Phylogeography of the Chinese False Gudgeon, Abbottina Rivularis, in East Asia, with Special Reference to the Origin and Artifcial Disturbance of Japanese Populations

Ichthyological Research (2019) 66:460–478 https://doi.org/10.1007/s10228-019-00686-w

FULL PAPER

Phylogeography of the Chinese false gudgeon, Abbottina rivularis, in East Asia, with special reference to the origin and artifcial disturbance of Japanese populations

Nian‑Hong Jang‑Liaw1,5 · Koji Tominaga1,6 · Chungung Zhang2 · Yahui Zhao2 · Jun Nakajima3 · Norio Onikura4 · Katsutoshi Watanabe1

Received: 7 September 2018 / Revised: 13 February 2019 / Accepted: 18 February 2019 / Published online: 8 March 2019 © The Ichthyological Society of Japan 2019

Abstract The Chinese false gudgeon, Abbottina rivularis, is a common cyprinid fsh that is widely distributed throughout continental East Asia, but exhibits a restricted, discontinuous distribution in western Japan, including Honshu and Kyushu islands. In this study, analyses of mitochondrial (cytochrome b) and nuclear (glyt, myh6, and RAG1) genes were conducted to investigate patterns and magnitudes of intraspecifc diferentiation among A. rivularis populations in Japan and adjacent continental areas. Phylogenetic analysis of the mitochondrial gene sequences resolved four major lineages—the Japan lineage (JL), a northern continental lineage (NCL), and two southern continental lineages (SCL1 and SCL2)—with uncorrected pairwise sequence distances of 9.4–15.2% (estimated divergence times, 7.9–17.1 Myr). Two lineages (JL and SCL1) occurred in both the Honshu and Kyushu districts of Japan. Compared with populations in continental areas, most Japanese populations exhibited less genetic diversity. The JL was divided into two well-diferentiated sub-lineages distributed on Honshu and Kyushu islands, respectively. Kyushu Island, as well as areas on Honshu where the species is known to have been introduced, also harbored the SCL1 lineage, which constituted most of the populations on Kyushu. The applied nuclear DNA data strongly suggest that hybridization between the Japan and continental lineages has occurred on Kyushu Island. The artifcial introduction hypothesis, instead of a two-origin scenario, best explains the origin of the SCL1 in Japan.

Keywords Abbottina rivularis · Freshwater fsh · Phylogeography · East Asia · Artifcial introduction · Mitochondrial DNA (mtDNA) · Nuclear DNA

Introduction

East Asia is a large region that includes several divergent geographic features. Land covers about 12,000,000 km2 (about 9% of all land on Earth) in this region, which includes Electronic supplementary material The online version of this part of the Russian Far East, China, the Korean Peninsula, article (https://doi.org/10.1007/s10228-019-00686-w) contains northern Vietnam, and surrounding islands, and several supplementary material, which is available to authorized users.

* Nian‑Hong Jang‑Liaw 3 Fukuoka Institute of Health and Environmental Sciences, 39 [email protected] Mukaizano, Dazaifu, Fukuoka 818‑0135, Japan * Katsutoshi Watanabe 4 Fishery Research Laboratory, Kyushu University, 2506 [email protected]‑u.ac.jp Tsuyazaki, Fukutsu, Fukuoka 811‑3304, Japan 5 Present Address: Conservation and Research Center, Taipei 1 Department of Zoology, Division of Biological Zoo, No. 30, Sec. 2, Xinguang Road, Wenshan District, Sciences, Graduate School of Science, Kyoto University, Taipei 11656, Taiwan Kitashirakawa‑Oiwake, Sakyo, Kyoto 606‑8502, Japan 6 Present Address: Kwansei Gakuin Senior High School, 1‑155 2 Institute of Zoology, Chinese Academy of Sciences, 1‑5 Uegahara‑ichibancho, Nishinomiya, Hyogo 662‑8501, Japan Beichen West Road, Chaoyang District, Beijing 100101, China

Vol:.(1234567890)1 3 Phylogeography of Abbottina rivularis 461 large rivers (e.g., the Amur River, Yellow River, Changjiang accompanied by the stocking of commercially valuable fsh River, and Pearl River) run through the continental area. In (Tsukahara 1954; Hosoya 2001; Matsuzawa and Senou addition, climate patterns vary with latitude and altitude. 2008). In Lake Biwa, the largest lake in Japan, located in the Due to the complicated topography and climatic character- Kinki Region, the indigenousness of this cyprinid is unclear istics, the biota of East Asia is complex and provides ample because of its limited occurrence there (Miura1966; Naka- subject matter for biogeographic studies (Li and Li 1997; mura 1969; Nakajima and Nakagawa 2007; Matsuzawa and Xiao et al. 2001; Kita and Kato 2004; Motokawa and Kaji- Senou 2008). hara 2017). In addition to continental East Asia, thousands Over the past few decades, phylogenetic/phylogeographic of islands lie along the Pacifc coast, where high endemism studies using nucleotide sequence information have become is often observed (e.g., Ota 1998; Hsieh 2002; Shih and the most useful approach for the investigation of genetic Suzuki 2008). backgrounds in terms of population biogeography, evolu- Japan is an island country with more than 3,000 islands tionary systematics, and conservation (Avise 2000; Wata- extending along the Pacifc coast of East Asia, stretching nabe et al. 2006; Beheregary 2008). To reconstruct evolu- from the Sea of Okhotsk in the north to the East China Sea tionary lineages in animals, mitochondrial (mt) DNA is a and Taiwan in the south. The Japanese freshwater fsh fauna suitable candidate marker (Avise 1994; Kocher and Stepien exhibits close relationships to those in adjacent areas, as 1997) and is widely used for various purposes, including the these fsh communities formed as a result of repeated con- study of natural and artifcial processes driving the distribu- nections and separations between continental Asian and tion of animals (Beheregary 2008). The cytochrome b gene Japanese freshwater systems (Lindberg 1972; Nishimura (cytb) has been one of the most frequently utilized segments 1980; Watanabe et al. 2017). The four largest islands (Hok- of mtDNA because it is generally polymorphic within spe- kaido, Honshu, Shikoku, and Kyushu, from north to south; cies, easy to align, and has been characterized in various Fig. 1) together account for 97% of Japan’s land area. The vertebrates, including many fsh species (e.g., Doadrio and four major islands are separated by narrow straits that form Domìnguez 2004; Tang et al. 2006; Yang et al. 2006; Tomi- migration barriers, as do extensive mountain systems in the naga et al. 2016). However, mtDNA alone is often insuf- interiors of these islands. Due to the complex topography cient for phylogenetic/phylogeographic analyses, especially in Japan, the freshwater ichthyofauna shows remarkable in studies of complex evolutionary processes including geographic heterogeneity (Watanabe 2012; Watanabe et al. hybridization or selection (Chan and Levin 2005). Thus, 2017). Using phylogenetic and phylogeographic analyses of nuclear (nc) DNA sequences for phylogenetic reconstruction the widely distributed freshwater species, several attempts have also been increasingly used to address phylogenetic/ have been made to reconstruct the historical formation of the phylogeographic issues (e.g., Li and Ortí 2007; Kawahara Japanese freshwater fauna (Watanabe et al. 2006; Watanabe et al. 2009; Watanabe et al. 2010a). et al. 2017). The present study investigated intraspecifc variations The Chinese false gudgeon, Abbottina rivularis, is a good of A. rivularis in Japan and adjacent areas by sequencing candidate for phylogeographic surveys in Japan and East mtDNA and nuclear genes with three objectives: (1) to Asia. The fsh is a small cyprinid that inhabits shallow zones identify clear phylogenetic relationships within/between of slow or lentic ditches, rivers, and lakes with sandy or Japanese and continental populations, with inference of muddy bottoms (Hosoya 2001). The species occurs widely divergence times; (2) to clarify the native distribution pat- in the East Asian region, including Japan and continental tern of A. rivularis in Japan; and (3) to propose possible East Asia from the Amur River in the north to Fujian and dispersal scenarios of A. rivularis on the Japanese islands. Yunnan provinces of China in the south (Bǎnǎrescu and For a widely distributed species like A. rivularis, cryptic Nalbant 1973; Chu and Chen 1989; Neely et al. 2008); this lineage divergence is often detected by phylogeographic fsh was also introduced into the Mekong River and other analysis (Avise 2000; Watanabe et al. 2017). Knowledge of regions (Vidthayanon and Kottelat 1995; Kottelat 2001; the natural distribution and genetic structure of A. rivularis Tang and He 2015). Although A. rivularis is a common will be helpful in understanding the natural history of the species in China, it is somewhat rare in Japan and is lim- freshwater ichthyofauna of Japan and continental East Asia. ited to several discontinuous areas: the Kanto Plain, Nobi Plain, Kinki Region, and Sanyo Region on Honshu Island, and northwestern Kyushu Island (Miura 1966; Hosoya 2001; Materials and methods Nakajima and Nakagawa 2007; Fig. 1). Few studies have examined the ecology or biogeography of this fsh in Japan Specimens. In total, 209 specimens of Abbottina rivula- (Tsukahara 1954; Nakajima and Nakagawa 2007; Hayashi ris were collected from Japan and continental East Asia et al. 2013), and researchers generally accept that A. rivula- for analyses; in addition, data for eight specimens were ris of the Kanto Plain was artifcially introduced probably obtained from DDBJ/EMBL/GenBank (Table 1; Liu et al.

1 3 462 N.-H. Jang-Liaw et al.

Fig. 1 Locations of sampling sites and lineages for Abbottina rivula- b Complete map of the study area. c, d Larger maps of Kyushu and ris in this study. a Map depicting the eastern part of the Asian Con- central Honshu, Japan. Site numbers and locality codes are listed in tinent. The red broken line indicates the known distribution range Table 1 of this species. The gray-shaded area is shown in more detail in b.

