Phylogenetic relationships of Acheilognathidae (Cypriniformes: Cyprinoidea) as revealed from evidence of both nuclear and mitochondrial gene sequence variation: evidence for necessary taxonomic revision in the family and the identification of cryptic species

Jae-Seong Lee

Bitterlings are relatively small cypriniform species and extremely interesting evolutionarily due to their unusual reproductive behaviors and their coevolutionary relationships with freshwater mussels. As a group, they have attracted a great deal of attention in biological studies. Understanding the origin and evolution of their mating system demands a well-corroborated hypothesis of their evolutionary relationships. In this study, we provide the most comprehensive phylogenetic reconstruction of species relationships of the group based on partitioned maximum likelihood and Bayesian methods using DNA sequence variation of nuclear and mitochondrial genes on 41 species, several subspecies and three undescribed species. Our findings support the monophyly of the Acheilognathidae. Two of the three currently recognized genera are not monophyletic and the family can be subdivided into six clades. These clades are further regarded as genera based on both their phylogenetic relationships and ...

Molecular Phylogenetics and Evolution 81 (2014) 182–194 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Phylogenetic relationships of Acheilognathidae (Cypriniformes: Cyprinoidea) as revealed from evidence of both nuclear and mitochondrial gene sequence variation: Evidence for necessary taxonomic revision in the family and the identification of cryptic species Chia-Hao Chang a,b,c, Fan Li d,e, Kwang-Tsao Shao a, Yeong-Shin Lin b,f, Takahiro Morosawa g, Sungmin Kim h, Hyeyoung Koo i, Won Kim h, Jae-Seong Lee j, Shunping He k, Carl Smith l,m, Martin Reichard m, Masaki Miya n, Tetsuya Sado n, Kazuhiko Uehara o, Sébastien Lavoué p, Wei-Jen Chen p,⇑, Richard L. Mayden c a Biodiversity Research Center, Academia Sinica, Taipei 11529, Taiwan Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan Department of Biology, Saint Louis University, St. Louis, MO 63103, USA d Department of Oceanography, National Sun Yet-sen University, Kaohsiung 80424, Taiwan e Institute of Biodiversity Science, Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Fudan University, Shanghai 200433, China f Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu 30068, Taiwan g Japan Wildlife Research Center, Tokyo 130-8606, Japan h School of Biological Sciences, Seoul National University, Seoul 151-747, Republic of Korea i Department of Biological Science, Sangji University, Wonju 220-702, Republic of Korea j Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea k Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China l School of Biology, University of St Andrews, St Andrews, Fife KY16 8LB, UK m Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Květná 8, 603 65 Brno, Czech Republic n Natural History Museum & Institute, Chiba 260-8682, Japan o Aquatic Life Conservation Research Center, Research Institute of Environment, Agriculture and Fisheries, Osaka 572-0088, Japan p Institute of Oceanography, National Taiwan University, Taipei 10617, Taiwan b c a r t i c l e i n f o Article history: Received 9 July 2014 Revised 28 August 2014 Accepted 29 August 2014 Available online 17 September 2014 Keywords: Acheilognathinae Cyprinidae Cryptic species Nuclear loci Cytochrome b European bitterling a b s t r a c t Bitterlings are relatively small cypriniform species and extremely interesting evolutionarily due to their unusual reproductive behaviors and their coevolutionary relationships with freshwater mussels. As a group, they have attracted a great deal of attention in biological studies. Understanding the origin and evolution of their mating system demands a well-corroborated hypothesis of their evolutionary relationships. In this study, we provide the most comprehensive phylogenetic reconstruction of species relationships of the group based on partitioned maximum likelihood and Bayesian methods using DNA sequence variation of nuclear and mitochondrial genes on 41 species, several subspecies and three undescribed species. Our findings support the monophyly of the Acheilognathidae. Two of the three currently recognized genera are not monophyletic and the family can be subdivided into six clades. These clades are further regarded as genera based on both their phylogenetic relationships and a reappraisal of morphological characters. We present a revised classification for the Acheilognathidae with five genera/lineages: Rhodeus, Acheilognathus (new constitution), Tanakia (new constitution), Paratanakia gen. nov., and Pseudorhodeus gen. nov. and an unnamed clade containing five species currently referred to as ‘‘Acheilognathus’’. Gene trees of several bitterling species indicate that the taxa are not monophyletic. This result highlights a potentially dramatic underestimation of species diversity in this family. Using our new phylogenetic framework, we discuss the evolution of the Acheilognathidae relative to classification, taxonomy and biogeography. Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author at: Room 301, Institute of Oceanography, National Taiwan University, No. 1 Sec. 4 Roosevelt Road, Taipei 10617, Taiwan. Fax: +886 2 23637062. E-mail address: wjchen.actinops@gmail.com (W.-J. Chen). http://dx.doi.org/10.1016/j.ympev.2014.08.026 1055-7903/Ó 2014 Elsevier Inc. All rights reserved. C.-H. Chang et al. / Molecular Phylogenetics and Evolution 81 (2014) 182–194 183 1. Introduction 1.2. Previous hypotheses of acheilognathid phylogeny Bitterlings are small fishes, typically less than 150 mm in length (Chen, 1998) and are native to Europe (three species) and east and southeast Asia (Kottelat and Freyhof, 2007). The species are associated with a variety of lowland freshwater habitats, including lakes, ponds, rivers and irrigation ditches. Aside from their striking nuptial coloration, bitterlings exhibit a remarkable breeding biology involving oviposition in the gill chambers of freshwater mussels (Unionidae and Margaritiferidae). Fertilization occurs in the mussel gill cavity and development is completed in the host mussel gill chamber (Smith et al., 2004). This breeding association makes species of bitterlings a focus of research aimed at understanding coevolutionary dynamics, life-history evolution, sexual selection, sperm competition, development and mate choice (Agbali et al., 2011; Casalini et al., 2009; Kitamura et al., 2012; Mills et al., 2005; Reichard et al., 2006, 2007, 2012; Spence and Smith, 2013; Spence et al., 2013). Bitterlings are thought to form a monophyletic group and have traditionally been classified in the subfamily Acheilognathinae, one of the 11 subfamilies of Cyprinidae (Howes, 1991; Nelson, 2006). Recent molecular phylogenetic studies of cyprinid fishes have revealed significant findings regarding the relationships of the bitterling clade to other cyprinoids and a reclassification of Cyprinidae (Chen and Mayden, 2009; Mayden and Chen, 2010; Mayden et al., 2009; Saitoh et al., 2006; Tang et al., 2010, 2011; Yang, L. et al., 2012b). Current molecular-based analyses support the monophyly of Acheilognathinae as a subfamily within the Cyprinidae (Chen and Mayden, 2009; Tao et al., 2013; Saitoh et al., 2006; Wang et al., 2012b). However, Cyprinidae is paraphyletic with respect to Psilorhynchidae, as the latter family is nested within the former (Chen and Mayden, 2009; Mayden and Chen, 2010). The most closely related lineages to Acheilognathinae include: Tanichthys (mountain minnows), Tincinae (tench), Leuciscinae (minnows), and Gobioninae (gudgeons). These lineages are both temperate in distribution and include many species endemic to Eurasia and North America, and deeply nested within the ‘‘cyprinid’’ tree. Given the repeated recovery of monophyletic groups, congruent phylogenetic relationships, and the paraphyly of the ‘‘Cyprinidae’’, Chen and Mayden (2009) argued for elevating the Acheilognathinae and some other previously recognized subfamilies of Cyprinidae to family status, within the superfamily Cyprinoidea. Despite many molecular phylogenetic analyses of the Cyprinoidea (Chen and Mayden, 2009; Levin et al., 2012; Mayden et al., 2009; Perea et al., 2010; Tang et al., 2010, 2011; Tsigenopoulos et al., 2010; Yang, J. et al., 2012a; Yang, L. et al., 2012b), relationships within the Acheilognathidae have not been examined until very recently. Previous phylogenetic studies of bitterlings have been limited in character and/or taxon sampling (Bohlen et al., 2006; Chang et al., 2009; Kitamura et al., 2012; Yang, Q. et al., 2011; Zhu and Liu, 2006). Bohlen et al. (2006) proposed that Rhodeus in Europe was sister to the Asian species Rhodeus sericeus. However, their results failed to clarify whether the history of the multiple species in the genus involved one or more connections, or whether additional sister group relationships among European and Asian species need to be considered. Resolving this hypothesis of evolutionary and biogeographic events related to the origin of European bitterlings requires a greater sampling of species. Prior to two recent studies (Cheng et al., 2014; Kawamura et al., 2014), the most comprehensive molecular-based study on species was that by Okazaki et al. (2001); however, this study suffered in data analysis. Okazaki et al. (2001) reconstructed relationships of bitterlings based only on partial sequences of 12S rRNA for 27 bitterling species/subspecies using Neighbor-Joining (NJ) analysis, a distance-based method of analysis that is known to be inadequate for the inference of species relationships. In their inferred NJ tree, Acheilognathus was recovered as monphyletic and sister to the weakly supported clade including Rhodeus and Tanakia. However, the monophyly of the latter two genera was not retained. Arai and Kato (2003) examined relationships using combined morphological and molecular (12S rRNA) characters as a follow up to a classification by Arai and Akai (1988). The former authors suggested a progressive evolution in bitterlings, implying