Genes Genet. Syst. (2016) 91, p. 77–84 Toward conservation of genetic and phenotypic diversity in Japanese

Jun Kitano1* and Seiichi Mori2 1Division of Ecological Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, 2Biological Laboratories, Gifu-keizai University, Kitakata cho 5-50, Ogaki, Gifu 503-8550, Japan

(Received 16 December 2015, accepted 20 February 2016; J-STAGE Advance published date: 10 June 2016)

Stickleback have been established as a leading model system for studying the genetic mechanisms that underlie naturally occurring phenotypic diversification. Because of the tremendous diversification achieved by in various environments, different geographical populations have unique phenotypes and genotypes, which provide us with unique opportunities for evolu- tionary genetic research. Among sticklebacks, Japanese species have several unique characteristics that have not been found in other populations. The sym- patric marine threespine stickleback species Gasterosteus aculeatus and G. nipponicus (Japan Sea stickleback) are a good system for speciation research. Gasterosteus nipponicus also has several unique characteristics, such as neo-sex chromosomes and courtship behaviors, that differ from those of G. aculeatus. Several freshwater populations derived from G. aculeatus (Hariyo threespine stickleback) inhabit spring-fed ponds and streams in central and exhibit year-round reproduction, which has never been observed in other stickleback populations. Four species of ninespine stickleback, including tymensis and the freshwater, brackish water and Omono types of the P. pungitius-P. sinensis complex, are also excellent model systems for speciation research. Anthropogenic alteration of environments, however, has exposed several Japanese stickleback populations to the risk of extinction and has actually led to extinction of several populations and species. Pungitius kaibarae, which is endemic to East Asia, used to inhabit Kyoto and Hyogo prefectures, but is now extinct. Causes of extinction include depletion of spring water, landfill of habitats, and construction of river- mouth weirs. Here, we review the importance of Japanese sticklebacks as genetic resources, the status of several endangered stickleback populations and species, and the factors putting these populations at risk.

Key words: Hariyo stickleback, migration, Musashi stickleback, Pungitius kaibarae, tsunami

Gasterosteus and Pungitius), different geographical popu- INTRODUCTION lations have unique phenotypes, which makes it difficult Stickleback fishes have been established as a leading to count the exact number of species in this family (Nelson, model system for studying the genetic mechanisms that 2006). In this review, when intrinsic hybrid incompati- underlie naturally occurring phenotypic diversification bility, such as hybrid sterility and inviability, has been and speciation (Peichel, 2005; Cresko et al., 2007; Kingsley found between two groups of fishes, we call them different and Peichel, 2007). Fishes belonging to family Gaster- species; otherwise, we call them populations. osteidae (order ) are generally called Gasterosteus and Pungitius have achieved tremendous sticklebacks. Gasterosteidae contains five genera: phenotypic diversification and thus provide us unique Spinachia, , Gasterosteus, Pungitius and Culaea opportunities for evolutionary research (Wootton, 1976, (Nelson, 2006). Because of the tremendous phenotypic 1984; Bell and Foster, 1994; McKinnon and Rundle, diversification achieved by sticklebacks (particularly 2002). During the last two decades, the genetic architec- tures of phenotypic divergence between populations have Edited by Aya Takahashi been extensively studied (Peichel et al., 2001; Colosimo et * Corresponding author. E-mail: [email protected] al., 2004; Cresko et al., 2004; Albert et al., 2008; Kitano DOI: http://doi.org/10.1266/ggs.15-00082 et al., 2009; Shapiro et al., 2009; Greenwood et al., 2011, 78 J. KITANO and S. MORI