2010; Yang et al. 2006; Xiao and Zhang, unpublished data). Peninsula (LP; C6 and C7), Liao River (LIR; C8–C10), The combined 217 specimens included 118 specimens from Luan River (LUR; C11), Hai River (HR; C12–C14), Yel- 20 localities in Japan and 99 specimens from 24 localities low River (YR; C15), Yangtze River (YZR; C16, C17, and in continental East Asia. These sampling localities were C21), Tsao-Er River (TER; C18), Ming River (MR; C19), classifed into 14 geographic regions or river systems: the Pearl River (PR; C20), and Ayeyarwady River (AYR; C22) Honshu region (HS; sites J1–J9) and Kyushu region (KS; (Fig. 1). Two other cyprinids, Biwia zezera and Microphy- J10–J20) in Japan; the Korean Peninsula (KP; K1); and the sogobio fukiensis, collected from Aichi Prefecture (Japan) Amur River (AR; C1–C5 in China, R1 in Russia), Liaodong and Fujian Province (China), respectively, were used as

1 3 Phylogeography of Abbottina rivularis 463 g4+m7(4), g4+m9 g1+m5(2), g1+m12, g1+m13, g7+m5 glyt + myh6 glyt g3+m7(4) g3+m7(4) g1+m8 g3+m7(4) – g3+m7, g3+m9, – – g3+m10, g3+m11(6) g1+m2(4), g5+m2 g6+m5(2), g1+m2(4) g1+m13 – RAG1 R3(3) – R4 R3(2) R3 R3(3), R4(2) – – R4(6), R5 R6, R7 R8, R9 R9(4) – – m12, m13 myh6 m7(4) m7(4) m8 m7(4) m7 m7(5), m9(2) – – m10, m11(6) m2(5), m5(3), m5(3) m2(4) m13 – ncDNA haplotypes (no. of individuals) haplotypes ncDNA glyt g3(4) g3(4) g1 g3(5) – g3(2), g4(5) – – g3(8) g1(8), g5 g6(2), g7 g1(4) g1 g1 GEDIMAP ID P1605 P1606 P1607 P1608 P1609 P1610 P1611 P1612 P1613 P1614 P1615 P1616 P1617 P1618 2 10 π × 0 0 – 0 – 0.03 (0.02) – – 0.10 (0.03) 0.05 (0.02) 0 0 – – Hd 0 0 – 0 – 0.29 (0.20) – – 0.65 (0.08) 0.40 (0.14) 0 0 – – - Lineages/sub (no. of lineages individuals) JL1(8) JL1(8) SCL1d JL1(18) JL1 JL1 (7) JL1 JL1 JL1(16) SCL1d(18) SCL1d(4) SCL1d(6) SCL1d SCL1d h63, h64(7) h66(2), h67 Haplotypes (no. Haplotypes of individuals) h57(8) h57(8) h58 h59(18) h60 h57, h59(6) h57 h59 h61(7), h62, h48(14), h65, h48(4) h48(6) h68 h68 8 8 1 1 7 1 1 4 6 1 1 18 16 18 n cyt b data gata (HS) gata Ibaraki (HS) Ibaraki (HS) Aichi (HS) Aichi Shiga (HS) Shiga (HS) Wakayama Wakayama (HS) Okayama (HS) Okayama (KS) Fukuoka (KS) Fukuoka (KS) Fukuoka (KS) Fukuoka (KS) Nagaoka, Nii - Nagaoka, Hitachi-Ota, Katori, Chiba Ichinomiya, Ichinomiya, Yoro, Gifu (HS) Yoro, Lake Biwa, Biwa, Lake Kusatsu, Shiga Shiga Kusatsu, Wakayama, Wakayama, Kurashiki, Kurashiki, Onga, FukuokaOnga, Nakama, Nakama, Fukutsu, Shimizu, Taromaru, Taromaru, ), haplotypes, lineages and sub- lineages sizes ( n ), haplotypes, including sample b information cytochrome mtDNA information; and region rivularis localities of Abbottina Sampling systems , including river J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 1 Table - sys Locality and code (river tem or region) lineages, haplotype diversity (Hd), and nucleotide diversity (π); and haplotypes of three nuclear loci (glyt, myh6, and RAG1) myh6, of three nuclear loci (glyt, (π); and haplotypes diversity (Hd), and nucleotide diversity haplotype lineages, Japan

1 3 464 N.-H. Jang-Liaw et al. g1+m5, g1+m14, g1+m14(2) g1+m5(3), glyt + myh6 glyt – g1+m2(2), g7+m14, g8+m2, g9+m14 – g1+m2(2), g1+m2 g1+m2 – g1+m2(2), g2+m4 g1+m2, g1+m14 – – g1+m1 R11, R12 RAG1 – R4, R9, R10, – R9(3), R10 R1 – R9(3) – R9(2), R13, R14 R9 – – – m14(3) myh6 – m2(3), m5, – m2(2), m14(2) m2 m2 m3 m2(2) m4 m2, m5(3) m14 – – m1 ncDNA haplotypes (no. of individuals) haplotypes ncDNA glyt – g1(4), g7, g8, g9 – g1(4) g1 g1 g1(2), g9 g2 g1(4) g1(2) – – g1 GEDIMAP ID P2277 P1619 P1599 P1620 P1596 P1621 P1597 P1622 P1598 P1623 P1624 P1601 P1602 P1600 2 10 π × – 0.17 (0.04) – 0 0.38 (0.10) – 0.63 (0.19) 0 0.31 (0.09) 0 0 0.10 (0.05) 0.57 (0.24) 0.09 (0.03) Hd – 0.67 (0.16) – 0 0.78 (0.14) – 1.00 (0.27) 0 0.89 (0.06) 0 0 1.00 (0.50) 1.00 (0.27) 0.67 (0.14) - Lineages/sub (no. of lineages individuals) NCL JL2(7) SCL1d SCL1d(7) NCL(10) JL2(1) NCL(3) SCL1d(3) NCL(10) SCL1d(7) SCL1d(2) NCL(2) NCL(3) SCL1c(12) h71(2) h15, h16, h17, h18 h23, h24, h25, h26(2) h9, h10 Haplotypes (no. Haplotypes of individuals) h11 h69, h70(4), h12 h48(7) h13(5), h14, h72 h19, h20, h21 h48(3) h13(2), h22(3), h48(7) h48(2) h1, h2 h3, h4, h5 h6(7), h7, h8(2), 1 7 1 7 1 3 3 7 2 2 3 10 10 12 n cyt b data 1 Fukuoka (KS) (AR) Gaizhou, Liaoning (LP) Fukuoka (KS) Liaoning (LP) moto (KS) moto Mongolia (LIR) moto (KS) moto moto (KS) moto longjiang (AR) longjiang (AR) longjiang (AR) longjiang (AR) Tachiarai, Tachiarai, Fusong, Jilin Fusong, Taku, Saga (KS) Saga Taku, Yangshufang, Yangshufang, Yanagawa, Yanagawa, Wanfu, Gaizhou, Wanfu, - Kuma Arao, Horqin, Inner Horqin, - Kuma Nagasu, - Kuma Tamana, Jixian, Hei - Baoqing, Hei - Lanxi, Hei - Harbin, Hei - (continued) J15 C5 J16 C6 J17 C7 J18 C8 J19 J20 C1 C2 C3 C4 1 Table - sys Locality and code (river tem or region) Continental

1 3 Phylogeography of Abbottina rivularis 465 glyt + myh6 glyt – – – – – g1+m1, g1+m6 – – – – – g1+m5 g1+m3 g1+m6 – – RAG1 – – – – – – – – – – – R1(2) – R1, R2(2) – – myh6 – – – – – m1, m3 – – m2 – m2 m5 m3 m6(1) – – ncDNA haplotypes (no. of individuals) haplotypes ncDNA glyt – – – – – g1(2) – – – – – g1 g1 g1(2) – – GEDIMAP ID P2279 P2281 P2281 P2282 P2283 P1595 P2280 P2276 P1604 P1594 P1603 P1593 P1625 P1591 P1592 P2278 2 10 π × 0.19 (0.10) 9.98 (5.0) – – – 10.62 (4.60) 0.09 (0.03) 0.19 (0.07) 0 10.39 (4.90) 7.54 (3.47) 0.79 (0.32) 0.20 (0.04) 0.46 (0.33) – 0 Hd 1.00 (0.50) 1.00 (0.50) – – – 1.00 (0.27) 0.67 (0.31) 1.00 (0.27) 0 0.67 (0.31) 1.00 (0.27) 0.93 (0.08) 0.93 (0.08) 0.42 (0.19) – 0 NCL SCL2 SCL1c SCL1c - Lineages/sub (no. of lineages individuals) SCL1b(2) SCL2, SCL1c SCL1d SCL2 SCL2 SCL1a, SCL1b, SCL2(3) SCL1d(3) SCL1d(4) SCL1b(2), NCL SCL1d(2), SCL1a(7), NCL (10) NCL SCL1a(8), SCL1a SCL1a(2) h32(2), h33(2), h34, h35 h74, h75, h76, h77, h78, h79 Haplotypes (no. Haplotypes of individuals) h41, h42 h45, h46 h47 h51 h52 h27, h28, h33 h43(2), h44 h48, h49, h50 h53(4) h28(2), h29 h54, h55, h56 h30, h31, h13(3), h73, h36(7), h37, h38 h39 h40(2) 2 2 1 1 1 3 3 3 4 3 3 8 9 1 2 10 n cyt b data 5 3 4 2 6 Inner Mongo - lia (LIR) (YZR) (YZR) ang (TER) (PR) (YZR) Sichuan Mongolia (LIR) (YZR) (MR) Fujian nan (AYR) (LUR) Gyonggi-do Gyonggi-do (KP) (HR) (HR) jing (HR) (YR) Balin Youqi, Balin Youqi, Yueyang, Hunan Yueyang, River Yangtze Zheji - Shaoxing, Guilin, Guangxi Huidong, Chifeng, Inner Chifeng, Wuhan, Hubei Wuhan, Wuyishan, - Mongshi, Yun Laoting, Hebei Laoting, Yonchon-gun, Yonchon-gun, Huairou, Beijing Beijing Huairou, Taoyu, Beijing Beijing Taoyu, Mentogo, Bei - Mentogo, Luonan, Shanxi Luonan, (continued) C9 C16 C17-2 C18 C20 C21 C10 C17-1 C19 C22 C11 K1 C12 C13 C14 C15 1 Table - sys Locality and code (river tem or region)

