Phylogenetic Relationships of Bitterling Fishes (Teleostei: Cypriniformes: Acheilognathinae), Inferred from Mitochondrial Cytochrome b Sequences Author(s): Kouichi Kawamura , Takayoshi Ueda , Ryoichi Arai and Carl Smith Source: Zoological Science, 31():321-329. 2014. Published By: Zoological Society of Japan DOI: http://dx.doi.org/10.2108/zs130233 URL: http://www.bioone.org/doi/full/10.2108/zs130233

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

ZOOLOGICAL SCIENCE 31: 321–329 (2014)

¤ 2014 Zoological Society of Japan

Phylogenetic Relationships of Bitterling Fishes (Teleostei: Cypriniformes: Acheilognathinae), Inferred from Mitochondrial Cytochrome b Sequences Kouichi Kawamura1*, Takayoshi Ueda2, Ryoichi Arai3, and Carl Smith4 1

Faculty of Bioresources, Mie University, Kurimamachiya 1577, Tsu, Mie 514-8507, Japan 2 Department of Biology, Faculty of Education, Utsunomiya University, 350 Mine, Utsunomiya 321-8505, Japan 3 Department of Zoology, University Museum, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan 4 School of Biology, University of St Andrews, St Andrews, Fife KY16 8LB, United Kingdom

Bitterling (Teleostei: Acheilognathinae) are small cyprinid fishes with a discrete distribution in East Asia and Europe. We used a complete mitochondrial cytochrome b sequence (1141 bp) from 49 species or subspecies in three genera (Tanakia, Rhodeus, and Acheilognathus), sampled across the major part of their distribution, to elucidate their phylogeny and biogeography, focusing particularly on their origin and dispersal. Based on high support value, the monophyletic Acheilognathinae separated into two major clades, Acheilognathus and Tanakia-Rhodeus. In the latter clade, the monophyly of Rhodeus was poorly supported, though it was topologically nested in Tanakia. On the basis of molecular-clock calibration, both clades diverged in the middle Miocene, with TanakiaRhodeus diverging slightly earlier than Acheilognathus. The Tanakia-Rhodeus clade expanded its distribution westward from the Far East, eventually reaching Europe, while Acheilognathus dispersed in the temperate regions of East Asia. A feature common to both clades is that most extant species, including Japanese endemics, appeared by the end of the Pliocene, corresponding with the present delineation of the Japanese archipelago. Autumn-spawning species with an embryonic diapause, unique to bitterling among cyprinid fishes, formed two distinct lineages (barbatulusrhombeus and longipinnis-typus) within Acheilognathus. The estimated time of divergence of the two lineages was approximately from the late Pliocene, a period characterized by glaciations. The timing of divergence suggests that the shift of spawning from spring to autumn, coupled with embryonic diapause, convergently emerged twice in the evolution of bitterling, possibly as an adaptation to the climate of the late Pliocene. Key words:

Acheilognathinae, biogeography, cytochrome b, convergence, diapause, dispersal

INTRODUCTION Bitterling (subfamily Acheilognathinae) are small cyprinid fishes from temperate regions of East Asia and Europe, ˘ arescu, ˘ including Japan and Taiwan (Ban 1990). This group comprises approximately 80 species or subspecies worldwide (Froese and Pauly, 2013), which are classified into three genera, Tanakia, Rhodeus, and Acheilognathus (Arai and Akai, 1988). They are characterized by a highly specialized spawning behavior that involves depositing their eggs on the gills of living freshwater bivalves (Wiepkema, 1961; Smith et al., 2004), and an unusual style of embryonic and larval development (Nakamura, 1969). While most bitterling * Corresponding author. Tel. : +81-59-231-9549; Fax : +81-59-231-9540; E-mail: [email protected] Supplemental material for this article is available online. doi:10.2108/zs130233

species spawn in spring, a few species in Acheilognathus spawn in autumn, with their hatched embryos showing an arrest in development during winter, which has been suggested to be a genetically determined diapause (Kawamura and Uehara, 2005). In addition, bitterling are characterized among cyprinid fish by diversity in morphology and karyology (Arai, 1982). These conspicuous characteristic features strongly support the monophyly of the bitterling (Okazaki et al., 2001). Substantial interest has been paid to the species richness of bitterling fishes, including their discontinuous distribution across Asia and Europe, in order to address questions on their evolutionary history and conservation (Arai, 1988; Smith et al., 2004; Reichard et al., 2012). Okazaki et al. (2001) first attempted to elucidate the molecular phylogeny of bitterling, analyzing mtDNA 12S ribosomal DNA sequences of 27 species or subspecies in three genera. In their study, the Acheilognathinae separated into two major clades, Acheilognathus and Tanakia-Rhodeus. In addition, mapping

