Extensive karyotype variability of African fish genus Nothobranchius (Cyprinodontiformes)

Abstract Karyotypes of 65 species of the genus Nothobranchius Peters, 1868 were reviewed and of those 35 examined first time. The results of present study have shown that fishes of the genus Nothobranchius possessed highly diverse karyotypes. The diploid chromosome number (2n) ranged from 16 to 50. The most frequent 2n was 2n = 36 (in 35 species) while the second one 2n = 38 (in 13 species). Proportion of biarmed chromosomes varied from 0 to 95% between species. Diploid chromosome number variability apparently exists as a result of chromosomal fusions or fissions and extensive karyotypic formula alterations promoting by inversions. Multiple sex chromosomes of system X1X1X2X2/X1X2Y type were found only in karyotypes of 5 species. The extensive karyotype variability, unusual for teleosts, of genus Nothobranchius can be likely associated with the characteristics of its life cycle and inhabiting under unstable environment of East African savannah temporal pools.


Introduction
More than a half of teleost fish examined had diploid chromosomes number 2n = 48-50 (Mank andAvise 2006, Molina et al. 2014). Karyotypes containing either high or low proportions of acrocentrics tend to be more frequent than those with balanced numbers of acrocentric and metacentric chromosomes (Molina et al. 2014). According to Naruse et al. 2004, Galetti et al. 2006and Molina et al. 2014 the karyotype of teleost fishes is stable but intrachromosomal rearrangements such as inversions and centromere shift are common. The association of chromosome rearrangements with speciation is known, especially inversions which can promote the local adaptation due to suppression of recombination and thus accumulation of linked adaptive genes. These then favour the accumulation of genetic incompatibilities between species, reduce fertility of hybrids contributing to reproductive isolation and speciation (Navarro and Barton 2003, Kirkpatrick and Barton 2006, Noor et al. 2001, Rieseberg 2001, Hooper & Price 2017. A higher degree of karyotype variation for freshwater fish species inhabiting a more unstable environment compared to that of marine ones has been demonstrated (Nirchio et al. 2014).
Killishes of the genus Nothobranchius Peters, 1868 comprise 76 valid species (Eschmeyer et al. 2018, FishBase 2018. The main life-style characteristics of killifishes reside in fact that species and their populations inhabiting in ephemeral pools of East Africa are isolated both geographically and temporarily due to extremely short life cycle (Wildekamp 2004;Reichard 2016).
Phylogenetic data based on molecular markers demonstrated that the genus Nothobranchius is a monophyletic assemblage and it includes four geographically separated clades (Dorn et al. 2014). Costa (2018) performed taxonomy analysis of the genus on the basis of morphology and phylogenetic data. Six subgenera were recognised: Adiniops Myers, 1924, Cynobranchius Costa, 2018, Nothobranchius Peters, 1868, Paranothobranchius Seegers, 1985, Plesiobranchius Costa, 2018, and Zononothobranchius Radda, 1969 Karyotypes of 30 species were described earlier and high karyotype variability was revealed (summarized in Arai 2011). The diploid chromosome number (2n) of Nothobranchius species varies from 16 to 50 (Scheel 1990, Krysanov et al. 2016. Two species N. guentheri (Pfeffer, 1893) and N. brieni Poll, 1938 had multiple chromosome system (Ewulonu et al. 1985, Krysanov et al. 2016. Thus, the representatives of the genus Nothobranchius is a good model for studying karyotype differentiation due to high karyotype variability and features of the life cycle. The aim of the study was to characterize karyotype diversity of the genus Nothobranchius and conduct cytogenetic comparison among different species. In present study, we i) reviewed all available data dealing with cytogenetic study of Nothobranchius species and ii) analyzed 35 other species not studied as yet for 2n and karyotype composition using conventional cytogenetic protocol.

Specimens collection
Individuals of Nothobranchius species were collected either from wild populations of East Africa or provided by killifish hobbyists. Geographical data and coordinates are given in supplements.