1 3 466 N.-H. Jang-Liaw et al.

outgroups (Yang et al. 2006; He et al. 2017). Furthermore, data from fve specimens of Pseudogobio esocinus and one of Pseudogobio vaillanti were obtained from DDBJ/EMBL/ GenBank (AB793798, AB793858, AB793946, AB793958, and AB793964, Tominaga et al. 2016; AY882900, Xia et al. glyt + myh6 glyt – 2005) and used for the estimation of divergence times. These outgroup taxa of Gobioninae are all closely related to A. rivularis (e.g., Rüber et al. 2007). DNA extraction, amplification, and sequencing. The mitochondrial cytb segment was sequenced for all 209 A. RAG1 – rivularis and the two outgroup specimens. One, two, or three nuclear genes (a total of 204 sequences) for portions of those specimens were sequenced; DNA amplifcation failed for EU934494

6 some specimens, most likely due to insufcient quality of Hai River, KP HS Honshu, HR Hai River, River, Ayeyarwady tissue/DNA samples (or due to a primer mismatch). myh6 – Total genomic DNA was isolated from a piece of mus- cle or fn preserved in 99% ethanol, using a genomic DNA EU934495; 5 purifcation kit (Promega, Tokyo, Japan). ExTaq polymerase (TaKaRa, Otsu, Japan) was used for polymerase chain reaction (PCR) amplifcation of the cytb gene segment, with ncDNA haplotypes (no. of individuals) haplotypes ncDNA glyt –

AY953020; AY953020; ′

4 two oligonucleotide primers of L14724 (5 -TGA CTT GAA RAA CCA YCG YYG-3′; Palumbi et al. 1991) and H15915 (5′-ACC TCC GAT CTY CGG ATT ACA AGA C-3′; Aoy- ama et al. 1999). The PCR conditions consisted of 30 cycles GEDIMAP ID P1626 of denaturation (at 94°C for 15 s), annealing (at 48°C for 15 s), and extension (at 72°C for 30 s) on a PC808 thermal

2 cycler (ASTEC, Fukuoka, Japan). After the PCR products EU934493, AF051856; 10 3 were purifed by treatment with ExoSAP-It (USB Corp., π × – Cleveland, OH, USA), they were sequenced on an automated DNA sequencer (ABI Prism GA3130xl; Applied Biosys-

AYR AR Amur River, to: in parentheses refer 1 ), abbreviations tems, Foster City, CA, USA) using amplifcation primers

Yangtze River YZR Yangtze River, YR Yellow River, TER Tsao-Er River, PR Pearl MR Ming River, River, Luan and the BigDye Terminator Cycle Sequencing FS Ready Hd – Reaction Kit ver. 3.1 (Applied Biosystems). All obtained DNA sequences were deposited in DDBJ/EMBL/GenBank EU934496, EU934497; 2 (accession nos. JX137485–137687, KU052311–052318). Haplotype frequencies of each population were deposited in the GEDIMAP database (accession nos. P1591–1626, - Lineages/sub (no. of lineages individuals) NCL

EU934499; P2276–2283; Watanabe et al. 2010b; http://gedimap.zool. 1 northern SCL southern continental lineage, continental lineage kyoto-u.ac.jp/). Three ncDNA loci [glycosyltransferase (glyt; 709 bp; 77 sequences; accession nos. JX137408–137484), myo- sin heavy polypeptide 6 (myh6; 673 bp; 76 sequences; Haplotypes (no. Haplotypes of individuals) h80 JX137332–137407), and recombination activating gene 1 1 n cyt b data (RAG1; 1482 bp; 51 sequences; JX137281–137331); see López et al. (2004) and Li and Ortí (2007) for detailed PCR conditions and primer information] were determined mainly

Liaodong Peninsula, LUR LP Liaodong Peninsula, LIR Liao River, Kyushu, from Japanese populations (Table 1). Heterozygotic nucleotide sites (only one site in RAG1 sequences) were removed

skoye (AR) skoye from the analysis and treated as missing data. Ozero Sindin - Ozero Genetic diversity and phylogenetic analysis. mtDNA and (continued) ncDNA sequences were aligned with the aid of Molecular Sequences that were downloaded from GenBank: from downloaded that were Sequences Evolutionary Genetics Analysis software (MEGA version 6; R1 1−6 1 Table - sys Locality and code (river tem or region) For each population sampled (alphanumeric locality codes refer to those to in Fig. (alphanumeric population sampled each locality codes refer For Korean Peninsula, KS Peninsula, Korean Japanese lineage, NCL JL Japanese lineage, lineages: for Abbreviations Tamura et al. 2013), and the output was later improved by

1 3 Phylogeography of Abbottina rivularis 467 eye. For cytb data, haplotype diversity (Hd) and nucleotide replicates were used for nodal evaluation as Bayesian pos- diversity (π) within populations and regions were calculated terior probabilities (BPPs) and were sampled every 500 using DnaSP version 5 (Librado and Rozas 2009). Genetic generations. About 25% of sampling trees were discarded distances within and between lineages were calculated using as burn-in. PAUP*4.10 (Swoford 2002), with the best-ftting model of To provide insight into the demographic history of A. TIM3 + G, which was selected using Akaike’s information rivularis from the DNA data, a minimum spanning tree criterion (AIC), obtained from jMODELTEST 2.1.5 (Posada (MST) among cytb haplotypes was generated from the 2008). matrix of pairwise distances calculated between all pairs of The numbers of nucleotide substitutions between cytb haplotypes using a modifcation of the algorithm described haplotypes in the pairwise comparisons were counted using in Rohlf (1973). The MST analysis was performed using a MEGA. Moreover, geographic patterns of genetic difer- program embedded in Arlequin. Furthermore, ncDNA MSTs entiation were evaluated using an analysis of molecular for the glyt, myh6, and RAG1 genes were, respectively, variance (AMOVA), calculated with Arlequin version 3.5 reconstructed. In addition, a combined dataset of ncDNA (Excofer and Lischer 2010), which assessed the extent to sequences was applied to analyze DNA polymorphisms and which genetic variations were attributable to three hierar- the genetic structure. Because few RAG1 segments were chical levels of subdivision: between Japanese and Asian successfully sequenced, sequences from two loci (glyt and continental regions, among populations (sampling locali- myh6) were selected and applied for the combined dataset. ties) within regions, and within populations. In this analysis, A Bayesian tree based on 22 composite haplotypes identifed Φ-statistics were calculated based on uncorrected sequence from 72 combined ncDNA sequences was constructed using diferences (uncorrected p) among haplotypes. The degree MrBayes. The model selection method and management of of diferentiation between the two regions was expressed as generations and burn-in options were the same as those ΦCT, the degree of diferentiation among populations within described above for the cytb analysis. The ncDNA combined regions was ΦSC, and the degree of diferentiation among all dataset was analyzed by Bayesian analysis, using the model populations was ΦST. Whether a value given above difered estimated with jMODELTEST under the BIC [likelihood signifcantly from zero was tested using a nonparametric settings from the best-ftting model (K80 + I); Lset base = permutation method (104 permutations) in Arlequin, with a equal; nst = 2; tratio = 4.4; rates = equal; pinvar = 0.9760]. signifcance level of 0.05. Pairwise F-statistic (FST) values Divergence times between lineages. We estimated the were also computed using the “population comparisons” set- time-calibrated tree based on the cytb sequence data with ting of Arlequin, with a signifcance level of 0.05. the prior assumptions for molecular substitution rate and Phylogenetic trees for cytb haplotypes were constructed divergence times reported in previous studies, calculated using neighbor-joining (NJ), maximum-likelihood (ML), according to the relaxed molecular clock model (Drummond and Bayesian inference (BI) analyses. The NJ and ML et al. 2006). We used molecular substitution rates estimated analyses were performed using PAUP*. The NJ tree was for teleost cytb, ranging from roughly 0.3% to 1.5% per constructed with ties broken randomly using the best-ft- million years (Myr) per lineage (e.g., Burridge et al. 2008; ting model TIM3 + G. ML settings from the best-ftting Watanabe and Takahashi 2010), with a mean of 0.76% model TIM3 + G (gamma shape = 0.3090) with base fre- (for European cyprinids; Zardoya and Doadrio 1999). We quencies of A = 0.2730, C = 0.3028, G = 0.1482, and T = adopted the uncorrelated log-normal relaxed clock model 0.2760, selected using the AIC, obtained from jMODEL- with a normal prior distribution for the mean molecular TEST. The ML analysis was conducted using a heuristic substitution rate, using 0.76% [standard deviation (SD) = algorithm with tree bisection–reconnection (TBR) branch 0.5%] for cytb; this lax constraint covers <0.3–1.6% in a swapping. The same model as the NJ analysis was applied 95% interval. In addition, we used three node constraints in for the ML analysis. Two bootstrap values (1,000 repli- the outgroup Pseudogobio esocinus species complex, which cates for the NJ and 100 replicates for the ML analysis includes three divergent mtDNA lineages, A, B, and C, in with full-heuristic search and TBR branch swapping) were Japan (Tominaga et al. 2016). The dates of these three nodes calculated to obtain a measure of node support for the were estimated in a time-tree analysis using geographic data resulting tree (Felsenstein 1985). Bayesian analyses were (dates of mountain uplift) from Tominaga et al. (2016); two conducted with MrBayes 3.2.1 (Ronquist and Huelsenbeck nodes were associated with vicariance by the Suzuka Moun- 2003), using models estimated with jMODELTEST under tains in central Honshu Island (normal distribution with 1.14 the Bayesian information criterion [BIC; likelihood set- ± 0.18 SD for divergence in lineage A and 1.15 ± 0.20 SD tings from the best-ftting model (TrN + G); Lset base = for divergence in B), and the third node was associated with (0.27, 0.31, 0.15, 0.27); nst = 6; rmat = (1, 12.95, 1, 1, vicariance by the Central Highlands in central Honshu Island 5.39, 1); rates = gamma; shape = 0.3], with 2 × 10 6 gen- (normal distribution with 4.90 ± 1.20 SD for divergence erations of Markov chain Monte Carlo (MCMC) analyses; between B and C).