2013; Wark et al., 2012; Laine et al., 2013, 2014; Arnegard Gasterosteus nipponicus Gasterosteus aculeatus et al., 2014; Miller et al., 2014). In several cases, genes or even genetic changes that cause morphological diver- gence have been identified (Colosimo et al., 2005; Miller et al., 2007; Chan et al., 2010; Cleves et al., 2014; O’Brown et al., 2015; Indjeian et al., 2016). cis-Regulatory changes in key developmental genes, such as Eda, Pitx1 130ºE 140ºE and GDF6, contribute to morphological divergence between populations inhabiting contrasting environments (Chan et al., 2010; O’Brown et al., 2015; Indjeian et al., N 2016). Furthermore, recent advances in genomic tech- nologies make it possible to find regions under divergent selection between populations in multiple pairs of eco- Hyotan Pond types (Mäkinen et al., 2008a, 2008b; Hohenlohe et al., 2010; Jones et al., 2012a, 2012b; Roesti et al., 2014, 2015; Lake Harutori 40ºN Feulner et al., 2015). Among sticklebacks, Japanese species have several Gensui River, Otsuchi Jintsu River unique characteristics that have not been found in other populations (Mori, 1997; Goto and Mori, 2003), as City of Ono reviewed in detail below. Anthropogenic alteration of environments, however, has exposed several stickleback populations to the risk of extinction and has actually led Tsuya River to extinction of several populations and species (Mori, Pacific Ocean 1997, 2003). Therefore, it is urgent to conserve these genetically important resources for evolutionary genetics Fig. 1. Upper panels indicate that caudal plates are shorter in research. In this paper, we review several unique char- G. nipponicus (Japan Sea stickleback) than in the Pacific Ocean acteristics of Japanese sticklebacks as well as the current population of G. aculeatus. Lateral plates were stained with status and conservation needs of endangered stickleback Alizarin red. Pictures are of first-generation, lab-raised populations. . Lower panel indicates the distribution of these two spe- cies, as well as other locations that are mentioned in this review.

GASTEROSTEUS NIPPONICUS (JAPAN SEA seasonal isolation (Kume et al., 2005), sexual isolation STICKLEBACK) (Kitano et al., 2009), ecological selection against migrants Japanese marine threespine sticklebacks (Gasterosteus) (Kume et al., 2010), hybrid male sterility (Kitano et al., can be genetically classified into two species (Higuchi and 2007, 2009) and ecological selection against hybrids Goto, 1996; Kitano et al., 2007, 2009; Higuchi et al., (Kitano et al., 2009). 2014): Gasterosteus aculeatus and G. nipponicus (Japan The Japan Sea sticklebacks are invaluable genetic Sea stickleback) (Higuchi et al., 2014). Ikeda (1933) was resources for investigating the genetic basis of hybrid the first to report that marine threespine sticklebacks col- male sterility (Kitano et al., 2009). As widely observed lected from the east and west coasts of Japan differed in Drosophila (Coyne and Orr, 2004; Presgraves, 2008), X morphologically in the heights of their caudal lateral chromosomes play substantial roles in hybrid male steril- plates (Fig. 1). While marine G. aculeatus are mainly ity between G. aculeatus and G. nipponicus (Kitano et al., found along the coasts of the Pacific Ocean, G. nipponicus 2009). In addition, G. nipponicus has several other char- is endemic to the Sea of Japan and the acteristics that differentiate it from G. aculeatus. First, and is occasionally found in several coastal regions in the G. nipponicus has a neo-sex chromosome system, which northwest of Japan (Fig. 1) (Higuchi and Goto, 1996; was created by Y-autosome fusion (Kitano et al., 2009; Yamada et al., 2007; Cassidy et al., 2013; Higuchi et al., Yoshida et al., 2014). Second, the courtship dance per- 2014). formed by G. nipponicus males is different from the zig- The presence of reproductive isolation in sympatry also zag dance that is typical of G. aculeatus males (Ishikawa supports the notion that the two taxa are distinct species and Mori, 2000; Kitano et al., 2007, 2008) (Fig. 2). Third, (Higuchi and Goto, 1996; Kitano et al., 2007, 2009). In no freshwater populations derived from G. nipponicus eastern , for example, G. nipponicus and G. have been reported thus far (Higuchi and Goto, 1996; aculeatus are sympatric, but are reproductively isolated Cassidy et al., 2013; Ravinet et al., 2014), whereas there by multiple isolating barriers, including eco-geographical are many G. aculeatus-derived freshwater populations isolation (Kume et al., 2005, 2010; Kitano et al., 2009), across the world (Wootton, 1976, 1984; Bell and Foster, Conserving Japanese sticklebacks 79

Gasterosteus aculeatus Gasterosteus nipponicus (Threespine stickleback) (Japan Sea stickleback) Male Female Male Female

Zigzag approach Rolling approach Head-up posture Head-up posture Weak or no Intense and frequent dorsal pricking dorsal pricking Waiting Pushed back

Nest care Waiting away from the nest Nest care Zigzag dance Follows Rolling dance Showing the nest Follows Enters the nest Showing the nest Enters the nest

Fig. 2. Simplified schema of behavioral chain reactions between males and females during courtship.

