Received 24 April 2016; accepted 10 June 2016; published 13 June 2016
Bitterlings are freshwater fish species ascribed to the subfamily Acheilognathinae (Cyprinidae), and are distributed throughout Eurasia, and more widely in East Asia. Three valid genera, Acheilognathus, Rhodeus, and Tanakia  , grouping approximately 80 species/subspecies  , have been recognized. It is known fact that all bitterlings are characterized by peculiar reproductive behavior which involves egg and sperm deposition in the mantle cavity of living freshwater bivalves.
Due to the abundance of species and the ease of artificial fertilization, many hybridization experiments in bitterlings were tried for the purpose of clarification on the phylogenetic relationships of bitterlings, the mechanism of species differentiation, and others. On the one hand  -  have reported some fertile hybrids, but on the other hand they have found that the sex ratio of bitterling hybrids was biased toward males.  observed similar phenomena and made mention of masculinization mechanism of bitterling hybrids. In any case, they had only limited information of chromosomes.
We have been studying hybridization experiments in bitterlings to make clear on the mechanisms of species differentiation and karyotype evolution, and to develop a new variety.
In the present report, the chromosome analysis of the masculinized hybrid between female T. limbata and male T. signifer in bitterlings was done.
2. Materials and Methods
2.1. F1 Hybrids between Female Tanakia limbata and Male T. signifer
Thirty eggs from a female of T. limbata and sperms from a male of T. signifer were fertilized artificially. Hatchability was 83% (25 hatched embryos/30 eggs). Survival rate at about one year old after hatching was 32% (8 fishes/25 hatched embryos), and all these 8 hybrids had vivid colors just like a male.
Sperms were gotten from a F1 hybrid. Chromosomal slides of this fish were made from kidney and testis cells. And also chromosomal slides of other three F1 hybrids and their parents were obtained from kidney cells.
2.2. F2 Hybrids between Female Rhodeus ocellatus and Male F1 Hybrid
Thirty-five eggs from a female R. o ocellatus and sperms from a male F1 hybrid (T. limbata ♀ × T. signifer ♂) were fertilized artificially. Fertilization rate was 57% (20 fertilized eggs/35 eggs used). Chromosomal slides of eight F2 hybrids were obtained from gastrula cells. Hatching of remaining embryos has not been found.
2.3. Meiosis of Rhdeus atremius Fangi
Chromosomal slides of R. a. atremius were made from kidney and testis cells to compare with F1 hybrid.
2.4. Chromosomal Slides
All of these chromosomal slides were made by direct air-drying method and chromosomes were stained with Giemsa. Karyotytpes of F1 hybrids were analyzed from twenty metaphases in each individual.
3. Results and Discussion
Karyotypes of T. limbata, T. signifer, and R. o. ocellatus, as shown in Figures 1-3 respectively, had 2n = 48 including 8 metacentrics (M), 20 submetacentrics (SM) and 20 subtelocentrics (ST) or acrocentrics (A). Three karyotypes were quite similar and there was not distinct difference among them.
The karyotype of F1 hybrid, shown in Figure 4, had 48 chromosomes and the same chromosomal constitution (8M + 20SM + 20ST/A) of their parents, in all metaphases observed. The distinction on the karyotype among them and the chromosomal aberration were not recognized, and it was estimated that F1 hybrid had the intermediate karyotype between the parents. And, unusual metaphase chromosomal figures at the first cleavage of meiosis were observed (Figure 5a) compared to a normal figure observed in R. a. fangi (Figure 5b). R. a. fangi (2n = 46, Figure 5c) had 23 bivalent chromosomes in meiosis (Figure 5b). And in the metaphase figure of F1 hybrids, many univalent chromosomes were found besides some bivalent chromosomes (Figure 5a). And then, the chromosomal number (bivalents + 2 × univalents) in each metaphase was 48. So, the omission of the chromosome was not recognized in that stage.
Metaphase figures from 8 embryos in F2 hybrid (R. o. ocellatus ♀ × F1 ♂) were observed (Figure 6). The distribution of the chromosomal numbers in F2 hybrid is shown in Table 1. All embryos had wide distribution and the mode varied from individual to individual. In some metaphase figures, the structural chromosomal aberration was found (Figure 6b). It is presumed that the variation of the mode was due to the difference of the chromosomal number in each sperm of F1, probably resulting from the incomplete pairing of homologous chromosomes in meiosis. In some lethal hybrids of salmon, trout and char, similar phenomena, i.e. the wide distribution of chromosomal numbers and the occurrence of the structural chromosomal aberration, have been observed  -  . It has been speculated that those phenomena were due to the abnormal cell division during early
Figure 1. Karyotypes of T. limbata.
Figure 2. Karyotype of T. signifer.
Figure 3. Karyotype of R. o. ocellatus.
Figure 4. Karyotype of F1 hybrid (T. limbata ♀ × T. signifer ♂).
Figure 5. Two metaphase figures of F1 hybrid (a) and R. a. fangi (b) at the first cleavage of meiosis, and a metaphase figure from a kidney cell of R. a. fangi (c).
Figure 6. Two metaphase figures of F2 hybrid. Arrows indicate obvious structural chromosomal aberrations.
Table 1. Distribution of chromosomal numbers in F2 hybrid.
development, resulting from the chromosomal discord between the parents. The present case can be interpreted in the similar way.
When we classify species, the important point is whether or not they can form hybrid. The species differentiation results from an accumulation of more than one reproductive isolation, and the evolution of the hybrid sterility may be the final important factor for the determination of species differentiation. The factor of the species differentiation may be concerned with that of the hybrid sterility. The study of the factor of the hybrid sterility in bitterlings would lead to the clarification of the mechanism about species differentiation and karyotype differentiation, and also to developing a new variety. The investigation at the gene-level is required to clarify the mechanism in addition to the chromosome-level.