1 3 468 N.-H. Jang-Liaw et al.

The Bayesian phylogenetic tree and divergence time (the TreeAnnotator in the BEAST package, and the tree was visu- time of the most recent common ancestor; tMRCA) were alized using FigTree v.1.4.3 (Rambaut 2016). Node support estimated using the above assumptions and a Yule (specia- was evaluated based on BPP. tion) tree prior with BEAST 2.4.6 (Bouckaert et al. 2014). The substitution models were selected using the BIC in jMODELTEST (number of substitution schemes = 3): GTR + G with Lset base = (0.2857, 0.3035, 0.1344), nst = 6, rmat Results = (0.7964, 11.8749, 0.5893, 1.1515, 4.9505), gamma shape = 0.2740, ncat = 4, and pinvar = 0. All other model param- Overall cytb variations and genetic diversity. Among the eters were set to the default priors. For MCMC analyses, we 217 Abbottina rivularis specimens analyzed, 381 (36.2%) performed two independent runs of 10 million generations nucleotides were variable and 308 (29.3%) nucleotides were to confrm the consistency of the results. We sampled every parsimoniously informative in the 1,052-bp cytb sequences. 2,000 generations and removed the frst 10% of samples The G + C content was 46%; no insertion, deletion, or stop as burn-in. The convergence of the chains to the station- codon was observed. Overall Hd was 0.93 (SD = 0.01), and ary distribution and large efective sample size (>200) were π was 8.70% (SD = 0.16%) (Table 2). Hd and π values within confrmed using Tracer v.1.6 (Rambaut et al. 2013). The each sampling locality ranged widely at 0.00–1.00 and consensus tree with median node heights was calculated by 0.00–10.62%, respectively (Table 1), and those within each

Table 2 Intra-lineage variation Groups and lineages Sample size No. of hap- No. of poly- Haplotype Nucleotide for geographical groups and lotypes morphic sites diversity (Hd) diversity π × lineages/sub-lineages based 102 on cytochrome b sequences of Abbottina rivularis in this study Groups Japan 118 17 171 0.80 (0.03) 6.71 (0.16) HS 61 8 144 0.73 (0.04) 0.76 (0.40) KS 57 9 141 0.43 (0.08) 3.19 (0.86) Continent 99 64 286 0.97 (0.01) 8.60 (0.23) AR 20 13 172 0.88 (0.07) 7.27 (1.06) HR 12 5 26 0.67 (0.02) 0.55 (0.25) KP 10 8 8 0.93 (0.60) 0.20 (0.04) LIR 16 10 177 0.93 (0.04) 6.41 (1.73) LP 13 9 24 0.87 (0.09) 0.46 (0.10) LUR 8 6 39 0.93 (0.08) 8.52 (0.13) YR 2 2 2 1.00 (0.50) 0.19 (0.10) YZR 10 6 113 0.84 (0.10) 5.41 (0.88) TER 3 3 3 1.00 (0.27) 0.19 (0.07) MR 1 – – – – PR 1 – – – – AYR 3 3 119 1.00 (0.03) 7.54 (4.54) Lineage/sub-lineage JL 68 11 49 0.78 (0.03) 1.00 (0.19) JL1 60 7 13 0.72 (0.04) 0.34 (0.04) JL2 8 4 6 0.75 (0.14) 0.22 (0.05) NCL 42 30 51 0.94 (0.03) 0.42 (0.05) SCL1+2 107 39 181 0.82 (0.04) 2.15 (0.38) SCL1 100 33 68 0.80(0.04) 1.04(0.08) SCL1a 24 12 28 0.90 (0.05) 0.70 (0.10) SCL1b 15 8 11 0.79 (0.11) 0.17 (0.04) SCL1c 61 13 16 0.48 (0.08) 0.11 (0.02) SCL2 7 6 78 0.95 (0.10) 2.34 (1.00) Total 217 80 308 0.93 (0.01) 8.70 (0.16)

Standard deviations are in parentheses; for defnitions of abbreviations, see Table 1

1 3 Phylogeography of Abbottina rivularis 469 region/river system ranged at 0.43–1.00 and 0.19–8.52%, including seven and four haplotypes, respectively, and sepa- respectively (Table 2). rately occupying HS (JL1) and KS (JL2). A total of 80 cytb haplotypes were detected from all A. The NCL was defned by 30 haplotypes from 42 speci- rivularis specimens (Table 1). Fifty-nine of these haplotypes mens, restrictedly found in northern populations of conti- were unique, represented by single individuals; 8 were from nental East Asia, including those from the Liao River (LIR), 6 localities in Japan, 44 were from 20 localities in China LP (on which sites C6 and C7 were located), KP, and the and Russia, and 7 were from a single locality in the Korean Amur River (AR). Peninsula. Fourteen haplotypes were detected from more The SCL1 consisted of 33 haplotypes from 100 speci- than one individual and only at a single sampling site. Seven mens that were widely distributed from AR to AYR in China haplotypes were detected at more than one locality. A high [except C19, Ming River (MR) and C20, Pearl River (PR) in proportion of Japanese A. rivularis (86 of 118 individuals; southern China], as well as on KS and at an isolated locality 72.9%) was identifed as having three haplotypes (h48, 43 on HS, Japan (J3; Fig. 1). SCL1 was further divided into four individuals; h57, 18 individuals; and h59, 25 individuals; sub-lineages (SCLla, SCLlb, SCLlc, and SCL1d), the dis- Table 1). tributions of which overlapped, with no obvious geographic The AMOVA for local samples from the two geographic isolation. Only SCL1a and SCL1c were supported by high regions (Japan and continental Asia) indicated highly signif- support values (Fig. 2). SCL1 haplotypes co-occurred with cant divergence among populations within regions (77.1%) NCL haplotypes in the AR and LIR basins. [Electronic supplementary material (ESM) Table S1]. The The SCL2 consisted of six haplotypes from seven speci- genetic variance between the two regions was signifcant mens that were distributed broadly in China from YZR to (17.3%; P < 0.05), but lesser than that within regions. The AYR, including MR and PR (Fig. 1a, b). Compared with FST value for cytb between the two regions was 0.230 and SCL1, SCL2 occupied more southern rivers in China; it was those between each major Japanese regional population and sympatric with SCL1 in YZR and AYR. Both lineages were the overall continental Asian population were 0.550 (Hon- supported by high support values, as was the SCL1 + SCL2 shu; HS) and 0.252 (Kyushu; KS) (ESM Table S2). The KS group (Fig. 2). showed the lowest FST value with the Tsao-Er River (TER) Among the four lineages, the Hd value was highest population (–0.086), followed by the the Ayeyarwady River for SCL2 (0.95) and lowest for the JL (0.78). The π value (AYR) population (0.151). Among all FST values, values was highest in SCL2 (2.34%) and lowest in NCL (0.42%) were relatively low between two peninsular populations (Table 2). Pairwise TIM3 + G distances between haplo- [Liaodong Peninsula (LP) and Korean Peninsula (KP): types within lineages ranged from 0.0054 (NCL) to 0.0270 0.039], between the Hai River (HR) and Luan River (LUR) (SCL2), and those between lineages ranged from 0.0941 (JL populations (0.082), and those between AYR and the Yellow vs. NCL; uncorrected p = 9.4%) to 0.2754 (NCL vs. SCL2; River (YR: 0.049), AYR and the the Yangtze River (YZR) uncorrected p = 15.2%) (Table 3). (–0.078), and AYR and TER (0.003) populations (ESM Network analysis and estimation of divergence times. Table S2). In the MST, cytb haplotypes of the four major lineages (JL, Cytb phylogeny, lineages, and their geographic dis- NCL, SCL1, and SCL2) were clearly separated (by 91, 133, tributions. The NJ, ML, and BI analyses of the 80 cytb and 96 steps for NCL–JL, JL–SCL1, and SCL1–SCL2, haplotypes of A. rivularis with outgroups yielded trees with respectively) and were in agreement with the major topolo- almost concordant topologies supported by high bootstrap gies inferred from the NJ, ML, and BI trees (Fig. 3a). In the (NJ and ML) and BPP (BI) values (Fig. 2). These trees JL, sub-lineages JL1 and JL2 showed obvious isolation by revealed four major lineages, i.e., the Japan lineage (JL), a 34 steps. Among the JL1 haplotypes, h57 and h59 were con- northern continental lineage (NCL), and two southern con- spicuously dominant, represented by a total of 43 (95.5%) tinental lineages (SCL1, and SCL2) that were highly sup- individuals from central Honshu (sites J4–J9). Unlike other ported in all analyses (Fig. 2). The appellation of lineages lineages showing well-developed bush-like network topolo- was based on phylogenetic relationships between haplotype gies, the topology for JL1 and JL2 was simple and short of groups and their geographical distributions (see below). diferentiation. However, some lineage appellations did not completely The divergence times of mtDNA lineages of A. rivula- refect their current distributions (e.g., the SCL1 haplotypes ris were estimated using the relaxed molecular clock model h48 and h65–h68 in KS and h58 in HS, Japan). Such mis- (Table 4; Fig. 4). The Bayesian estimation of tMRCAs sug- matches are examined in the “Discussion” section. gested that the divergence of A. rivularis began 17.1 million The JL was defned by 11 haplotypes from 68 specimens, years ago (Mya) (95% credible interval, 10.7–25.0 Mya). For representing populations from almost all localities in HS the Japanese lineage, the estimated divergence time of the (except J3 in Kanto) and two localities in KS of Japan. The JL from the continental NCL was 7.9 (4.7–11.7) Mya, and JL was further divided into two sub-lineages (JL1 and JL2), the divergence between JL1 and JL2 occurred 2.5 (1.4–3.9)

1 3 470 N.-H. Jang-Liaw et al.

