany) equipped with CCD camera and “KaryoImage” Metasystems Software (Germany). In each specimen the chromosome number and type was determined on metaphase plate. Chromosome morphology was determined according to Levan et al. (1964). The chromosomes were classified as metacentric (M), submetacentric (SM), and acrocentric (A). To determine the fundamental number (NF), chromosomes of the M and SM groups were considered bi-armed and those of group A as uni-armed.

The described karyotypes stand apart from those already reported for species of genus Nothobranchius. The karyotype of N. brieni has the chromosomal number 2n = 49/50 and 25 pairs of uni-armed chromosomes in female (50A) and 23 pairs of uni-armed homomorphic and three heteromorphic chromosomes in male (1M + 48A). The karyotype of Nothobranchius sp. ‘Kasenga’ has the same diploid number 2n = 49/50 but a different karyotype structure possessing metacentric, sub-metacentric, and uni-armed chromosomes with 4M + 14SM + 32A for females and 5M + 14SM + 30A for males, while other species of the genus have a considerably lower modal diploid number of only 36 chromosomes (Table 1).

The reduced diploid numbers and heteromorphic chromosomes in males suggest the occurrence of a multiple-sex chromosome system. A trivalent observation in the first meiotic chromosomes in N. brieni and the presence of a bi-armed chromosome exclusively in the male karyotype indicate a multiple-sex chromosome system of the type X₁X₂Y/X₁X₁X₂X₂. One bi-armed neo-Y chromosome has most likely resulted from the Robertsonian fusion between the Y chromosome and an autosome, as has been described for other fish species (e.g., Kitano and Peichel 2012). In N. brieni and Nothobranchius sp. ‘Kasenga’ the Y chromosomes is a large metacentric one, and X₁ and X₂ chromosomes are acrocentric of different sizes. The same-sex chromosome system has been reported only for N. guentheri (Ewulonu et al. 1985) among the 23 previously karyotyped species.

In the genus Nothobranchius and the related Aphyosemyon Mayers, 1924 the evolutionary trend to reduce the total number of chromosomes via acrocentric chromosome fusion was specified (Scheel 1990, Völker et al. 2005). This assumption has been confirmed by the data presented in Table 1. According to this hypothesis, basal taxa have higher chromosome numbers and more acrocentric chromosomes while derived taxa have lower numbers of chromosomes with metacentric chromosomes (Agnese et al. 2006). It is widely accepted that the hypothetical ancestral karyotype of teleostean fishes consisted of 2n = 48-50 acrocentric chromosomes (Ohno et al. 1969, Nakatani et al. 2007). The two species presented in this study have numbers and a structure of karyotype conservative for Actinopterygii fishes (Mank and Avise 2006, Molina et al. 2014). It is supposed that karyotype of N. brieni is similar to that of the hypothetical ancestor of the genus Nothobranchius. There is a lack of molecular genetic data on this species, therefore we are not able to consider its phylogenetic position within the clade.