1994). This makes G. nipponicus an excellent model for investigation of the genetic basis for constraints of fresh- water colonization (Ravinet et al., 2014; Ishikawa et al., 2016).

THREESPINE STICKLEBACKS IN SPRING-FED HABITATS In the southern part of Japan, freshwater habitats of G. aculeatus are restricted to spring-fed ponds and streams (Mori, 1997). Although the upper lethal temperatures vary among stickleback populations and can be influ- enced by the environment’s salinity, sticklebacks are gen- erally cold-water fishes, and optimal temperatures for stickleback growth and reproduction are usually below 20 °C (Ikeda, 1933; Wootton, 1984). Because the south- ern parts of Honshu become hot during summer, the majority of ponds and streams are not suitable for stick- lebacks (Mori, 1997); however, some of those fed by cold underground spring water are inhabitable (Mori, 1985, 1997). For example, in tributaries of Tsuya River in Gifu pre- fecture (Fig. 3), which are at the southern limit of the nat- ural distribution of freshwater G. aculeatus, the water temperature is constantly near 15 °C (Mori, 1985). Freshwater populations in this area are called the Hariyo threespine stickleback (Fig. 3). The constant environ- ment of this area is likely responsible for enabling the Fig. 3. Tsuya River (upper panel) and a freshwater threespine threespine sticklebacks to breed almost throughout the stickleback, which is called the Hariyo threespine stickleback, in a spring-fed pond in a tributary of Tsuya River (lower panel). year (Mori, 1985); this year-round reproduction is unique to Japanese freshwater populations in spring-fed habitats in Honshu and has never been reported in other Furthermore, the Hariyo stickleback is a good model threespine stickleback populations (Baker, 1994). As a system for investigating the genetic basis of convergent result, the populations comprise an interesting model sys- evolution. The divergence time between the Hariyo pop- tem with which to explore the genetic and physiological ulations and their marine ancestors was estimated to be mechanisms underlying variation in the timing and dura- 0.37–0.43 million years ago based on the analysis of par- tion of reproduction. tial sequences of the mitochondrial cytochrome b gene 80 J. KITANO and S. MORI

(Watanabe et al., 2003), which is older than that of other with continuous rows of lateral plates were classified as freshwater G. aculeatus populations in the world P. sinensis and fish with middle gaps in the plate rows (Hagland et al., 1992; Bell and Foster, 1994; McKinnon were classified as P. pungitius. However, genetic data and Rundle, 2002). Although similar phenotypic changes indicate that the P. pungitius-P. sinensis complex may be are usually observed, such as reduced numbers of armor more appropriately separated into freshwater, brackish plates, reduced thyroid hormone levels, shortened spine water and Omono types (Takata, 1987; Takahashi and lengths and increased body depth, the genetic bases for Goto, 2003). Among these, the brackish water type the parallel phenotypic changes often differ between seems to be most genetically similar to North American Hariyo and other freshwater stickleback populations and European populations of P. pungitius (Takahashi and (Schluter et al., 2004; Colosimo et al., 2005; Kitano et al., Goto, 2003). 2010; Jones et al., 2012a; O’Brown et al., 2015). For Genetic distinction of these four species is supported by example, although a reduction of armor plate number is the presence of reproductive isolation between sympatric caused by a mutation at the same nucleotide position in species. In eastern Hokkaido, for example, the brackish a regulatory region of Eda (Schluter et al., 2004; O’Brown water type, the freshwater type and P. tymensis often et al., 2015), mutations might occur independently in the inhabit the downstream, midstream and upstream of a Hariyo and other freshwater populations, because Hariyo single river, respectively, but their distributions often does not have the typical freshwater Eda haplotype that overlap. In those areas, the frequencies of hybrids are is widely observed in other freshwater populations low, and reproductive isolation is achieved through a com- (Colosimo et al., 2005). bination of eco-geographical isolation, ecological selection against migrants and hybrid male sterility (Takahashi et al., 2005; Tsuruta et al., 2008; Ishikawa et al., 2013). In NINESPINE STICKLEBACK SPECIES COMPLEX several areas along Omono River in prefecture, the Japanese ninespine sticklebacks are also excellent Omono type and the freshwater type are sympatric, but model systems for speciation research. In Japan, there strong assortative mating maintains a low frequency of are currently four species of ninespine sticklebacks (Fig. hybridization (Tsuruta et al., 2002). 4): Pungitius tymensis, and the freshwater, the brackish Importantly, hybrids between the brackish water type water and the Omono types of the P. pungitius-P. sinensis and the freshwater type show hybrid male sterility complex (Takata, 1987). Another species, P. kaibarae, (Takahashi et al., 2005). Hybrids between P. tymensis which is endemic to East Asia, used to inhabit Kyoto and and the freshwater type or the brackish water type also Hyogo prefectures, but is now extinct (Mori, 1997). show hybrid sterility (Kobayashi, 1959) (J. Kitano, per- Pungitius pungitius and P. sinensis were previously dis- sonal observations). Thus, these systems offer unique tinguished according to lateral plate morphology: fish opportunities to test the generality of large-X effects on hybrid abnormality in sticklebacks. In Honshu, similar to threespine sticklebacks, nine- spine sticklebacks inhabit only spring-fed streams and ponds and tend to have longer breeding seasons than those in Hokkaido (Sugiyama and Mori, 2009). One iso- lated population of the freshwater type can be found in a P. tymensis tributary of Arakawa River in Kanto region and is called the Musashi ninespine stickleback (Kanazawa, 2009). This population lacks lateral plates and has only caudal scutes (Igarashi, 1968). The genetic basis for phenotypic changes observed in the Musashi sticklebacks has not yet Freshwater type been investigated.