Fig. 2 Bayesian phylogeny inferred for Abbottina rivularis cytochrome b haplotypes. Numbers near branches are bootstrap and Bayesian posterior probability values (NJ/ ML/BI) for the adjacent nodes. Abbreviations of lineages: NCL northern continental lineage, JL Japanese lineage, SCL southern continental lineages. See Table 1 for abbreviations of sampling locality/river system information for lineages and sub-lineages

Mya (Fig. 4). Two southern continental lineages (SCL1 and the mtDNA cytb data; only JL1 was well identifed and SCL 2) were estimated to have diverged 9.6 (5.9–14.2) Mya. was grouped tightly by the glyt, myh6, and combined data The substitution rate of cytb was estimated at 0.76–0.92% (Figs. 3b–d, 5). Values of π ranged from 0.32% to 0.34% (mean 0.79%)/Myr/lineage. among all sequences of the glyt, myh6, or RAG1 genes Intraspecifc variation and networks for ncDNA genes. (ESM Table S3); these values were much lower than that Data for the three ncDNA genes showed little divergence for the cytb data (8.7%; Table 2). Low nucleotide divergence and unclear boundaries between lineages inferred from was also shown for the topologies of the MSTs (Fig. 3b–d).

1 3 Phylogeography of Abbottina rivularis 471 0.0270 ± 0.0034 105.69 109.21 103.97 105.92 – 159.64 148.42 147.93 148.11 SCL2 0.0097 0.0009 0.1005 ± 0.0032 ± 12.93 22.26 – – 154.50 136.52 137.22 136.97 SCL1c 0.0009 0.1038 ± 0.099 0.0123 ± 0.031 0.0030 ± 21.63 – – 157.97 139.50 139.14 139.27 SCL1b 0.0039 0.0038 0.0016 0.0988 ± 0.098 0.0212 ± 0.0206 ± 0.0080 ± – – 161.72 138.83 140.15 139.67 SCL1a 0.0101 0.0021 0.1445 ± – – – 0.0140 ± – 157.96 138.08 138.75 138.51 SCL1 0.0036 – – – – – 0.0375 ± 158.22 139.67 140.16 139.99 SCL1+2 0.0131 0.0129 0.0131 0.0133 0.0373 0.0125 0.0009 0.1517 ± 0.1469 ± 0.1502 ± 0.1537 ± 0.2754 ± 0.1504 ± 0.0054 ± 101.12 97.75 98.98 NCL 0.0120 0.0113 0.0114 0.0114 0.0047 0.0112 0.0100 0.0013 0.1411 ± 0.1298 ± 0.1326 ± 0.1320 ± 0.2271 ± 0.1328 ± 0.0961 ± 0.0030 ± 37.25 – JL2 0.0118 0.0111 0.0112 0.0114 0.0049 0.0111 0.0097 0.0055 0.0017 0.1406 ± 0.1304 ± 0.1323 ± 0.1332 ± 0.2285 ± 0.1332 ± 0.0929 ± 0.0354 ± 0.0056 ± – JL1 0.1408 ± 0.0119 0.1302 ± 0.0113 0.1324 ± 0.0113 0.1328 ± 0.0115 0.2274 ± 0.0127 0.1331 ± 0.0112 0.0941 ± 0.0094 – – 0.0205 ± 0.003 JL using the TIM3 + G model between major lineages lineages major rivularis of Abbottina b haplotypes partial using the TIM3 + G model between pairwise with cytochrome Estimated standard error for distance values inferred estimates SCL2 SCL1c SCL1b SCL1a SCL1 SCL1+2 NCL JL2 JL1 JL 3 Table or sub-lineages within haplotypes lineages from distances inferred along the average diagonal are in boldface Values the diagonal, respectively and below in 1,052 bp and pairwise lie above diferences The numbers of nucleotide TIM3 + G distance values = 0.1098 ± 0.0009 haplotypes from inferred value Distant overall

1 3 472 N.-H. Jang-Liaw et al.

Fig. 3 Minimum spanning trees (MSTs) based on variation between number of individuals having the corresponding haplotype. Each line haplotype sequences of populations of Abbottina rivularis inferred connecting two circles represents a single mutational step between from (a) mitochondrial DNA cytochrome b, (b) nuclear DNA glyt one haplotype and another. Small open circles signify possible miss- gene, (c) nuclear DNA myh6 gene, and (d) nuclear DNA glyt gene ing haplotypes, and numbers at nodes indicate the numbers of nucle- data. For ncDNA MSTs, the sampling sites and numbers of speci- otide changes between haplotypes. See Table 1 for abbreviations of mens are noted next to haplotypes, and the relationships with the lineages and sampling locality/river system information cytochrome b dataset are shown as pie charts. Circle size refects the

Table 4 Estimated times of the most recent common ancestor (tMRCA) within/between lineages/sub-lineages of Abbottina rivularis, based on cytochrome b sequences from the Bayesian analysis with the uncorrelated lognormal relaxed clock model

JL1 JL2 JL SCL1a SCL1b SCL1c SCL1 SCL2 SCL1+2 NCL NCL–JL All haplotypes

Median (Myr) 0.63 0.26 2.50 1.10 0.37 0.38 1.56 4.08 9.61 0.75 7.85 17.06 95% HPD lower 0.27 0.08 1.37 0.59 0.16 0.19 0.90 2.41 5.93 0.42 4.74 10.69 95% HPD upper 1.14 0.53 3.86 1.80 0.65 0.63 2.41 6.18 14.19 1.21 11.74 25.03

The median values and the upper and lower values of the 95% highest posterior density (HPD) are listed For abbreviations, see Table 1

For the glyt gene, only nine haplotypes were detected one from Kyushu), and R9 (16 specimens from Kyushu). from 77 specimens, including two major haplotypes (g1, Notably, haplotype R4 specimens included all three cytb represented by 40 specimens from Honshu, Kyushu, north- (sub-)lineages (JL1, JL2, and SCL1) that occurred in Japan. ern China, and Korea; and g3, represented by 23 specimens The ncDNA data, coupled with mtDNA data, strongly from Honshu). For the myh6 gene, 14 haplotypes were suggested that some hybridization had occurred between detected from 76 specimens. Among these, 27 specimens Japanese and continental lineages on Kyushu Island. For collected from Honshu, which belonged to JL1 based on the the glyt + myh6 combined data (22 composite haplotypes; cytb data, were grouped closely together (m7, 9–11). Three cb1–22), the Kyushu specimens were paraphyletically major haplotypes were represented by 50 specimens: m2 positioned in the phylogenetic tree (Fig. 5); their ncDNA (18 from Kyushu and three from China), m5 (represented sequences were classifed into at least three groups: one by one specimen from LUR and 10 from Kyushu), and m7 (cb18, 20, and 22) was related to Honshu sequences (99% (18 from Honshu only). For the RAG1 gene, 14 haplotypes BPP), another (cb2, 4, and 14–17) was the same as or close were detected from 51 specimens. Only three haplotypes to the continental sequences, and the last (cb19 and 21) was were found in more than three specimens: R3 (nine speci- unique but similar to the continental sequences. All three mens from Honshu), R4 (nine specimens from Honshu and diferentiated groups shared both the JL2 and SCL1 mtDNA

1 3 Phylogeography of Abbottina rivularis 473

Fig. 4 Bayesian time-calibrated tree of Abbottina rivularis and (≥0.85); asterisks indicate 1.00 probability. Nodes denoted by closed closely related species based on mtDNA cytochrome b sequences. circles are those at which date calibration was applied based on Bars along nodes indicate 95% credible intervals of node heights. Tominaga et al. (2016). Photograph: A. rivularis collected from Numbers at internodes represent Bayesian posterior probability Okayama, Japan (J9), uncatalogued haplotypes. In contrast, Honshu specimens with JL1 mtDNA Asian lineages by the sea, which is an efective isolation haplotypes formed a monophyletic group (cb7 and cb9–13) mechanism for freshwater fauna on both sides of the barrier without exception (100% BPP). All continental specimens (Okazaki et al. 2002; Sasaki et al. 2007; Tominaga et al. with the NCL or SCL1 mtDNA haplotypes also shared simi- 2016). The geographical range of the NCL, which was phy- lar sequences at basal positions in the ncDNA tree (Fig. 5). logenetically closest to the JL, includes the Korean Penin- sula, which appears to support the traditional hypothesis that the Korean Peninsula served as a migration route for Discussion freshwater fshes from continental Asia into Japan during the glacial periods (e.g., Aoyagi 1957; Nishimura 1980). Diferentiation and biogeography of major lineages. The However, the estimated divergence time between the NCL present phylogenetic analyses, conducted using mitochon- and JL (4.7–11.7 Mya; around the Late Miocene; Table 4) drial cytb sequence data, revealed four well-diferentiated was much greater than that expected based on the traditional lineages (NCL, SCL1, SCL2, and JL) in Abbottina rivularis scenario (during the Quaternary). Although further examina- in East Asia, including continental and Japanese areas. All tion of molecular dating is necessary, the present study of four lineages were supported by high bootstrap/BPP values. A. rivularis DNA variations provides little support for the They were roughly allopatrically distributed, although some recent origin hypothesis of the Japanese endemic lineage. geographical overlap of ranges was observed. Although our Previous molecular fndings for other freshwater fshes (e.g., ncDNA data did not clearly support these lineages, this is Okazaki et al. 2002; Shirai et al. 2003; Takehana et al. 2009; highly probably due to low divergence in the ncDNA genes. Tominaga et al. 2016) also contradict this hypothesis. The Japanese lineage, JL, was distinctly separated from The northern lineage, NCL, occurs in the northernmost the continental lineages (NCL, SCL1, and SCL2) and was area of the distribution range of this species, partially with considered to be an endemic evolutionary lineage of Japan. the southern SCL1 lineage (Fig. 1). The network topology of We can reasonably suggest that the JL was isolated from the NCL suggests a possible dispersal scenario. A dominant