322

K. Kawamura et al.

morphological characters on a phylogenetic tree, Okazaki et al. (2001) suggested the two distinct evolutionary pathways in karyology (from 2n = 48 to either 2n = 44 or 2n = 46) and autapomorphy of wing-like yolk-sacs among Rhodeus species. Despite these important preliminary findings, the details of the phylogenetic relationships of bitterling, especially at the species level, remain ambiguous. In recent decades, the expansion of molecular analyses and interest in phylogeography (Avise, 2000; Beheregaray, 2008), as well as growth in interest in using bitterling as models in behavioral and evolutionary studies (Reichard et al., 2009; Agbali et al., 2010; Pateman-Jones et al., 2011; Reichard et al., 2012), has led to an increasing number of phylogenetic studies targeting specific species of bitterling, such as Tanakia himantegus (Chang et al., 2009), Tanakia tanago (Kubota et al., 2010), Rhodeus amarus (Bohlen et al., 2006; Bryja et al., 2010) and Acheilognathus macropterus (Zhu and Liu, 2007). In addition, the evolution of characters specific to bitterling have been investigated in some species (Reichard et al., 2011; Kitamura et al., 2012). While these studies enhance our understanding of bitterling biology, a fuller understanding of phylogenetic relationships among bitterling is necessary to facilitate deeper insights into bitterling evolution. In the present study a comprehensive analysis of the phylogenetic relationships of bitterling fishes were inferred using mtDNA cytochrome b (Cyt b) genes. Because Cyt b shows a higher rate of sequence divergence than 12S ribosomal DNA, and is also conserved at the species level (Miya and Nishida, 2000; Nei and Kumar, 2000), it is an appropriate marker for the phylogenetic analysis of bitterling. In this study, the aim was to first clarFig. 1. Sampling locations of bitterling species used in the present study. Figify the phylogeny and time of divergence of bitterures indicate locations shown in Table 1. ling with a molecular clock. Second, to identify the evolution of characters specific to bitterling, particDNA extraction and sequencing ularly autumn spawning associated with embryonic diapause. MATERIALS AND METHDOS Sample collection and DNA extraction 48 species or subspecies of the subfamily Acheilognathinae (25 in Acheilognathus, 15 in Rhodeus, and eight in Tanakia) were used in this study. Sixty-eight samples of 41 species or subspecies were collected from 51 localities in Japan, Korea, China, Taiwan, Vietnam, Laos, and Poland (Fig. 1, Table 1). Fish were identified from diagnostic characters, following classification of genera by Arai and Akai (1988). In order to capture the full diversity of bitterling, representative species or subspecies were included in the analysis, while species with ambiguous taxonomic status were excluded. In addition, multiple samples were analyzed for those species with extensive distributions. For samples that were not available in our data collection, 15 sequences of 13 species or subspecies published on GenBank were additionally used. The cyprinids Biwia zezera, Hemibarbus barbus, and Zacco platypus were used as outgroups for bitterling. The nucleotide sequences of 41 species or subspecies of bitterling and one outgroup species (Z. platypus) are deposited in DDBJ/EMBL/GenBank (see Table 1 for accession numbers).

Fin clip or muscle tissue was used as sources of genomic DNA. Tissue was preserved in 100% ethanol or stored at 4°C until DNA extraction. Genomic DNA was extracted using standard phenol/ chloroform extraction procedures (Lansman et al., 1981). A 1141 bp sequence of the complete Cyt b sequence was amplified via PCR, using the following primer pairs: GLU-L (5′-TGATATGAAAAACCATCGTTG-3′) and CB6THR-H (5′-CTCCAGTCTTCGRCTTACAAG-3′) (Palumbi et al., 1991) and PCR protocols described by Kawamura et al. (2001). The PCR products were sequenced by the ABI Prism BigDye Terminator Cycle Sequencing Kit 1.1 standard protocol (using the same primers) with ABI Prism 3100 DNA Analyzer (Applied Biosystems). Phylogenetic analyses A total of 69 sequences were aligned using DNASYS v3.1 (Hitachi Software). Base compositional bias was calculated across taxa using PAUP* 4.0b10 (Swofford, 2003). The relationship between the absolute number of transitions (Ti) and transversions (Tv) against corrected distances based on the GTR + I + G model was plotted to analyze nucleotide saturation. Calculation of genetic distance was conducted using PAUP*. Phylogenetic analyses were performed using maximum likeli-

MtDNA phylogeny of bitterling Table 1.

323

Numbers and names of sampling locations, and GenBank accession numbers of samples used in this study.

Species name Acheilognathus barbatulus 1 A. barbatulus 2 A. barbatulus 3 A. barbatus A. changtingensis A. chankaensis 1 A. chankaensis 2 A. chankaensis 3 A. chankaensis 4 A. chankaensis 5 A. cyanostigma A. gracilis A. imberbis A. lanchiensis A. longibarbatus A. longipinnis A. longispinnis A. macropterus 1 A. macropterus 2 A. macropterus 3 A. macropterus 4 A. macropterus 5 A. macropterus 6 A. majusculus A. melanogaster A. meridianus 1 A. meridianus 2 A. omeiensis 1 A. omeiensis 2 A. polylepis A. rhombeus 1 A. rhombeus 2 A. rhombeus 3 A. rhombeus 4 A. tabira erythropterus A. tabira jordani A. tabira nakamurae A. tabira tabira A. tonkinensis 1 A. tonkinensis 2 A. tonkinensis 3 A. typus A. yamatsutae 1 A. yamatsutae 2 Rhodeus amarus R. atremius atremius R. atremius suigensis R. colchicus R. fangi R. laoensis R. meridionalis R. notatus 1 R. notatus 2 R. ocellatus kurumeus R. ocellatus ocellatus 1 R. ocellatus ocellatus 2 R. ocellatus ocellatus 3