ENDANGERED STATUS OF THESE INVALUABLE Brackish water type GENETIC RESOURCES Unfortunately, many stickleback populations are now endangered. For example, capture rates of Japan Sea sticklebacks have been decreasing in rivers and estuarine Omono type lakes, including Shinano River, Tedori River and Jintsu Fig. 4. Four ninespine stickleback species (genus Pungitius): P. River (Goto, 2003). This is mainly due to several weirs tymensis and freshwater, brackish water and Omono types of that were constructed near the mouths of rivers and that the P. pungitius-P. sinensis complex. Scale bars, 10 mm. block the access of adult sticklebacks to spawning Conserving Japanese sticklebacks 81 grounds (Goto and Mori, 2003). The weirs may also CONCLUSIONS block descending migration of Japan Sea juveniles, and because Japan Sea sticklebacks have lower abilities to Recent advances in genome sequencing technologies survive in freshwater environments (A. Ishikawa and J. enable us to investigate the genetic and genomic basis for Kitano, unpublished observations), trapping in freshwa- adaptive evolution and speciation at the molecular level ter environments may be lethal for them. Urbanization (Nadeau and Jiggins, 2010; Seehausen et al., 2014). Fur- and landfill of spawning habitats also occur in many thermore, genome editing technologies enable us to con- places. duct genetic engineering not only in model organisms but In Honshu, the abundance of freshwater habitats for also in natural populations to test phenotypic effects of both threespine and ninespine sticklebacks is also decreas- genetic changes (Jinek et al., 2012). Japanese stickle- ing (Mori, 1997). One notable example is P. kaibarae, backs provide us with excellent models to investigate the which is now extinct in Japan. Two of the main causal genetic mechanisms that underlie phenotypic diversifica- factors are the depletion of spring water and the loss of tion and speciation. habitats due to landfill. Because urban development It should be noted that we could not review all of the often results in disturbances, such as bank protection and Japanese stickleback populations in this paper. For overuse of groundwater, the number of inhabitable example, some populations of Japanese G. aculeatus, such springs with constantly cold water temperatures is as the Hyotan Pond and Lake Harutori populations, show decreasing, and, as a result, the Hariyo threespine stick- partial migration (Mori, 1990; Kitamura et al., 2006): leback, the Omono ninespine stickleback and the Musashi some fish within a population migrate, while others do ninespine stickleback have all become endangered (Mori, not. These populations are a valuable resource for inves- 1997; Kanazawa, 2009; Sugiyama and Mori, 2009). It tigation of the genetic and physiological basis of variation should be noted that spring water may also be important in migratory behavior (Mori, 1990; Kitamura et al., 2006; for the recovery of stickleback habitats that are threat- Kitano et al., 2012). Although the reasons are unknown, ened by natural disasters. For example, after a tsunami Japanese marine forms of G. aculeatus have also tended struck the stickleback habitats of Otsuchi City in Tohoku to decrease in number. region on March 11, 2011 (Fig. 5), spring water was It is urgent to conserve these invaluable populations observed to supply clean water, which helped to flush out and species. For conserving the Japan Sea sticklebacks, oil and debris that were brought in by the tsunami (Mori, it will be necessary to design weirs and dams that enable 2013). them to easily migrate between the sea and spawning Another risk factor for stickleback populations is the grounds. Ecological restoration of spawning habitats is straightening of rivers, which generally reduces ecological also essential. The best way to conserve freshwater pop- diversity (Nakamura et al., 2014). As reviewed above, ulations in spring-fed habitats is to maintain spring different species exploit different ecological niches, so it is waters. Electric pumping of ground water has success- essential to preserve ecologically diverse environments in fully helped to sustain several Japanese freshwater pop- both coastal lakes and rivers in order for multiple stick- ulations (Mori, 1997). At the same time, it is necessary leback species to co-exist sympatrically (Kume et al., to explain the importance of sticklebacks to local people: 2010; Ishikawa et al., 2013).