1 3 474 N.-H. Jang-Liaw et al.

have served as a refuge for freshwater fshes during glacial periods. One of the southern lineages, SCL1, was distributed mainly from the Liao River to Yunnan Province, China, and was also found from specimens in the Amur River, and Kyushu and Honshu Islands of Japan (Table 1; Fig. 1). The SCL1 co-occurred with NCL in northeastern continental area (the Amur River and Liao River basins) and this may be explained by historical vicariance followed by secondary contact, probably through river capture events (Qiu et al. 2012). Similar phylogeographic pattern is also reported in the goby Perccottus glenii, distributed in these basins (Xu et al. 2014). However, the discontinuous distribution of SCL1 haplotypes partly seems unnatural, suggesting the infuence of human disturbance. The distribution pattern of SCL1 in Japan is discussed in more detail in the next section. In the context of the present sampling strategy that focused on Japan and northern East Asia, the analyzed specimens from southern East Asia were insufcient to provide an overview of the phylogeography of A. rivularis in this area. In particular, the detailed distribution and divergence patterns of SCL2 remain unclear because of the limited number of specimens, although SCL2 haplotypes were detected over a wide range of central to southern China (the Yangtze River, the Ming River, the Pearl River, and the Ayeyarwady River). Phylogeographic and taxonomic studies based on detailed genetic and morphological examinations are needed for this species. Natural distribution of A. rivularis in Japan and implications of artifcial disturbance. Of the two distinct mtDNA lineages (JL and SCL1) observed in Japan, the JL Fig. 5 Bayesian 50% majority rule consensus tree obtained through was found exclusively in specimens from Japan, and hence analysis of Abbottina rivularis combined nuclear DNA data (glyt + is considered to be a native lineage endemic to Japan. It was myh6). Bayesian posterior probability values are shown at adjacent further divided into two sub-lineages, JL1 and JL2, which nodes. For abbreviations of lineages, refer to Table 1 were found exclusively on Honshu and Kyushu, respectively. The two sub-lineages were estimated to have diferentiated haplotype (h13) occupied the central position of the network about 2.5 Mya (1.4–3.9 Mya; the Pliocene to early Pleisto- and was possessed by specimens collected from southern cene). This estimate is consistent with results from several sites in its range: Liaodong Peninsula (site C6), the upper previous studies that focused on molecular dating of Japa- reach of the Liao River (C8), and the Korean Peninsula (K1); nese freshwater fshes, such as bitterlings Tanakia lanceo- all river systems to which these sites belong run into the lata and Tanakia limbata (see Hashiguchi et al. 2006), the Yellow Sea. The Yellow Sea is a semi-enclosed shelf sea Carassius auratus complex (see Takada et al. 2010), and between Korea and China, with water depths of <100 m Pseudogobio esocinus (see Tominaga et al. 2016). These (Kim and Kennett 1998). During periods of low sea lev- researchers, who used a strict or mean evolutionary rate of els, e.g., during the Last Glacial Maximum, the Yellow Sea 0.76%/Myr for cytb or other geographic calibrations, esti- was exposed subaerially and covered by a network of river mated divergence times of 2.5, 2.7, 1.8, and 2.2 Myr between channels (Qin et al. 1986). The formation of land and river Kyushu (and the westernmost tip of Honshu) and western systems on the foor of the Yellow Sea provided a putative Honshu populations of their respective study species. These migration route for terrestrial animals (e.g., wood-feeding similar estimates, together with our results for A. rivularis, cockroaches; Maekawa and Nalepa 2011). The present could be attributable to common geographic events, such results for the NCL imply that the Yellow Sea provided a as the formation of mountains and a strait between the two dispersal route for A. rivularis in this area and would also regions.

1 3 Phylogeography of Abbottina rivularis 475

Within the JL1 sub-lineage, two populations in eastern relationship is similar to cases for some other fshes [e.g., Honshu (sites J1 and J2) exhibited the same cytb haplotype Biwia zezera (see Watanabe et al. 2010a); and the botiid (h57) as the central Honshu populations (sites J6–J8). This loach Parabotia curtus (see Watanabe et al. 2009)] and is result was unexpected because a substantial geographic bar- consistent with the presence of a paleo-river system that rier, the Fossa Magna, lies between central and eastern Hon- appeared in the Seto Inland Sea (the area surrounded by shu; this barrier is well known to divide fsh fauna and popu- Honshu, Shikoku, and Kyushu islands) during glacial regres- lations (Nishimura 1980; Watanabe 2012; Watanabe et al. sion periods (Nishimura 1980; Kimizuka and Kobayasi 2017). The result is very consistent with an artifcial origin 1983; Kimura 1985). Namely, the genetic data support the of A. rivularis in eastern Honshu, which has been hypoth- native distribution of A. rivularis in the Sanyo Region. Also, esized by several authors (e.g., Hosoya 2001; Matsuzawa the close relationship between the Sanyo and central Hon- and Senou 2008). Another haplotype that may have origi- shu (Kinki Region and Nobi Plain) populations suggest that nated by diferent introduction events also occurs in eastern the Kinki Region populations, rather than the Nobi Plain Honshu (site J3), i.e., an SCL1 haplotype (h58), which is populations, are indigenous. Abbottina rivularis on Honshu close to both the Kyushu haplotype h68 (which occurred at Island prefer foodplain and wetland environments and are sites J13 and J14), with a single nucleotide substitution, and limited to the lower reaches of large rivers (Sanyo and Nobi) to an Amur River haplotype h12 (site C5), with two nucleo- and marshes around lakes (Lake Biwa). Some fsh species, tide substitutions. Again, the “recent artifcial introduction” including the critically endangered loach Parabotia curtus, hypothesis can be applied to explain this separate, unusual depend on such environments, remain in a narrow area of distant distribution among these SCL1 haplotypes; thus, we the Sanyo Region, and overlap with the range of A. rivularis can also infer that the current distribution of A. rivularis in (Hosoya 2002; Watanabe et al. 2009). These environmental eastern Honshu may have been caused by multiple artifcial conditions may have maintained the relatively high genetic introductions. diversity and population size of the A. rivularis population In contrast, A. rivularis populations in central Honshu at site J9 (Table 1), compared with those of other Honshu are believed to be native, although the indigenousness of the populations. population around Lake Biwa is controversial (Nakamura Despite the relatively large sampling efort on Kyushu 1969; Hosoya 2001; Nakajima and Nakagawa 2007; Matsu- Island (which yielded 57 specimens from 11 localities), JL2 zawa and Senou 2008). The present data support its native haplotypes were found in only two localities (sites J15 and distribution in central Honshu, because haplotypes h57, h59, J17). The JL2 sub-lineage was found exclusively in northern and h60 were found exclusively in this region (sites J4–J8; Kyushu, suggesting that there have been A. rivularis popu- except for eastern Japan; Fig. 1d). However, no population lations indigenous to Kyushu. On the other hand, another divergence was observed between the Kinki Region (Lake lineage, SCL1, was found at most (9 of 11) sampling sites. Biwa) and Nobi Plain populations, despite the expectation Populations with SCL1 showed very low nucleotide diver- of signifcant divergence due to the geographic isolation sity (Table 1) and close relationships to some populations by the Suzuka Mountains, similar to observations for other of continental Asia. These results indicate that these pop- freshwater fshes (e.g., Tominaga et al. 2016; Watanabe ulations in Kyushu were recently established and rapidly et al. 2010a, 2014, 2017; Kitanishi et al. 2016). This fnding expanded in northwestern Kyushu. In addition, when com- implies that some artifcial translocations, probably from the paring mtDNA and ncDNA data, no (or at least incomplete) Kinki Region to the Nobi Plain (see below), occurred within reproductive isolation is expected between individuals with central Honshu, although a conclusive determination cannot mtDNA JL2 and SCL1. be made about the indigenousness of the Lake Biwa popula- One possible hypothesis for the Kyushu SCL1 haplo- tion from the present data. On the other hand, compared to types is very recent (e.g., during the Last Glacial Period) another gobionin species, Biwia zezera, which exhibits high natural dispersal through a land bridge between Kyushu and genetic diversity (e.g., h = 0.969 and π = 0.6% for pooled continental areas. This recent dispersal is inferred to have samples from Lake Biwa) and large population sizes in cen- occurred in other species, such as the black-spotted frog, tral Honshu (Watanabe et al. 2010a), the very low genetic Pelophylax nigromaculata (see Zhang et al. 2008). How- diversity in A. rivularis populations in this area suggests a ever, such a recent interchange of freshwater fshes is not recent population bottleneck or founder event involving a supported by the presence of distinct genetic divergence single or a few mtDNA haplotypes. between populations of species distributed in both areas In addition to central and eastern (introduced) Honshu, (Shirai et al. 2003; Kitagawa et al. 2005; Takehana et al. another JL1 population was found at site J9 in the Sanyo 2009; Tominaga et al. 2016; Watanabe et al. 2018). Also, Region, western Honshu. Haplotypes occurring at site the recent natural dispersal scenario may not explain why J9 (h61–h64) were closely related to, but distinct from, NCL haplotypes, occurring widely in northeastern continen- those in central Honshu (h57, h59, and h60) (Fig. 2). This tal area, are not found in Kyushu. Although the possibility

