Short Communication
Short Communication
Divergent karyotypes of the annual killifish genus Nothobranchius (Cyprinodontiformes, Nothobranchiidae)
expand article infoEugene Krysanov, Tatiana Demidova, Bela Nagy§
‡ Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia
§ France Killifish Association, Paris, France
Open Access


Karyotypes of two species of the African annual killifish genus Nothobranchius Peters, 1868, N. brieni Poll, 1938 and Nothobranchius sp. from Kasenga (D.R. Congo) are described. Both species displayed diploid chromosome number 2n = 49/50 for males and females respectively with multiple-sex chromosome system type X1X2Y/X1X1X2X2. The karyotypes of studied species are considerably different from those previously reported for the genus Nothobranchius and similar to the Actinopterygii conservative karyotype.


Africa, chromosome number, karyotype, killifish, Nothobranchius


Annual killifishes belonging to the genus Nothobranchius Peters, 1868 are mainly distributed in eastern Africa but several species are found in central Africa (Wildekamp 2004). They inhabit temporary pools that dry out during the dry season and have specific adaptations for extreme environments. Annual fishes are characterised by specific life history traits of extremely short lifespan and diapause in embryonic development (Furness 2015, Nagy 2015). Their unique biology makes them a model taxon with which to investigate aging, embryonic development, ecology, and natural selection (Cellerino et al. 2015).

Killifishes of the genus Nothobranchius comprise 71 valid species (FishBase 2015). In this genus karyologicaly were described only 23 species (Arai 2011). These species have variable karyotypes with diploid chromosome numbers (2n) ranging from 2n = 16 for N. rachovii Ahl, 1926 to 2n = 43 for N. thierryi (Ahl, 1924) (Scheel 1990). More than 60% of karyotypes in Nothobranchius are characterised by a modal diploid number of 2n = 36-38.

A multiple-sex chromosome system of X1X1X2X2/X1X2Y type has been reported for only one species of Nothobranchius, N. guentheri (Pfeffer, 1893) with a female karyotype consisting of 36 chromosomes and the male karyotype consisting of 35 chromosomes (Ewulonu et al. 1985).

In this paper, the karyotypes of two species, Nothobranchius brieni Poll, 1938 and Nothobranchius sp. from Kasenga, were studied, bringing the number of species studied to 25.

Material and methods

Specimens of N. brieni were collected from a large ephemeral swamp in the Lualaba drainage, near the village of Bukama in Katanga province (Democratic Republic of Congo, 09°11.374'S 25°51.334'E) on 2 April 2013 by E. Abwe, B. Katemo Manda, and B. Nagy, whereas specimens of Nothobranchius sp. from Kasenga (Nothobranchius sp. ‘Kasenga’) were collected in an ephemeral swamp in the Luapula drainage, near Kasenga, a village in Katanga province (D.R. Congo, 10°31.360'S, 28°27.368'E) on 17 April 2015, by E. Abwe, A. Chocha Manda, B. Katemo Manda, and T. Popp (Fig. 1).

Figure 1.

Localities of specimen collections in the Democratic Republic of Congo (1 Nothobranchius brieni 2 Nothobranchius sp. ‘Kasenga’).

Cytogenetic analysis

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 diploid chromosome numbers of N. brieni were 2n = 49 for males and 2n = 50 for females with NF = 50/50 respectively. The female karyotype consisted of 25 pairs of acrocentric chromosomes gradually decreasing in size (Fig. 2a). The male karyotype consisted of 23 pairs of acrocentric chromosome and one bi-armed pair and two unpaired acrocentric chromosomes (Fig. 2b). In the first meiotic chromosomes during spermatogenesis 23 bivalents and a trivalent were observed at diakinesis (Fig. 2c).

Figure 2.

Karyotypes of Nothobranchius brieni a somatic chromosomes of female b somatic chromosomes of male c meiotic metaphase I (testicular). (A – acrocentric chromosomes). Note trivalent chromosome (arrowed). Scale bar: 10 µm.

The karyotype Nothobranchius sp. ‘Kasenga’ had diploid number 2n = 49 for males and 2n = 50 for females with NF = 68/68 respectively. The female karyotype had two pairs of metacentric, seven pairs of sub-metacentric, and 16 pair of acrocentric chromosomes varying in size from large to small (Fig. 3a). The male karyotype had 23 pair of chromosomes similar to the female with one bi-armed and two unpaired acrocentric chromosomes (Fig. 3b).

Figure 3.

Karyotypes of Nothobranchius sp. ‘Kasenga’ a somatic chromosomes of female b somatic chromosomes of male (M – metacentric chromosomes, SM – submetacentric chromosomes, A – acrocentric chromosomes). Scale bar: 10 µm.


Cytogenetic characteristics

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 diploid number (2n) of Nothobranchius species (from Arai 2011 with modifications). *sex chromosome system of the type X1X2Y/X1X1X2X2.