Location No. 31 15 33

30 41 20 37 6 35 39 8 46 39 25 36 34

28 1 43

26 4 12 19 2 10 13 4 35 47 44 1 16 24 51 12 9 40 50 29 30 7 48 42 38

Collection location (Drainage) Qingpu, Shanghai, China (Yangtze River) Tieling, Liaoning, China (Liao River) Wuhan, Hubei, China (Yangtze River) Yichang, Hubei, China (Yangtze River) Changting, Fujian, China (Hanjiang River) Taian, Shandong, China (Yellow River) Putian, Fujian, China (Mulanxi R.) Nae-ri, Gyeonggi-do, Korea (Anseong River) Leshan, Sichuan, China (Yangtze River) Duchang, Jiangxi, China (Yangtze River) Shiga, Japan (Lake Biwa) Nanchang, Jiangxi, China (Yangtze River) Daye, Hubei, China (Yangtze River) Lanxi, Zhejiang, China (Qiantang River) Cao Bang market, Viet Nam Osaka, Japan (Yodo R.) Jiuzhou, Hainan Island, China Lanxi, Zhejiang, China (Qiantang River) Galcheon-ri, Gyeongsangnam-do, Korea (Nakdong River) Yuanjiang, Hunan, China (Lake Dongting) Baxian, Chongqing, Sichuan, China (Yangtze River) Jinxian, Jiangxi, China (Yangtze River) Fuyuan, Heilongjiang, China (Amur River) Banghyeon-ri, Jeollabuk-do, Korea (Seomjin River) Miyagi, Japan (Kitakami River) Yangshuo, Guangxi, China (Pearl River) Wuyuan, Jiangxi, China (Yangtze River) Leshan, Sichuan, China (Yangtze River) Leshan, Sichuan, China (Yangtze River) Huangshan, Anhui, China (Fuchunjiang River) Mijeom-ri, Gyeongsangnam-do, Korea (Seomjin River) Mie, Japan (Kushida River) Saga, Japan (Ushizu River) Dochang-ri, Gangwon-do, Korea (Imjin River) Tochigi, Japan (Kuna River) Shimane, Japan (Ohara River) Fukuoka, Japna (Chikugo River) Mie, Japan (Kushida River) Daye, Hubei, China (Yangtze River) Dingan, Hainan Island, China Chongzuo, Guangxi, China (Pearl River) Miyagi, Japan (Kitakami River) Fengcheng, Liaoning, China (Yalujiang River) Sangrim-ri, Gyeongsangbuk-do, Korea (Nakdong River) Wloclawek, Poland (Wloclawek reservoir, Vistula River) Saga, Japan (Baba River) Hiroshima, Japan (Ashida River) Notabeni, Georgia (Notabeni, River) Minhou, Fujian, China (Min River) Bang Kung Keng, Khammouan, Laos Axioupolis, Vardar, Greece (Vardar River) Banam-ri, Jeollabuk-do, Korea (Incheon R.) Taian, Shandong, China (Yellow River) Osaka, Japan (Yamato River) Qionghai, Hainan Island, China Hengchun, Pingtung, Taiwan (Lake Longluan) Xiuning, Anhui, China (Qiantang River)

Accession No. AB366443 AB366444 AB366445 HQ113261* EF571662* AB366446 AB366448 AB366447 AB366480 HQ113249* AB239347 HQ113253* AB366450 AB366481 AY952335* AB366451 AB366453 AB366454 AB366456 AB366457 AB366458 EF571665* EF571655* AB366461 AB366462 AB366464 HQ113238* HQ113256* HQ113257* HQ113251* AB366465 AB239403 AB366466 AB366467 AB366469 AB366468 AB366471 AB239404 AB366473 AB366452 AB366478 AB366474 AB366475 AB366477 AB366519 AB366485 AB366494 DQ396678* AB366497 AB369282 DQ396682* AB366490 AB366502 AB366504 AB366508 AB366510 AB366511

Reference This study This study This study Yang et al. (2011) Yang et al. (2011) This study This study This study This study Yang et al. (2011) This study Yang et al. (2011) This study This study Unpublished This study This study This study This study This study This study Yang et al. (2011) Yang et al. (2011) This study This study This study Yang et al. (2011) Yang et al. (2011) Yang et al. (2011) Yang et al. (2011) This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Bohlen et al. (2006) This study This study Bohlen et al. (2006) This study This study This study This study This study This study

324

K. Kawamura et al.

Table 1.

Continued.

Species name R. ocellatus ocellatus 4 R. ocellatus ocellatus 5 R. ocellatus ocellatus 6 R. ocellatus ocellatus 7 R. pseudosericeus R. rheinardti R. sericeus R. sinensis 1 R. sinensis 2 R. sinensis 3 R. spinalis 1 R. spinalis 2 Tanakia himantegus chii 1 T. himantegus chii 2 T. himantegus himantegus T. koreensis 1 T. koreensis 2 T. lanceolata 1 T. lanceolata 2 T. limbata 1 T. limbata 2 T. signifer T. somjinensis T. tanago Biwia zezera Hemibarbus barbus Zacco platypus

Location No. 31 22 36 32 21 49 14 30 17 31 45 44 31 42 23 27 16 11 4 11 18 28 3

5

Accession No.

Collection location (Drainage) Qingpu, Shanghai, China (Yangtze River) Damsan-ri, Chungcheongnam-do, Korea (Gwangcheon River) Yuanjiang, Hunan, China (Lake Dongting) Hefei, Anhui, China (Yangtze River) Hoengseong-eup, Gangwon-do, Korea (Namhan River) Hue, Vietnam (Perfume River) Nehe, Heilongjiang, China (Amur River) Taian, Shandong, China (Yellow River) Songhak-ri, Gyeonggi-do, Korea (Namhan River) Qingpu, Shanghai, China (Yangtze River) Qiongshan, Hainan Island, China Chongzuo, Guangxi, China (Pearl River) Qingpu, Shanghai, China (Yangtze River) Tsuichih, Hsichih, Taiwan Hengchun, Pingtung, Taiwan Hwajeon-ri, Gyeongsangbuk-do, Korea (Nakdong River) Noha-ri, Jeollabuk-do, Korea (Geum River) Fengcheng, Liaoning, China (Yalujiang River) Fukuoka, Japan Mie, Japan (Kushida River) Fukuoka, Japan (Naka River) Toseong-ri, Gangwon-do, Korea (Imjin River) Banghyeon-ri, Jeollabuk-do, Korea (Seomjin River) Kanagawa, Japan (Tsurumi River) Shiga, Japan (Lake Biwa) Unknown Watarai, Mie, Japan (Miya R. system)