Fig. 6. Hongan Shozu, a spring-fed habitat of threespine stick- Fig. 5. A stickleback habitat in Otsuchi City struck by a tsu- leback in the city of Ono, and the Stickleback Conservation Cen- nami in 2011. ter. 82 J. KITANO and S. MORI for example, a center for conservation and observation of Cresko, W. A., Amores, A., Wilson, C., Murphy, J., Currey, M., a freshwater population of threespine stickleback was Phillips, P., Bell, M. A., Kimmel, C. B., and Postlethwait, J. built in the city of Ono in Fukui prefecture (Fig. 6), where H. (2004) Parallel genetic basis for repeated evolution of armor loss in Alaskan threespine stickleback populations. local people, including elementary and high school stu- Proc. Natl. Acad. Sci. USA 101, 6050–6055. dents, can visit. To conserve these invaluable resources, Cresko, W. A., McGuigan, K. L., Phillips, P. C., and Postlethwait, it will be necessary to conduct further investigation into J. H. (2007) Studies of threespine stickleback developmental population risk factors and to coordinate conservation evolution: progress and promise. Genetica 129, 105–126. plans with local communities and policymakers. Feulner, P. G. D., Chain, F. J. J., Panchal, M., Huang, Y., Eizaguirre, C., Kalbe, M., Lenz, T. L., Samonte, I. E., Stoll, M., Bornberg-Bauer, E., et al. (2015) Genomics of divergence This research was supported by Grants-in-Aid for Scientific along a continuum of parapatric population differentiation. Research on Innovative Areas from the Ministry of Education, PLoS Genet. 11, e1004966. Culture, Sports, Science and Technology to J.K. (23113007 and Goto, A. (2003) Biodiversity and conservation research on 23113001), the Environment Research and Technology Develop- sticklebacks. In: The Natural History of Sticklebacks (eds.: ment Fund from the Ministry of the Environment (4ZD-1203) to Goto, A., and Mori, S.), pp. 233–249. Hokkaido University S.M. and J.K., and the NIG Cooperative Research Program Press, Sapporo. (2011- A69 and 2012-A63) to S.M. We thank all members of the Goto, A., and Mori, S. (2003) The Natural History of Stickle- Kitano Lab and the Mori Lab for discussion, and two anonymous backs. Hokkaido University Press, Sapporo. reviewers for helpful comments on the manuscript. Greenwood, A. K., Jones, F. C., Chan, Y. F., Brady, S. D., Absher, D. M., Grimwood, J., Schmutz, J., Myers, R. M., Kingsley, D. M., and Peichel, C. L. (2011) The genetic basis REFERENCES of divergent pigment patterns in juvenile threespine stickle- backs. Heredity 107, 155–166. Albert, A. Y., Sawaya, S., Vines, T. H., Knecht, A. K., Miller, C. Greenwood, A. K., Wark, A. R., Yoshida, K., and Peichel, C. L. T., Summers, B. R., Balabhadra, S., Kingsley, D. M., and (2013) Genetic and neural modularity underlie the evolution Schluter, D. 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