Species 2n References
N. brieni Poll, 1938* 49♂/50♀ Current study
N. eggersi Seegers, 1982 36 Scheel 1990
N. elongatus Wildekamp, 1982 38 Scheel 1990
N. foerschi Wildekamp & Berkenkamp, 1979 34 Ewulonu et al. 1985, Scheel 1990
N. furzeri Jubb, 1971 38 Reichwald et al. 2009
N. guentheri (Pfeffer, 1893)* 35♂/36♀ Ewulonu et al. 1985, Scheel 1990
N. hengstleri Valdesalici, 2007 38 Wildekamp et al. 2009
N. janpapi Wildekamp, 1977 38 Scheel 1990
N. jubbi Wildekamp & Berkenkamp, 1979 34 Scheel 1990
N. kirki Jubb, 1969 36 Scheel 1990
N. korthausae Meinken, 1973 36 Scheel 1990
N. krysanovi Shidlovskiy, Watters & Wildekamp, 2010 18 Shidlovskiy et al. 2010
N. kuhntae (Ahl, 1926) 38 Scheel 1990
N. lucius Shidlovskiy, Watters & Wildekamp, 2010 36 Wildekamp et al. 2009
N. makondorum Shidlovskiy, Watters & Wildekamp, 2010 38 Wildekamp et al. 2009
N. melanospilus (Pfeffer, 1896) 38 Ewulonu et al. 1985
N. microlepis (Vinciguerra, 1897) 24 Scheel 1990
N. palmqvisti (Lönnberg, 1907) 36 Ewulonu et al. 1985, Scheel 1990
N. polli Wildekamp, 1978 36 Ewulonu et al. 1985
N. patrizii (Vinciguerra, 1897) 36 Ewulonu et al. 1985
N. pienaari Shidlovskiy, Watters & Wildekamp, 2010 34 Shidlovskiy et al. 2010
N. rachovii Ahl, 1926 16 Ewulonu et al. 1985, Krysanov 1992
N. steinforti Wildekamp, 1977 36 Scheel 1990
N. thierryi (Ahl, 1924) 43 Scheel 1990
Nothobranchius sp. ‘Kasenga’* 49♂/50♀ Current study

Sex chromosomes

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.

Karyotype evolution

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.


This research work was supported by RFBR (Russian Foundation for Basic Research) #16-04-01102.


  • Agnèse JF, Zentz F, Legros O, Sellos D (2006) Phylogenetic relationships and phylogeography of the Killifish species of the subgenus Chromaphyosemion (Radda, 1971) in West Africa, inferred from mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 40(2): 332–346. doi: 10.1016/j.ympev.2006.03.018
  • Cellerino A, Valenzano DR, Reichard M (2015) From the bush to the bench: the annual Nothobranchius fishes as a new model system in biology. Biological Reviews 34(2): 511–533. doi: 10.1111/brv.12183
  • Dorn A, Musilova Z, Platzer M, Reichwald K, Cellerino A (2014) The strange case of East African annual fish: aridification correlates with diversification for a savannah aquatic group? BMC Evolutionary Biology 14: 210. doi: 10.1186/s12862-014-0210-3
  • Ewulonu UV, Haas R, Turner BJ (1985) A multiple sex chromosome system in the annual killifish, Nothobranchius guentheri. Copeia 2: 503–508. doi: 10.2307/1444868
  • Furness AI (2015) The evolution of an annual life cycle in killifish: adaptation to ephemeral aquatic environments through embryonic diapause. Biological reviews of the Cambridge Philosophical Society 91(3): 796–812. doi: 10.1111/brv.12194
  • Kitano J, Peichel C (2012) Turnover of sex chromosomes and speciation in fishes. Environmental Biology of Fishes 94: 549–558. doi: 10.1007/s10641-011-9853-8
  • Kligerman AD, Bloom SE (1977) Rapid chromosome preparations from solid tissues of fishes. Journal of the Fisheries Research Board of Canada 34(2): 266–269. doi: 10.1139/f77-039
  • Krysanov EYu (1992) Aneuploidy in postnatal ontogenesis of fishes. Acta Zoologica Fennica 191: 177–182.
  • Nagy B (2015) Life history and reproduction of Nothobranchius fishes. Journal of the American Killifish Association 47(4/6): 182–192.
  • Nakatani Y, Takeda H, Kohara Y, Morishita S (2007) Reconstruction of the vertebrate ancestral genome reveals dynamic genome reorganization in early vertebrates. Genome Research 17(9): 1254–1265. doi: 10.1101/gr.6316407
  • Ohno S, Muramoto J, Klein J, Atkin NB (1969) Diploid-tetraploid relationship in clupeoid and salmon fish. Chromozómes Today 2: 139–147.
  • Scheel JJ (1990) Atlas of killifishes of the Old World. TFH. Publications, Neptune, 448 pp.
  • Shidlowsky KM, Watters BR, Wildekamp RH (2010) Notes on the annual killifish species Nothobranchius rachovii (Cyprinodontiformes; Nothobranchiidae) with the description of two new species. Zootaxa 2724: 37–57.
  • Völker M, Ráb P, Kullmann H (2005) Karyotype differentiation in Chromaphyosemion killifishes (Cyprinodontiformes, Nothobranchiidae). I: Chromosome banding patterns of C. alpha, C. kouamense and C. lugens. Genetica 125(1): 33–41. doi: 10.1111/j.1095-8312.2008.00967.x
  • Wildekamp RH, Shidlovskiy KM, Watters BR (2009) Systematics of the Nothobranchius melanospilus species group (Cyprinodontiformes: Nothobranchiidae) with description of two new species from Tanzania and Mozambique. Ichthyological Exploration of Freshwaters 20: 237–254.
  • Wildekamp RH (2004) A world of killies – Atlas of the oviparous cyprinodontiform fishes of the world (Vol. 4). The American Killifish Association, Elyria, Ohio, 398 pp.