AB366512 AB366514 AB366515 AB366516 AB366517 AB366527 AB366518 AB366520 AB366521 AB366522 AB366523 AB366524 AB366529 DQ178384* AB366528 AB366530 AB366531 AB366532 AB366534 AB239406 AB366536 AB366537 AB366538 AB366539 AB250108 AB070241 AB366543

Reference This study This study This study This study This study This study This study This study This study This study This study This study This study Chang et al. (2009) This study This study This study This study This study This study This study This study This study This study Horikawa et al. (2007) Saitoh et al. (2006) This study

Asterisks (*) denote sequences downloaded from GenBank.

hood (ML) (Felsenstein, 1981) and partitioned Bayesian inference (BI). The most appropriate sequence evolution was selected under the Akaike Information Criterion (AIC) (Akaike, 1974) for ML and Bayesian Information Criterion (BIC) (Schwarz, 1978) for BI, using Modeltest 3.7 (Posada and Crandall, 1998). For ML analysis, the GTR + I + G model (I = 0.514, G = 0.889) was determined to be the most appropriate under AIC. The optimal ML tree was constructed with a heuristic search as implemented in PAUP*, with TBR branch-swapping and 10 random sequence additions. Support for recovered clades was assessed using the bootstrap analysis with 1000 total pseudoreplicates using PHYML (Guindon and Gascuel, 2003). Bayesian mixed inference was run in MrBayes 3.2 (Ronquist and Huelsenbeck, 2003), employing partition-specific modeling. For Cyt b the most appropriate model was TrNef + I + G (I = 0.655, G = 0.801) for the first codon, TPM2uf + I (I = 0.766) for the second codon and TIM2 + I + G (I = 0.014, G = 3.1540) for the third codon. MrBayes was run with 106 generations Markov chain. Starting trees were random, one cold and three heated chains were run simultaneously. Trees were saved every 100 generations for a total size of 104 in the initial sample. The first 103 trees were discarded as burnin and a majority rule (50%) consensus tree calculated from the 9000 remaining trees was used to determine the posterior probability of clades. Estimation of divergence time and character mapping Homogeneity of nucleotide substitution rate was checked with a likelihood test comparing the log-likelihood of the most probable trees, with and without enforcement by a molecular clock. This test rejected the null hypothesis of rate constancy (χ2 = 215.9, df = 82,

P < 0.001). Therefore, divergence times were estimated using a Bayesian relaxed molecular clock method, which was performed using BEAST v1.6.2 (Drummond and Rambaut, 2007). The tree topology constructed by BI analysis, based upon the GTR + I + G model, was used for inferring divergence time. BEAST was run with 5 × 107 generations Markov chain, initiated with random starting trees. Trees were sampled every 1,000 generations, with the exclusion of the first 5 × 106 generations as burn-in. Divergence time was calibrated using the first appearance of fossil records of Acheilognathinae in the early Miocene (20 mya), found from the Early Miocene Hachiya Formation in the Kani Group of Gifu in Japan (Yasuno, 1984; Nomura, 1986; Yabumoto and Uyeno, 2009). To understand the evolution of traits, changes in three discrete characters (distribution, karyotype and diapause) were reconstructed on the estimated BI tree, using MacClade v4.08 (Maddison and Maddison, 2003). Karyological information was cited from Arai (2011) and Okazaki et al. (2001) (see Supplementary Table S1 online).

RESULTS Gene sequences and variations Cyt b gene sequences obtained from the 69 fish samples (including outgroup sample) were diagnosed as distinct haplotypes (Table 1). No insertions or deletions were observed in any sequences. In total, 84 sequences including 15 citations from GenBank were used for phylogenetic analysis. Of the 1141 bp nucleotide sites, 515 sites were variable, of which 460 were parsimony informative. Average percentage sequence divergence (uncorrected p distance) within the Acheilognathinae was 17.8%, with the highest

MtDNA phylogeny of bitterling

325

sequence divergence of 19.9% between Acheilognathus chankaensis 4 and Rhodeus sinensis 1. Mean base composition was found to be reasonably uniform among the taxa examined (26.4% A, 27.4% C, 15.6% G, and 30.6% T). The third codon position revealed a significant reduction in the frequency of guanine (8.2%). The overall transition to transversion (Ti/Tv) ratio was 3.1. The relationships between corrected distance based on the GTR + I + G model and number of transition and transversion substitutions of Cyt b sequences were plotted for all pairwise comparisons (including outgroup taxa), for all positions and for third positions. All plots indicated that, even at the third position, no saturation was observed in Cyt b genes (plots not shown). P h yl og ene t ic r e la ti on ships The ML and BI trees were almost identical in topology, except for limited variation in some support values. Of these trees, the ML tree is shown in Fig. 2, along with bootstrap probabilities (BP) from ML and posterior probability (PP) values from BI at each node (shown only for BP > 50% in ML, and PP > 0.6 in BI). In the ML tree, bitterling are clearly separated into two clades, Acheilognathus and Tanakia-Rhodeus. Although the Acheilognathus clade was unambiguously monophyletic in both analyses (93% in ML and 1.0 in BI), statistical support for the Fig. 2. Maximum likelihood (ML) tree showing interrelationships of bitterling, inferred from mitochondrial Tanakia-Rhodeus clade was cytochrome b genes (constructed under the GTR + I + G model). Bootstrap confidence (BP) values from not as high in the ML analyML analysis, and the posterior probability (PP) value from Bayesian inference, are given in order at each sis, contrary to the outcome node. ‘–’ indicates that BP values are lower than 60. of the BI analysis (58% in (groups A–E) were evident. However, the interrelationships ML and 0.87 in BI). among all the lineages (groups A–F) were poorly resolved. In the Acheilognathus clade, one large lineage containing 13 species (group F), which is most derived, was presIn group F, discordance of taxonomy and phylogeny was ent (85% in ML and 1.0 in BI). In addition, five small lineages observed in subgroup F1. Acheilognathus rhombeus in

