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1 January 2005 Hybrid Male Sterility between the Fresh- and Brackish-water Types of Ninespine Stickleback Pungitius pungitius (Pisces, Gasterosteidae)
Hiroshi Takahashi, Terumi Nagai, Akira Goto
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Two ecologically distinct forms, fresh- and brackish-water types, of ninespine stickleback coexist in several freshwater systems on the coast of eastern Hokkaido. Recent genetic analyses of 13 allozyme loci revealed genetic separation between the two types even though their spawning grounds were in close proximity. On the other hand, there is only a small difference in mitochondrial DNA (mtDNA) sequence between the two types suggesting that they diverged quite recently or that mtDNA introgression occurred between them. To test for postzygotic reproductive isolating mechanisms and hybrid mediated gene flow, we examined the viability and reproductive performance of reciprocal F1 hybrids. The hybrids grew to the adult size normally and both sexes expressed secondary sexual characters in the reciprocal crosses. The female hybrids were reciprocally fertile, while the male hybrids were reciprocally sterile. Histological and flow-cytometric analyses of the hybrid testis revealed that the sterility pattern was classified as ‘gametic sterility,’ with gonads of normal size but abnormal spermatogenesis. To our knowledge, the present finding is a novel example of one sex hybrid sterility in the stickleback family (Gasterosteidae).


The ninespine stickleback Pungitius pungitius is a small euryhaline fish belonging to the family Gasterosteidae (Pisces) known as sticklebacks, an important model system in evolutionary biology (Mattern, 2004). This species has a nearly continuous circumpolar distribution, occurring in fresh and coastal waters of northern Eurasia and North America (Münzing, 1969; Wootton, 1976). Although its widespread distribution and morphological variability are comparable to those of the threespine stickleback Gasterosteus aculeatus, the ninespine stickleback has received less attention from biologists than the latter species (Wootton, 1976).

Takata et al. (1987) revealed that there are two ecologically and morphologically distinct forms, which co-occur abundantly in several freshwater systems on the coast of eastern Hokkaido, Japan. The two forms are identified as “freshwater type” and “brackish-water type,” though taxonomically undefined, based on the spawning habitat (Takata et al., 1987). They differ from one another in three meristic characters; the freshwater type has a high number of dorsal spines and gill-rakers, and a low number of vertebrae, when compared with the brackish-water type (Takata et al., 1987). The body color of the brackish-water type is typically silvery, and the freshwater type is usually yellowish or greenish brown. Although their habitats frequently overlap in lower reaches, discrete habitat preferences are generally maintained throughout the year. The freshwater type exclusively occupies freshwater areas within river systems, whereas the brackish-water type occupies brackish-water areas, such as estuaries and lagoons. Since these two forms are reciprocally monophyletic (Takahashi et al., 2003), the evolutionary background is differ from that of the anadromous–freshwater system in the threespine stickleback of which freshwater forms were considered to have multiple, independent origins (McKinnon and Rundle, 2002).

Takata et al. (1987) examined allozyme variations between the two types in the Biwase River, eastern Hokkaido, and revealed complete allelic displacement at three of 13 loci examined. They claimed that the two types should be regarded as independent species, according to the biological species concept (Mayr, 1963). On the other hand, Takahashi and Goto (2001) suggested that the fresh- and brackish-water types had diverged quite recently or otherwise exchanged mitochondrial DNA (mtDNA) through introgressive hybridization, on the grounds that there was no obvious difference in their mtDNA control region sequences. Similar examples of discordant patterns of nuclear and mtDNA are abundant in sticklebacks (e.g., Taylor and McPhail, 1999, 2000; Takahashi and Takata, 2000). These studies suggested that mtDNA introgression has erased mtDNA history of the recipient population. Although information about the postzygotic reproductive isolating mechanisms will provide insight into the discrepancy between the allozyme and mtDNA data (e.g., Takahashi and Takata, 2000), little is known about such mechanisms between the two types.

As a first step to examine postzygotic reproductive isolating mechanisms between the two stickleback types, we examined the viability, growth potential, and reproductive performance of their artificial hybrids. It should be noted that these fitness components are part of postzygotic reproductive isolating mechanisms. Although, postzygotic reproductive isolating mechanisms are classified into extrinsic and intrinsic barriers (Coyne and Orr, 2004), the former such as ecological inviability, behavioral sterility (e.g., Rundle and Whitlock, 2001; Vamosi and Schluter, 1999) were not tested in the present study. To elucidate the cause of hybrid sterility found in the present study, we also made histological observation of gonads and comparison of the DNA contents between the gonad and somatic cells.


Rearing of hybrid stocks

Mature fishes of the fresh- and brackish water types of Pungitius pungitius were collected from the Bekanbeushi River, eastern Hokkaido, Japan, in June 2000. Three females and six males of each type were used as parents of artificial hybrids and of controls. We used the semidry method for artificial insemination, because of limited amounts of sperm in sticklebacks. The eggs were pressed out with fingers from a single mature female and halved into two Petri dishes. The testes were surgically removed from a single male and cut with scissors in a drop of normal saline. The halves of the eggs were fertilized with sperm suspensions obtained from a single different type male and with a single same type male as a control. The fertilization rate was estimated by the frequency of eggs that had undergone cleavage at 3 h after fertilization (2- or 4-cell stages). The fertilized eggs gathering in a cluster were detached from each other to avoid suffocation, and then maintained in a Petri dish at 15°C until they hatched. The hatching rate was calculated as the relative percentage of the initial eggs incubated.

Each full sib stock was kept in a separate aquarium (15×10 cm and 10 cm high) for about two months, and then 20 fishes were chosen randomly and transferred to a larger aquarium (60