1 3 476 N.-H. Jang-Liaw et al. of recent natural dispersal cannot be completely ruled out, between two species of freshwater eels, Anguilla celebesensis and A. interioris the discontinuous distribution of SCL1 in Japanese (Kyushu ? Ichthyol Res 47:157–161 Avise JC (1994) Molecular markers, natural history and evolution. and a portion of eastern Honshu) and Chinese populations Chapman & Hall, New York strongly suggests an alternative hypothesis, i.e., the artifcial Avise JC (2000) Phylogeography: the history and formation of species. introduction of continental A. rivularis to Japan. Harvard University Press, Cambridge ǎ ǎ Evidence from other fish species likely supports the B n rescu PM, Nalbant TT (1973) Pisces, Teleostei. Cyprinidae (Gobi- oninae). Das Tierreich 93:1–304 artifcial introduction hypothesis for SCL1 on Kyushu. In Beheregary LB (2008) Twenty years of phylogeography: the state of the cyprinid Biwia zezera and Opsariichthys platypus, both the feld and the challenges for the Southern Hemisphere. Mol indigenous and introduced individuals are widely found Ecol 17:3754–3774 in Kyushu (Horikawa et al. 2007; Kitanishi et al. 2016), Bouckaert R, Heled J, Kühnert D, Vaughan T, Wu C-H, Xie D, Suchard MA, Rambaut A, Drummond AJ (2014). BEAST 2: a software although the introduced individuals originate from western platform for Bayesian evolutionary analysis. PLoS Comput Biol Honshu, instead of the continent. These artifcial distur- 10: e1003537 bances in the faunal and genetic diversity of Kyushu may Burridge CP, Craw D, Fletcher D, Waters JM (2008) Geological dates have been caused by inland fshery activities, including and molecular rates: fsh DNA sheds light on time dependency. Plecoglossus altivelis Mol Biol Evol 25:624–633 the wide-scale introduction of Ayu ( Chan KMA, Levin SA (2005) Leaky prezygotic isolation and porous altivelis), common carp (Cyprinus carpio), and crucian genomes: rapid introgression of maternally inherited DNA. Evo- carps (Carassius cuvieri and the Carassius auratus spe- lution 59:720–729 cies complex). In particular, the transport of A. rivularis Chu XL, Chen YR (1989) The fshes of Yunnan, China. Part I. Cyprini- dae. Science Press, Beijing into Kyushu and dispersal throughout the island appear to Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: be attributable to the translocation of the latter two groups more models, new heuristics and parallel computing. Nat Meth- (Maruyama et al. 1987; Statistics Department, Ministry of ods 9:772 Agriculture, Forestry, and Fisheries of Japan 2000), which Doadrio I, Domìnguez O (2004) Phylogenetic relationships within the fsh family Goodeidae based on cytochrome b sequence data. Mol share habitats and environments (e.g., standing water and Phylogenet Evol 31:416–430 ponds). Indeed, a phylogeographic study of crucian carp Drummond AJ, Ho SYW, Phillips MJ, Rambaut A (2006) Relaxed suggested the artifcial introduction(s) of Chinese crucian phylogenetics and dating with confdence. PLoS Biol 4: e88. https carp to Kyushu (Yamamoto et al. 2010). Furthermore, alien ://doi.org/10.1371/journal.pbio.0040088 Excofer L, Lischer HEL (2010) Arlequin suite ver 3.5: a new series fshes from China inhabiting similar habitats, such as the of programs to perform population genetics analyses under Linux bitterling Rhodeus ocellatus ocellatus and the Chinese lin- and Windows. Mol Ecol Resour 10:564–567 eage of Pseudorasbora parva, have been widespread in Felsenstein J (1985) Confdence limits on phylogenies: an approach Japan (Watanabe et al. 2000; Matsuzawa and Senou 2008). using the bootstrap. Evolution 39:783–791 Hashiguchi Y, Kado T, Kimura S, Tachida H (2006) Comparative phy- Collectively, the present results and those of previous stud- logeography of two bitterlings, Tanakia lanceolata and T. lim- ies provide supportive evidence that a large portion of the bata (Teleostei, Cyprinidae), in Kyushu and adjacent districts of Kyushu populations of A. rivularis likely originated and/or western Japan, based on mitochondrial DNA analysis. Zool Sci introgressed by introductions from continental Asia and that 23:309–322 Hayashi K, Kim EJ, Onikura N (2013) Growth and habitat use of the the Honshu populations may also have expanded eastward Chinese false gudgeon, Abbottina rivularis, in and irrigation chan- due to human activities. nel near the Ushizu River, northern Kyushu Island, Japan. Ichthyol Res 60:218–226 Acknowledgements We would like to express our sincerest apprecia- He H, Li YH, Cao K, Li MY, Fu CZ (2017) Taxonomy and phyloge- tion to A. Goto, S.-R. Jeon, S. Mori, T. Mukai, H. Sakai, and C. S. netic relationships of the genus Abbottina fshes in the subfamily Tzeng for their generous donation of tissue samples; and to T. Abe Gobioninae. Acta Hydrobiologica Sinica 41:843–852 and H. Egi for their kind help with feldwork. We are also grateful to Horikawa M, Nakajima J, Mukai T (2007) Distribution of indigenous I. Koizumi and two anonymous reviewers for their helpful comments and non-indigenous mtDNA haplotypes of Biwia zezera (Cyprini- and suggestions on the manuscript. This study was supported in part dae) in northern Kyushu, Japan. Jpn J Ichthyol 54:149–159 by JSPS KAKENHI (nos. 18570086, 19405011, 21370035, 26291079, Hosoya K (2001) Abbottina rivularis. In: Kawanabe H, Mizuno N, 26250044, and 17H03720) and the Global Center of Excellence Pro- Hosoya K (eds) Freshwater fshes of Japan. Yama-Kei Publish, gram “Formation of a Strategic Base for Biodiversity and Evolutionary Tokyo Research: from Genome to Ecosystem” of Kyoto University. Hosoya K (2002) Leptobotia curta. In: Nakabo T (ed) Fishes of Japan with pictorial keys to the species, English edn. Tokai University Press, Tokyo Hsieh CF (2002) Composition, endemism and phytogeographical afnities of the Taiwan fora. Taiwania 47:298–310 References Kawahara R, Miya M, Mabuchi K, Near TJ, Nishida M (2009) Stickle- back phylogenies resolved: Evidence from mitochondrial genomes Aoyagi H (1957) General notes on the freshwater fshes of the Japanese and 11 nuclear genes. Mol Phylogenet Evol 50:401–404 Archipelago. Taishukan, Tokyo Kim J-M, Kennett J P (1998) Paleoenvironmental changes associated Aoyama S, Watanabe S, Ishikawa S, Nishida M, Tsukamoto K (1999) with the Holocene marine transgression, Yellow Sea (Hwanghae). Are morphological characters distinctive enough to discriminate Mar Micropaleontol 34:71–89