326

K. Kawamura et al.

Japan (A. rhombeus 2, 3) was closer to Acheilognathus barbatulus than A. rhombeus in Korea (A. rhombeus 1, 4). Group F contained autumn-spawning species with embryonic diapause, which were not monophyletic, forming two distinct lineages: A. barbatulus and A. rhombeus in subgroup

F1 and Acheilognathus longipinnis and Acheilognathus typus in subgroup F2 (Fig. 3). In the Tanakia-Rhodeus clade, Rhodeus was topologically monophyletic and embedded in Tanakia, which is paraphyletic to Rhodeus. However, the monophyly of Rhodeus was poorly supported by ML (< 60% in ML), in contrast with BI (0.72 in BI). In Tanakia, a lineage (group I) was strongly supported (92% in BP and 1.0 in BI), which excluded T. tanago and T. himantegus; the phylogenetic positions of these two species were not resolved. Although Rhodeus comprised five lineages (groups J-N) (> 95% in BP and 1.0 in BI), support values for the interrelationships among these lineages were low in ML (< 70% in ML), in contrast to BI (> 0.87 in BI).

Fig. 3. Bayesian inference phylogeny with estimated divergence times. The node with the asterisk indicates the calibration point based on fossil records for the Acheilognathinae. Open rectangular bars on the nodes show the 95% credible interval. The first column (I) corresponds to the spawning season and the second (II) to geographic distribution. For information of karyology, see Supplementary Table S1 online. I (Spawning season): S, springearly summer; A, autumn; ?, unknown. II (Geographic distribution): ‫ع‬, Asia (China, Vietnam); ٨, Korean Peninsula; ٤, Japan; ‫غ‬, Taiwan; ً, Europe.

Divergence time estimates and character evolution In a molecular clock based on fossil calibration, the divergence rate of Cyt b in bitterling was estimated to be 0.52 ± 0.05% per million years, which differed from a commonly used estimate of 0.76% in cyprinids using the information of well-dated geological events (Zardoya and Doadrio, 1999). However, it coincided well with an estimate of 0.52% in Capoeta (Cyprininae) based on a fossil calibration (Levin et al., 2012). Divergence times of lineages estimated using a relaxed molecular clock are shown in Fig. 3. In both clades of Acheilognathus and Tanakia-

MtDNA phylogeny of bitterling

Rhodeus, most of the major lineages appeared in the middle Miocene. Speciation showed a peak in the late Miocene and the representatives of extant species appeared by the end of the Pliocene. With the exception of T. tanago, which emerged in the middle Miocene, most species in the three genera endemic to Japan appeared in the late Miocene and Pliocene. Divergence of the Rhodeus sericeus lineage occurred in Europe in the Pleistocene. Almost at the same time, A. rhombeus in Japan was isolated from conspecies on the Asian continent. Autumn spawning with embryonic diapause evolved in two different lineages (group F1 and F2) in the middle Pliocene. In karyology, species with the chromosome number of 2n = 46, a derived lineage in Rhodeus (group N), appeared by the end of Pliocene. DISCUSSION Molecular phylogeny and taxonomy of Acheilognathinae In the phylogenetic relationships of bitterling inferred from 12S ribosomal DNA, Okazaki et al. (2001) reported that the Acheilognathinae comprised two major clades, Acheilognathus and Tanakia-Rhodeus. The monophyly of the former clade was strongly supported by neighbor-joining and maximum parsimony analyses, while that of the latter was poorly supported. In the Tanakia-Rhodeus clade the monophyly of either Tanakia or Rhodeus was also not supported (Okazaki et al., 2001). In the present study, two major clades were recognized (Fig. 2), which corresponded with those identified by Okazaki et al. (2001). The Acheilognathus clade was well supported by both ML and BI analyses, while the monophyly of the Tanakia-Rhodeus clade was weakly supported in the ML analysis (58 in ML), but strongly supported by BI analysis (0.87 in BI). Similarly, the monophyly of Rhodeus was only supported by BI analysis (0.87 in BI), although Rhodeus was topologically embedded in Tanakia. Six lineages were recognized in the Acheilognathus (groups A–F) and eight in the Tanakia-Rhodeus (groups G– N) clades (Fig. 2). The interrelationships among these lineages were poorly resolved. An estimation of the divergence time of these lineages indicated that they separated within a short period (Fig. 4), at a point early in the middle Miocene in the case of Tanakia-Rhodeus and later in the middle Miocene for Acheilognathus. The ambiguity in the interrelationships of lineages in two clades may, thus, have been be due to a rapid radiation of the group in the middle Miocene. In future researches, full resolution of the interrelationships of these lineages must be done using nuclear markers. This is important for the resolution of Tanakia/Rhodeus clades in particular. Many currently extant species appeared in the late Miocene, both in the Acheilognathus and Tanakia-Rhodeus clades (Fig. 3). At the species level, a discordance between taxonomy and phylogeny was observed in four lineages (A. barbatulus and A. rhombeus in Group F1, Tanakia koreensis and Tanakia somjinensis in Group I, Rhodeus spinalis and other conspecies in Group L, and Rhodeus atremius and other related species in Group N). This finding raises questions about the validity of the current taxonomic status of these species, which are to be reevaluated in a future study.

