Genes Genet. Syst. (2016) 91, p. 77–84 Toward conservation of genetic and phenotypic diversity in Japanese sticklebacks Jun Kitano1* and Seiichi Mori2 1Division of Ecological Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan 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 fishes 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 stickleback species 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 Honshu and exhibit year-round reproduction, which has never been observed in other stickleback populations. Four species of ninespine stickleback, including Pungitius 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 Gasterosteiformes) are generally called Gasterosteus and Pungitius have achieved tremendous sticklebacks. Gasterosteidae contains five genera: phenotypic diversification and thus provide us unique Spinachia, Apeltes, 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., Sea of Japan 2010; Jones et al., 2012a, 2012b; Roesti et al., 2014, 2015; Lake Harutori 40ºN Feulner et al., 2015). Omono River Among sticklebacks, Japanese species have several Shinano River Gensui River, Otsuchi Jintsu River unique characteristics that have not been found in other Tedori River populations (Mori, 1997; Goto and Mori, 2003), as City of Ono Arakawa River 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. fish. 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 Sea of Okhotsk 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 Hokkaido, 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