1 3 Phylogeography of Abbottina rivularis 477

Kimizuka Y, Kobayasi H (1983) Geographic distributions of karyo- special reference to their paleogeographical implications. Popul logical races of Cobitis biwae (Cobitididae). Jpn J Ichthyol Ecol 40:189–204 30:308–312 Palumbi S, Martin A, Romano S, McMillian WO, Stice L, Grabowski Kimura T (1985) Nihon-retto, III. The Japanese Archipelago. Kokin- G (1991) The simple fool’s guide to PCR. University of Hawaii, shoin, Tokyo Honolulu Kita Y, Kato M (2004). Molecular phylogeny of Cladopus and Hydro- Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol bryum (Podostemaceae, Podostemoideae) with implications for Evol 25:1253–1256 their biogeography in East Asia. Syst Bot 29:921–932 Qin Y, Li F, Tang B, Milliman JD (1986) Buried paleoriver system in Kitagawa T, Jeon S-R, Kitagawa E, Yoshioka M, Kashiwagi M, Oka- western part of the South Huang Sea. Chin Sci Bull 31:1887–1889 zaki T (2005) Genetic relationships among the Japanese and Qiu SW, Wang XK, Zhang SQ, Liang Y, Zhang ZQ, Zhu JH (2012) The Korean striated spined loach complex (Cobitidae: Cobitis) and evolution of the large paleolake in Songliao Plain and its forma- their phylogenetic positions. Ichthyol Res 52:111–122 tion. Quatern Sci 32:1011–1021 Kitanishi S, Hayakawa A, Takamura K, Nakajima J, Kawaguchi Y, Rambaut A (2016) FigTree Version 1.4.3. http://tree.bio.ed.ac.uk/softw Onikura N, Mukai T (2016) Phylogeography of Opsariichthys are/fgtree/. Accessed 23 March 2017 platypus in Japan based on mitochondrial DNA sequences. Ich- Rambaut A, Suchard MA, Xie D, Drummond AJ (2013) Tracer v1.6, thyol Res 63:506–518 Available from http://beast.bio.ed.ac.uk/Tracer. Accessed 3 Kocher TD, Stepien CA (1997) Molecular systematics of fshes. Aca- August 2013 demic Press, New York Rohlf F J (1973) Algorithm 76. Hierarchical clustering using the mini- Kottelat M (2001) Fishes of Laos. WHT Publications (Pte.) Ltd, mum spanning tree. Comput J 16:93–95 Colombo Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic Li X, Li J (1997) The Tanaka-Kaiyong Line-an important foristic inference under mixed models. Bioinformatics 19:1572–1574 line for the study of the fora of East Asia. Ann Mo Bot Gard Rüber L, Kottelat M, Tan HH, Ng PKL, Britz R (2007) Evolution 84:888–892 of miniaturization and the phylogenetic position of Paedocypris, Li C, Ortí G (2007) Molecular phylogeny of Clupeiformes (Actinop- comprising the world’s smallest vertebrate. BMC Evol Biol 7:38 terygii) inferred from nuclear and mitochondrial DNA sequences. Sasaki T, Kartavtsev YP, Chiba SN, Uematsu T, Sviridov VV, Hanzawa Mol Phylogenet Evol 44:386–398 N (2007) Genetic divergence and phylogenetic independence of Librado P, Rozas J (2009) DnaSP v5: a software for comprehen- Far Eastern species in subfamily Leuciscinae (Pisces: Cyprinidae) sive analysis of DNA polymorphism data. Bioinformatics inferred from mitochondrial DNA analyses. Genes Genet Syst 25:1451–1452 82:329–340 Lindberg GU (1972) Large-scale fuctuations of sea level in the Qua- Shih HT, Suzuki H (2008) Taxonomy, phylogeny, and biogeography ternary period: hypothesis based on biogeographical evidence. of the endemic mudfat crab Helice/Chasmagnathus complex Nauka, Leningrad (Crustacea: Brachyura: Varunidae) from East Asia. Zool Stud Liu H, Yang J, Tang Q (2010) Estimated evolutionary tempo of East 47:114–125 Asian gobionid fshes (Teleostei: Cyprinidae) from mitochondrial Shirai SM, Yabumoto Y, Kim I, Zhang C (2003) Phylogeny of siniper- DNA sequence data. Chin Sci Bull 55:1501–1510 cid fshes and their relatives inferred from mtDNA cytochrome b López JA, Chen WJ, Ortí G (2004) Esociform phylogeny. Copeia gene: a preliminary study. Bull Kitakyushu Mus Ser A1:45–49 2004:449–464 Statistics Department, Ministry of Agriculture, Forestry, and Fisheries Maekawa K, Nalepa CA (2011) Biogeography and phylogeny of wood- of Japan. (2000) The 10th fsheries census. AAFS, Tokyo feeding cockroaches in the genus Cryptocercus. Insect 2:354–368 Swoford DL (2002) PAUP* 4.0b10a: phylogenetic analysis using par- Maruyama T, Fujii K, Kijima T, Maeda H (1987) Introductory process simony (and other methods). Sinauer, Sunderland of foreign new fsh species. Fisheries Agency, Tokyo Takada M, TachiharaK, Kon T, Yamamoto G, Iguchi KI, Miya M, Matsuzawa Y, Senou H (2008) Alien fshes of Japan. Bun-ichi, Tokyo Nishida M (2010) Biogeography and evolution of the Carassius Miura T (1966) Ecological notes of the fshes and the interspecifc rela- auratus-complex in East Asia. BMC Evol Biol 10:7. https://doi. tions among them in Lake Biwa. Jpn J Limnol 27:49–72 org/10.1186/1471-2148-10-7 Motokawa M, Kajihara H (2017) Species diversity of animals in Japan, Takehana Y, Uchiyama S, Matsuda M, Jeon S-R, Sakaizumi M (2009) diversity and commonality in animals. Springer, Tokyo Geographic variation and diversity of the cytochrome b gene in Nakajima J, Nakagawa M (2007) Spawning season of the Chinese wild populations of Medaka (Oryzias latipes) from Korea and false gudgeon, Abbottina rivularis, in Katada Naiko, Lake Biwa. China. Zool Sci 21:483–491 Humans and Nature 17:13–18 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Nakajima J, Onikura N, Matsui S, Oikawa S (2006) Geographical Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol distribution of genuine freshwater fshes in Fukuoka Prefecture, Evol 30:2725–2729 northern Kyushu, Japan. Jpn J Ichthyol 53:117–131 Tang WJ, He DK (2015) Investigation on alien fshes in Qinghai Prov- Nakamura M (1969) Cyprinid fshes of Japan. Studies on the life his- ince, China (2001–2014). J Lake Sci 27:502–510 tory of cyprinid fshes of Japan. Research Institute for Natural Tang Q, Liu H, Mayden R, Xiong B (2006) Comparison of evolution- Resources, Tokyo ary rates in the mitochondrial DNA cytochrome b gene and con- Neely DA, Perez MHS, Mendsaikhan B (2008) First Records of Rhi- trol region and their implications for phylogeny of the Cobitoidea nogobius lindbergi (Teleostei: Gobiidae) and Abbottina rivularis (Teleostei: Cypriniformes). Mol Phylogenet Evol 39:347–357 (Teleostei: Cyprinidae) in the Lake Buyr Drainage, Mongolia. J Tominaga K, Nakajima J, Watanabe K (2016) Cryptic divergence and Great Lakes Res 34:334–341 phylogeography of the pike gudgeon Pseudogobio esocinus (Tel- Nishimura S (1980) Formation of the Sea of Japan. Tsukiji-Shokan, eostei: Cyprinidae): a comprehensive case of freshwater phyloge- Tokyo ography in Japan. Ichthyol Res 63:79–93 Okazaki T, Jeon S-R, Kitagawa T (2002) Genetic diferentiation of Tsukahara H (1954) Breeding habits of the fresh-water sucker, Abbot- piscivorous chub (genus Opsariichthys) in Japan, Korea and Rus- tina rivularis (Basilewsky). Jpn J Ichthyol 3:139–143 sia. Zool Sci 19:601–610 Vidthayanon C, Kottelat M (1995) First record of Abbottina rivularis Ota H (1998) Geographic patterns of endemism and speciation in (Cyprinidae: Gobioninae) from the Mekong Basin. Jpn J Ichthyol amphibians and reptiles of the Ryukyu Archipelago, Japan, with 41:463–465

1 3 478 N.-H. Jang-Liaw et al.

Watanabe K (2012) Faunal structure of Japanese freshwater fshes and Xia X, Zhang X, Liu H (2005) Phylogeography and speciation of its artifcial disturbance. Environ Biol Fishes 94:533–547 Pseudogobio vaillanti in China: deep molecular divergence in Watanabe K, Takahashi H (2010) Natural history of freshwater fsh the absence of morphological change. Prog Nat Sci 15:1064–1069 biogeography (Tansuigyo-chiri no shizenshi). Hokkaido Univer- Xiao W, Zhang Y, Liu H (2001) Molecular systematics of Xenocypri- sity Press, Sapporo nae (Teleostei: Cyprinidae): taxonomy, biogeography, and coevo- Watanabe K, Iguchi K, Hosoya K, Nishida M (2000) Phylogenetic lution of a special group restricted in East Asia. Mol Phylogenet relationships of the Japanese minnows, Pseudorasbora (Cyprini- Evol 18:163–173 dae), as inferred from mitochondrial 16S rRNA gene sequences. Xu W, Yin W, Chen A, Li J, Lei G, Fu C (2014) Phylogeographical Ichthyol Res 47:43–50 analysis of a cold-temperate freshwater fsh, the Amur sleeper Watanabe K, Takahashi H, Kitamura A, Yokoyama R, Kitagawa T, (Perccottus glenii) in the Amur and Liaohe River basins of north- Takeshima H, Sato S, Yamamoto S, Takehana Y, Mukai T, Ohara east Asia. Zool Sci 31:671–679 K, Iguchi K (2006) Biogeographical history of Japanese fresh- Yamamoto G, Takada M, Iguchi K, Nishida M (2010) Genetic consti- water fshes: phylogeographic approaches and perspectives. Jpn tution and phylogenetic relationships of Japanese crucian carps J Ichthyol 53:1–38 (Carassius). Ichthyol Res 57:215–222 Watanabe K, Abe T, Iwata A (2009) Phylogenetic position and generic Yang J, He S, Freyhof J, Witte K, Liu H (2006) The phylogenetic status of the Japanese botiid loach. Ichthyol Res 56:421–425 relationships of the Gobioninae (Teleostei: Cyprinidae) inferred Watanabe K, Kawase S, Mukai T, Kakioka R, Miyazaki J-I, Hosoya K from mitochondrial cytochrome b gene sequences. Hydrobiologia (2010a) Population divergence of Biwia zezera (Cyprinidae: Gobi- 553:255–266 oninae) and the discovery of a cryptic species, based on mitochon- Zardoya R, Doadrio I (1999) Molecular evidence on the evolutionary drial and nuclear DNA sequence analyses. Zool Sci 27:647–655 and biogeographical patterns of European cyprinids. J Mol Evol Watanabe K, Kano Y, Takahashi H, Mukai T, Kakioka R, Tominaga K 49:227–237 (2010b) GEDIMAP: a database of genetic diversity for Japanese Zhang H, Yan J, Zhang G, Zhou K (2008) Phylogeography and freshwater fshes. Ichthyol Res 57:107–109 demographic history of Chinese black-spotted frog populations Watanabe K, Mori S, Tanaka T, Kanagawa N, Itai T, Kitamura J, (Pelophylax nigromaculata): evidence for independent refugia Suzuki N, Tominaga K, Kakioka R, Tabata R, Abe T, Tashiro Y, expansion and secondary contact. BMC Evol Biol 8:21. https:// Hashimoto Y, Nakajima J, Onikura N (2014) Genetic population doi.org/10.1186/1471-2148-8-21 structure of Hemigrammocypris rasborella (Cyprinidae) inferred from mtDNA sequences. Ichthyol Res 61:352–360 Publisher’s Note Springer Nature remains neutral with regard to Watanabe K, Tominaga K, Nakajima J, Kakioka R, Tabata R (2017) jurisdictional claims in published maps and institutional afliations. Japanese freshwater fshes: biogeography and cryptic diversity. In: Motokawa M, Kajihara H (eds) Species diversity of animals in Japan, diversity and commonality in animals. Springer, Tokyo, pp 183–227 Watanabe K, Sakai H, Sanada T, Nishida M (2018) Comparative phylogeography of diadromous and freshwater daces of the genus Tribolodon (Cyprinidae). Ichthyol Res 65:383–397

1 3