327

Biogeography of bitterling Although the Japanese islands are located at the eastern margin of the Asian continent, they support a notably diverse bitterling fauna (16 species or subspecies), most of which are endemic (Akai et al., 2009). The oldest known fossils of bitterling come from Japan and are dated to the early Miocene (ca. 20 mya) (Yasuno, 1984; Yabumoto and Uyeno, 2009). On the basis of paleontological information it appears that the Acheilognathinae, as well as other extant cyprinid subfamilies, first evolved in Japan in the early-middle Miocene, because any fossils of these subfamilies in this period have not been discovered in the Asian mainland, including Korean Peninsula. They later dispersed to the Asian mainland through a western land bridge in the late Miocene (Nakajima, 1986). Tanakia seems to have appeared earlier than the other two genera in the early Miocene (Fig. 3). The distribution of Tanakia, with the exception of T. himantegus, is restricted to Japan and Korea, while the two other genera are widely distributed throughout East Asia (Arai and Akai, 1988). Historical records and the restricted distribution of Tanakia lend support to the idea that the Acheilognathinae have their origins in Japan, followed by a later dispersal to the Asian mainland. The rapid radiation of most extant species, in Japan and elsewhere, was dated to the late Miocene or early Pliocene (Fig. 3). In contrast to conditions in the middle Miocene, which experienced relatively mild temperatures, the climate is believed to have become markedly harsher in the late Miocene, and is linked to the emergence of the Asian monsoon (Kitamura, 2010). The current contours of the Japanese archipelago also emerged during this period, while the migration of cyprinid fishes between the Japanese islands and the mainland occurred via the western land bridge (Watanabe, 2010). A possible scenario for the radiation of bitterling in the late Miocene and Pliocene may have been a consequence of a prominent change in climatic conditions, combined with the opportunity for widespread dispersal. In many species of the three genera of bitterling, differentiation of populations (subspecies formation) exclusively occurred in the Pleistocene (Fig. 3). Rhodeus sericeus appears to have diverged into three species/subspecies (R. amarus, Rhodeus colchicus and Rhodeus meridionalis) in a region that encompasses Eastern Europe and the Caucasus region in this era. The Pleistocene is known for its repeated glaciations (Cox and Moore, 2005), which would have facilitated isolation and speciation of R. sericeus in western Eurasia (Bohlen et al., 2006; Zaki et al., 2008; Bryja et al., 2010), as it did in other taxa (Hewitt, 1999). Character evolution in bitterling Autumn spawning with an embryonic diapause has been reported for only four species of Acheilognathus: A. barbatulus, A. longipinnis, A. rhombeus and A. typus (Nakamura, 1969; Li and Arai, unpubl. data; Matsuda and Arai, unpubl. data). In a previous study (Kawamura and Uehara, 2005), it was suggested that embryonic diapause in autumn-spawning bitterling was controlled by genetic factors, which are absent in spring-spawning bitterling. In the present study, A. barbatulus and A. rhombeus formed a lineage distinct from that of A. longipinnis and A. typus, although it is evident that they all diverged from a common ancestor of Acheilognathus (Fig.

328

K. Kawamura et al.

2). The estimated divergence time and tree topology suggest that these two lineages appeared in different regions (barbatulus-rhombeus on the Asian mainland and longipinnistypus in Japan) at approximately the same period in the Pliocene (Fig. 3). Thus the two lineages of Acheilognathus may have convergently evolved both autumn spawning and embryonic diapause as an adaptation to changed climatic conditions in the late Pliocene (Kawamura and Uehara, 2005), when the Neogene first experienced glaciations (Cox and Moore, 2005). Okazaki et al. (2001) noted that the chromosome number of 2n = 44 in Acheilognathus and 2n = 46 in Rhodeus may have originated from an ancestral chromosome number of 2n = 48. In Acheilognathus, Group F, for which monophyly was strongly supported in molecular trees (Fig. 3), and which includes autumn-spawning species, is characterized by scale-like projections on the body surface of the freeembryo (Suzuki and Hibiya, 1985a) and well-developed pharyngeal teeth in the adult (Suzuki and Hibiya, 1985b). The former character, coupled with conspicuous maggot-like undulatory movements of the free-embryo, function as adaptations to resist ejection by host mussels (Suzuki and Hibiya, 1985a). Different adaptations, but with the same function, are expressed by the free-embryos of Rhodeus. Thus, winglike yolk-sac projections, which are an autapomorphy of Rhodeus (Okazaki et al., 2001), limit embryo ejections by host mussels (Suzuki and Hibiya, 1985a). Differences in the traits that serve the same function of minimizing ejections from mussels, may reflect differences in the gill structure of the host mussels used by the two groups (Liu et al., 2006). The pharyngeal teeth of Rhodeus are not as well developed as those of Acheilognathus. In species with a chromosome number of 2n = 46 (Group N in Fig. 3), which show the most specialized karyological features (Ojima et al., 1973; Ueda et al., 2001), pharyngeal teeth are relatively underdeveloped (Suzuki and Hibiya, 1985b). In fish, it is recognized that feeding adaptations can be a major force in adaptive radiations, such as those seen in pupfish (Cyprinodontidae) and cichlids (Kocher, 2004; Martin and Wainwright, 2011). However, in bitterling it may be adaptations for the environment of the host mussel in the free-embryonic period has greater importance than feeding adaptations (Suzuki and Hibiya, 1985a; Liu et al., 2006). ACKNOWLEDGMENTS We are much indebted to the following researchers for collecting samples for this study: H. Wu, J. Zhong, S. Jeon, K. Shao, M. Kottelat, J. Freyhof, M. Przybylski, T. Ishinabe, Y. Kanoh, J. Kitamura, T. Nishimura, K. Saitoh, K. Uehara, S. Seki, J. Kitajima, H. Yamane, N. Aoyama. This study was supported in part by grants for Science Research from the Ministry of Education, Science, Sports and Culture, Japan (Nos. 10041156 and 12575009).