Cytogenetic analysis
Chromosomes were prepared according to the method of Kligerman and Bloom (1977). The chromosome preparations were obtained from anterior kidney tissue. Briefly, individuals were injected intraperitoneally with 0.1% colchicine solution for 3-4 hours. The hypotonization in 0.075 M KCl was 20-30 min at room temperature. Then tissue samples were fixed in 3:1 methanol: acetic acid for 24 hours.
Slides were air dried and then stained with 2% Giemsa solution in phosphate buffer a (pH 6.8) for 10 min. Chromosomes were analyzed under microscope "AxioImager" Karl Zeiss (Germany) equipped with CCD camera and "KaryoImage" Metasystems Software (Germany). Chromosome morphology was determined according to Levan et al. (1964) and classified as metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a). To determine the fundamental number (NF), chromosomes of the m and sm groups were considered biarmed and those of group st/a uniarmed.
Statistical analysis was done using IBM SPSS 20 package. Data were tested for normality. Regression between the rate of biarmed chromosomes and diploid chromosome number, and the Spearman correlation were calculated.

Subgenus Cynobranchius
Karyotypes of two species belonging to this subgenus were described by Scheel (1981Scheel ( , 1990). The karyotype of N. microlepis had the 2n = 24 and most chromosomes in the karyotype were uniarmed with only one pair of biarmed chromosomes (NF = 26). N. fasciatus had 2n = 34 with 22 uniarmed and 12 biarmed chromosomes.
The lowest 2n was found in two closely related species N. rachovii (2n = 16, NF = 30) and N. krysanovi (2n = 18, NF = 34). Most of chromosomes in their karyotypes were metacentric elements with only one pair of acrocentric chromosomes as described earlier (Scheel 1990, Shidlovskiy at al. 2010. Both species had similar karyotype structure and were distinguished by one additional pair of metacentric chromosomes in N. krysanovi.

Subgenus Paranothobranchius
The only species in the subgenus N. ocellatus has 2n = 30 and uniarmed chromosomes dominated in the karyotype (NF = 40).

Subgenus Adiniops
Eight species had the 2n = 38 with different ratio of uniarmed and biarmed chromosomes. Karyotypes of three species N. fuscotaeniatus, N. geminus and N. luekei possessed 36 uniarmed and only two biarmed chromosomes (NF = 40) while N. vosseleri (NF = 60) karyotype had only 16 uniarmed and 22 biarmed chromosomes. Other species had karyotypes with uniformly decreasing numbers of uniarmed chromosomes from 34 to 26 and numbers of biarmed chromosomes increased correspondingly. Females of N. janpapi had more chromosome than males 2n = 38/37 and multiple sex chromosome system X 1 X 1 X 2 X 2 /X 1 X 2 Y type was revealed. The modal diploid chromosome number 2n = 36 was found for 14 species. Four sister species N. albimarginatus, N. cardinalis, N. rubripinnis and N. ruudwildekampi had similar karyotypes with 34 uniarmed and only 2 biarmed chromosomes (NF = 38). Karyotypes of three species N. eggersi, N. korthausae, and N. wattersi possesed 32 uniarmed and 4 biarmed chromosomes (NF = 40). Females of N. guentheri had more chromosome than males 2n = 36/35 and multiple sex chromosome system X 1 X 1 X 2 X 2 / X 1 X 2 Y type was revealed.
Karyotypes of other species had uniformly decreasing numbers of uniarmed chromosomes from 30 to 14 and numbers of biarmed chromosomes increased correspondingly.
N. flammicomantis possessed the lowest diploid numbers in the subgenus 2n = 20. The karyotype of N. flammicomantis consisted mainly of biarmed chromosomes with one pair of uniarmed chromosomes (NF = 38).

Karyotype characteristics of representatives of the genus Nothobranchius
Karyotypes of 65 species of the genus Nothobranchius were overviewed and those of 35 species reported here for first time. The results of present work have shown that representatives of the genus Nothobranchius possess a highly diverse karyotype. The 2n ranged from 16 to 50. The most frequent was 2n = 36 (35 species) and the second was 2n = 38 (13 species). similar karyotype diversity was found only for one closely related genus Aphyosemion Myers, 1924 among the family Cyprinodontiformes (Völker et al. 2008).
It has been shown that karyotypes of teleost fish consisted mainly of uniarmed or biarmed chromosomes (Molina et al. 2014). We did not find a similar trend in karyotype structure within the genus Nothobranchius. Fully acrocentric or metacentric karyotypes occurred as frequently as intermediate type. Such a high diversity of 2n and karyotype structure could be the result of many inter-and intrachromosomal rearrangements. Scheel (1990) assumed that the karyotype evolution of the Old World Cyprinodontidae proceeded by decreasing the 2n while increasing the proportion of biarmed chromosomes in the karyotype by means of centric fusions. The correlation between the proportion of biarmed chromosomes and 2n was non-significant for the representatives of the genus Nothobranchius in contrast to those of the genus Aphyosemion (Agnèse et al. 2006) since pericentric inversions played essential role in the chromosome evolution of the genus.

Sex chromosomes
Most of the studied species did not display morphologically distinguished sex chromosomes. Sex chromosomes were found only in six species, namely N. guentheri (Ewulonu et al. 1985), N. brieni (Krysanov et al. 2016), N. lourensi, N. janpapi, N. ditte and F. thierryi (this study) where multiple sex chromosome system of X 1 X 1 X 2 X 2 /X 1 X 2 Y type was found. Neo-Y chromosome likely originated through Robertsonian fusion of the original Y chromosome and autosome as was shown for another fish species (Kitano and Peichel 2012). Nothobranchius species with multiple sex chromosomes were found in two subgenera Zononothobranchius (N. brieni) and Adiniops (N. guentheri, N. lourensi and N. janpapi) (Costa 2018). According to molecular data N. guentheri and N. janpapi are not closely related (Dorn et al. 2014). We suppose that multiple sex chromosomes originated in these species independently.

Chromosome evolution of Nothobranchius subgenera
Subgenera Cynobranchius and Plesiobranchius form basal Northern phylogenetic clade (sensu Dorn et al., 2014). It is noteworthy that the species with the most dis-tinctive 2n and karyotype structures, namely N. virgatus and N. microlepis belonged to the basal clade.
Subgenus Nothobranchius corresponds well with the Southern clade (sensu Dorn et al 2014). Karyotype alterations by pericentric inversions were main trends in the karyotype evolution of species with 2n = 38. Four species N. furzeri, N. kadleci, N. orthonotus and N. kuhntae distinguished from each other by the ratio of uniarmed and biarmed chromosomes.
Reductions of diploid chromosomes number by fusions were probably characteristic of species with 2n lower 38. Biarmed chromosomes dominated in the karyotypes of species (N. rachovii and N. krysanovi) with the lowest diploid numbers (16 and 18) in the genus.
Only the species N. ocellatus from the subgenus Paranothobranchius with a distinctive karyotype structure is included in the Southern clade.
Subgenus Zononothobranchius corresponds well with the Inland clade (sensu Dorn et al 2014). Karyotypes of all species except N. malaissei, N. brieni and N. milvertzi have 2n = 36 and ratio of biarmed and uniarmed chromosomes differs among species. The karyotype evolution of the species with the 2n = 36 probably proceeded mainly by pericentric inversions.
Therefore, karyotype evolution of the subgenus proceeded mainly by pericentric inversions or rarest chromosome fusions (or fissions).
Subgenus Adiniops corresponds well with the Coastal clade (sensu Dorn et al 2014). Most species of the subgenus have diploid chromosomes number 36 or 38. And four species have diploid number lower than 36. Karyotype diversity is a result of chromosome fusions, fissions and pericentric inversions. Moreover, three species N. guentheri, N. janpapi and N. lourensi have multiple sex chromosome system.
Thus, two main trends were revealed in chromosome evolution of the genus: chromosome fusions (or rare fissions) and pericentric inversions.

Conclusions
According to our data species of the genus Nothobranchius possess high variability of karyotype structure and diploid chromosome numbers. Such variability exists as a result of chromosome fusions or fissions and pericentric inversion, which is especially characteristic for the species with 2n equal 36 and 38. Centromere fusion apparently took place in formation of karyotypes with reduced 2n (less than 36). In our opinion, variability of Nothobranchius karyotypes is associated with the characteristics of its life cycle and inhabiting in ephemeral partly isolated pools of East African savannah. Karyotype flexibility of Nothobranchius individuals may play adaptive role for survival under unstable conditions.