REFERENCES Agbali M, Reichard M, Bryjova A, Bryja J, Smith C (2010) Mate choice for non-additive genetic benefits correlate with MHC dissimilarity in the rose bitterling (Rhodeus ocellatus). Evolution 64: 1683–1696 Akai Y, Akiyama N, Ueno T, Kuzushima K, Suzuki N, Masuda O, et al. (2009) Encyclopeida of Bitterling. Marine Enterprise, Tokyo Akaike H (1974) A new look at the statistical model identification. IEEE Trans Autom Contr 19: 716–723

Arai R (1982) A chromosome study on two cyprinid fishes, Acrossocheilus labiatus and Pseudorasbora pumila pumila, with notes on Eurasian cyprinids and their karyotypes. Bull Natn Sci Mus, Tokyo Ser A 8: 131–152 Arai R (1988) Fish systematics and cladistics. In “Ichthyology Currents 1988” Ed by T Uyeno, M Okiyama, Asakura Shoten, Tokyo, pp 4–33 Arai R (2011) Fish Karyotypes. Springer, Tokyo Arai R, Akai Y (1988) Acheilognathus melanogaster, a senior synonym of A. moriokae, with a revision of the genera of the subfamily Acheilognathinae (Cypriniformes, Cyprinidae). Bull Nat Sci Mus Tokyo (A) 14: 199–213 Avise JC (2000) Phylogeography: The History and Formation of Species. Harvard University Press, Cambridge, Massachusetts ˘ arescu ˘ Ban P (1990) Zoogeography of Fresh Waters. Aula-Verlag, Wiesbaden Beheregaray LB (2008) Twenty years of phylogeography: the state of the field and the challenges for the Southern Hemisphere. Mol Ecol 17: 3754–3774 Bohlen J, Slechtova V, Bogutskaya N, Freyhof J (2006) Across Siberia and over Europe: phylogenetic relationships of the freshwater fish genus Rhodeus in Europe and the phylogenetic position of R. sericeus from the River Amur. Mol Phylogenet Evol 40: 856–865 Bryja J, Smith C, Koneˇcný A, Reichard M (2010) Range-wide population genetic structure of the European bitterling (Rhodeus amarus) based on microsatellite and mitochondrial DNA analysis. Mol Ecol 19: 4708–4722 Chang CH, Lin WW, Shao YT, Arai R, Ishinabe T, Yeda T, et al. (2009) Molecular phylogeny and genetic differentiation of the Tanakia himantegus complex (Teleostei: Cyprinidae) in Taiwan and China. Zool Stud 48: 823–834 Cox C, Moore P (2005) Biogeography: An Ecological and Evolutionary Approach. Blackwell Publishing Limited, Malden, MA Drummond AJ, Rambaut A (2007) BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 7: 214 Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17: 368–376 Froese R, Pauly D (2013) FishBase. Available at http://www.fishbase. org/search.php Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696–704 Hewitt GM (1999) Post-glacial re-colonization of European biota. Biol J Linn Soc 68: 87–112 Horikawa M, Nakajima J, Mukai T (2007) Distribution of indigenous and non-indigenous mtDNA haplotypes of Biwia zezera (Cyprinidae) in northern Kyusyu, Japan. Jpn J Ichthyol 54: 149– 159 Kawamura K, Uehara K (2005) Effects of temperature on freeembryonic diapause in the autumn-spawning bitterling Acheilognathus rhombeus (Teleostei: Cyprinidae). J Fish Biol 67: 684–695 Kawamura K, Ueda T, Arai R, Nagata Y, Saitoh K, Ohtaka H, et al. (2001) Genetic introgression by the rose bitterling, Rhodeus ocellatus ocellatus, into the Japanese rose bitterling, R. o. kurumeus (Teleostei: Cyprinidae). Zool Sci 18: 1027–1039 Kitamura A (2010) Development of Japanese archipelago and paleoenvironment. In “Natural History of Freshwater Fishes in Biogeography” Ed by K Watanabe, H Takahashi, Hokkaido University Press, Sapporo, pp 13–28 Kitamura J, Nagata N, Nakajima J, Sota T (2012) Divergence of ovipositor length and egg shape in a brood parasitic bitterling fish through the use of different mussel hosts. J Evol Biol 25: 566– 573 Kocher TD (2004) Adaptive evolution and explosive speciation: the cichlid fish model. Nat Rev Genet 5: 288–298

MtDNA phylogeny of bitterling Kubota H, Watanabe K, Suguro N, Tabe M, Umezawa K, Watanabe K (2010) Genetic population structure and management units of the endangered Tokyo bitterling, Tanakia tanago (Cyprinidae). Conserv Genet 11: 2343–2355 Lansman RA, Shade RO, Shapira JF, Avise JC (1981) The use of restriction endonucleases to measure mitochondrial DNA sequence relatedness in natural populations. III. Techniques and potential applications. J Mol Evol 17: 214–226 Levin BA, Freyhof J, Lajbnerd Z, Perea S, Abdoli A, Gaffaroglu ˇ M, et al. (2012) Phylogenetic relationships of the algae scraping cyprinid genus Capoeta (Teleostei: Cyprinidae). Mol Phylogenet Evol 62: 542–549 Liu H, Yurong Z, Reichard M, Smith C (2006) Evidence of host specificity and congruence between phylogenies of bitterlings and freshwater mussels. Zool Stud 45: 428–434 Maddison DR, Maddison WP (2003) MacClade 4: analysis of phylogeny and character evolution, version 4.06. Available from http://macclade.org Martin CH, Wainwright PC (2011) Trophic novelty is linked to exceptional rates of morphological diversification in two adaptive radiations of Cyprinodon pupfish. Evolution 65: 2197–2212 Miya M, Nishida M (2000) Use of mitogenomic information in teleostean molecular phylogenetics: a tree-based exploration under the maximum-parsimony optimality criterion. Mol Phylogenet Evol 17: 437–455 Nakajima T (1986) Pliocene cyprinid pharyngeal teeth from Japan and west Asia Neogene cyprinid zoogeography. In “Proceedings of the Second International Conference on Indo-Pacific Fishes” Ed by T Uyeno, R Arai, T Taniuchi, K Matsuura, Ichthyological Society of Japan, Tokyo, pp 502–513 Nakamura M (1969) Cyprinid Fishes in Japan: Studies on the Life History of Cyprinid Fishes of Japan. Research Institute for National Resources, Tokyo Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics. Oxford University Press, New York Nomura T (1986) Preliminary report of the Miocene Hachiya Formation and its K-Ar ages. J Geol Soc Japan 92: 73–76 Ojima Y, Uyeno K, Hayashi M (1973) Karyotypes of the acheilognathine fishes (Cyprinidae) of Japan with a discussion of phylogenetic problems. Zool Mag Japan 82: 171–177 Okazaki M, Naruse K, Shima A, Arai R (2001) Phylogenetic relationships of bitterlings based on mitochondrial 12S ribosomal DNA sequences. J Fish Biol 58: 89–106 Palumbi SR, Martin A, Romano S, McMillan WO, Stice L, Grabowski G (1991) The Simple Fool’s Guide to PCR. Version 2. Dept. of Zoology, Univ. of Hawaii, Honolulu Pateman-Jones C, Rasotto MB, Reichard M, Liao C, Liu H, ZiE˛Ba G, et al. (2011) Variation in male reproductive traits among three bitterling fishes (Acheilognathinae: Cyprinidae) in relation to the mating system. Biol J Linn Soc 103: 622–632 Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817–818 Reichard M, Ondracová ˇ M, Bryjova A, Smith C, Bryja J (2009) Breeding resource distribution affects selection gradients on male phenotypic traits: experimental study on lifetime reproductive success in the bitterling fish (Rhodeus amarus). Evolution 63: 377–390 Reichard M, Bryja J, Polacik ˇ M, Smith C (2011) No evidence for host specialization or host-race formation in the European bitterling (Rhodeus amarus), a fish that parasitizes freshwater mussels. Mol Ecol 20: 3631–3643 Reichard M, Vrtílek M, Douda K, Smith C (2012) An invasive spe-

329

cies causes role reversal in a host-parasite relationship. Biol Lett 8: 601–604 Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572– 1574 Saitoh K, Sado T, Mayden RL, Hanzawa N, Nakamura K, Nishida M, et al. (2006) Mitogenomic evolution and interrelationships of the Cypriniformes (Actinopterygii: Ostariophysi): The first evidence toward resolution of higher-level relationships of the world’s largest freshwater fish clade based on 59 whole mitogenome sequences. J Mol Evol 63: 826–841 Saitoh K, Sado T, Doosey MH, Baer HLJ, Inoue JG, Nishida M, et al. (2011) Evidence from mitochondrial genomics supports the lower Mesozoic of South Asia as the time and place of basal divergence of cypriniform fishes (Actinopterygii: Ostariophysi). Zool J Linn Soc 161: 633–662 Schwarz G (1978) Estimating the dimension of a model. Ann Stat 6: 461–464 Smith C, Reichard M, Jurajda P, Przybylski M (2004) The reproductive ecology of the European bitterling (Rhodeus sericeus). J Zool 262: 107–124 Suzuki N, Hibiya T (1985a) Minute tubercles on the skin surface of larvae of Acheilognathus and Pseudoperilampus (Cyprinidae). Jpn J Ichthyol 32: 335–344 Suzuki N, Hibiya T (1985b) Pharyngeal teeth and masticatory process of the basioccipital bone in Japanese bitterlings (Cyprinidae). Jpn J Ichthyol 32: 180–188 Swofford DL (2003) PAUP*: Phylogenetic Analysis Using Parsimony (* and Other Methods). Version 4.0. Sinauer Associates, Sunderland, MA Ueda T, Naoi H, Arai R (2001) Flexibility on the karyotype evolution in bitterlings (Pisces, Cyprinidae). Genetica 111: 423–432 Watanabe K (2010) Faunal transition of freshwater fish in Japanese archipelagos, based on the fossils of freshwater fish in the Neogene In “Natural Hiistory of Freshwater Fishes in Biogeography” Ed by K Watanabe, H Takahashi, Hokkaido University Press, Sapporo, pp 185–202 Wiepkema PR (1961) An ethological analysis of the reproductive behaviour of the bitterling (Rhodeus amarus Bloch). Arch Neerl Zool 14: 103–199 Yabumoto Y, Uyeno T (2009) Fossils of bitterling. In “Encyclopedia of Bitterling” Ed by Y Akai et al., Marine Enterprise, Tokyo, pp 14–17 Yang Q, Zhu Y, Xiong B, Liu H (2011) Acheilognathus changtingensis sp. nov., a new species of the cyprinid genus Acheilognathus (Teleostei: Cyprinidae) from Southeastern China based on morphological and molecular evidence. Zool Sci 28: 158–167 Yasuno K (1984) Fossil pharyngeal teeth of the Rhodeinae fish from the Miocene Katabira Formation of the Kani Group, Gifu Prefecture, Japan. Bull Mizunami Fossil Mus 11: 101–105 Zaki SAH, Jordan WC, Reichard M, Przybylski M, Smith C (2008) A morphological and genetic analysis of the European bitterling species complex. Biol J Linn Soc 95: 337–347 Zardoya R, Doadrio I (1999) Molecular evidence on the evolutionary and biogeographical patterns of European cyprinids. J Mol Evol 49: 227–237 Zhu Y, Liu H (2006) Genetic diversity and biogeographical process of Acheilognathus macropterus revealed by sequence variations of mitochondrial cytochrome b gene. Acta Hydrobiol Sin 39: 134–140 (Received November 13, 2013 / Accepted January 21, 2014)