a ‘‘trend’’ of bitterling evolution wherein Tanakia was the ‘‘ancestral’’ group, with both Acheilognathus and Rhodeus evolving from Tanakia. A more complete analysis by Chen and Mayden (2009), incorporating more taxa and characters in the cyprinoid phylogeny, contradicted these proposed evolutionary trends in bitterlings, and instead resolved Acheilognathus sister to other species. The two most recent molecular studies on bitterlings by Cheng et al. (2014) and Kawamura et al. (2014) both increased taxonomic sampling (44 and 82 taxa included, respectively) relative to previous studies. However, both studies were limited in character sampling in using primarily cytochrome b sequences. Kawamura et al. (2014), while having increased taxonomic sampling, did not diversify species, with most coming from East Asia (particularly Korea and Japan) and only one sample was from Europe; no Middle Eastern species were included. Limited sampling from specific regions will tend to limit the resolution of evolutionary and biogeographic events. Despite these limitations, the studies by Cheng et al. (2014) and Kawamura et al. (2014) consistently resolved Acheilognathidae as monophyletic with two major clades, Acheilognathus and TanakiaRhodeus. No previous studies (when multiple species from the three current genera were sampled) including the most recent ones have recovered Tanakia as monophyletic (Arai and Kato, 2003; Cheng et al., 2014; Kawamura et al., 2014; Okazaki et al., 2001). Thus, the monophyly of the genera Tanakia and Rhodeus remains questionable. 1.1. Taxonomy Bitterlings have a complicated taxonomic history. The Acheilognathidae currently includes about 74 species (from 117 available species names; Eschmeyer and Fong, 2014) and several undescribed species (Arai, 1988; Liu et al., 2006; Smith et al., 2004). The classification inclusive of three genera, Acheilognathus, Rhodeus, and Tanakia, has been particularly unstable and has been retained largely as convention. Up to seven genera have been used for the group (Acanthorhodeus, Acheilognathus, Rhodeops, Rhodeus, Paracheilognathus, Pseudoperilampus, and Tanakia). Although classifications have included three genera some studies have not embraced this classification (Fujiwara et al., 2009; Hwang et al., 2014; Wang et al., 2012a). For instance, Arai and Akai (1988) used Acheiloghnathus macropterus; whereas Hwang et al. (2014) identified the species Acanthorhodeus. However, more recent studies (Duc et al., 2013; Li and Arai, 2010; Yang, Q. et al., 2010, 2011) generally agree in recognizing the ‘‘three genera scenario’’ (Arai and Akai, 1988). Diagnoses of the genera include characters related to karyotypes, color patterns on dorsal fins, and features of the lateralis system. 1.3. Objectives In the present study we re-examined the phylogenetic relationships within Acheilognathidae using the largest molecular dataset assembled to date, with six nuclear gene loci (recombination activating gene 1 [RAG1], rhodopsin [RH], interphotoreceptor 184 C.-H. Chang et al. / Molecular Phylogenetics and Evolution 81 (2014) 182–194 retinoid-binding protein gene 2 [IRBP2], early growth response protein genes [EGR] 1, 2B, and 3) (Chen et al., 2008) and one mitochondrial gene (cytochrome b, [Cyt b]) for 41 bitterling species represented by 117 individuals. These data and resulting analyses are the most comprehensive for this family and are capable of testing both previous phylogenetic hypotheses and hypotheses of a classification involving only three acheilognathid genera. The early evolution of Acheilognathidae is examined, providing new insight within the relationships among European and Asian species. Emphasis was also placed on greater geographic sampling of the most widely distributed species/subspecies (A. macropterus, A. rhombeus, A. tabira, A. barbatus, T. himantegus, R. ocellatus, R. sinensis) to examine the possibility of undetected or cryptic species in the family. 2. Materials and methods 2.1. Sample collection A total of 117 individuals of each bitterling species (41 species, 2 undescribed species, several subspecies) were sampled, where possible, across their native ranges. Specimens were sampled from natural habitats, commercial aquarists and known stocks from Japanese conservation research centers (e.g., Tanakia tanago). Following Eschmeyer’s (2014) Catalog of Fishes, our sampling includes 50% of the listed species of Acheilognathus, 58% of Rhodeus, and 83% of Tanakia. Most specimens were identified either by those providing samples or by the first author (CHC) using several identification resources, including information on local faunas, such as Chen et al. (1998) and Nakabo (2013). Species were identified as belonging to Acheilognathus, Rhodeus, or Tanakia following the classification of Arai and Akai (1988). Two small juvenile specimens could not be identified to either genus or species and are referred to as ‘‘Acheilognathidae gen. sp.’’. Similarly, it was not possible to identify some adult specimens to the level of species using current species diagnoses; these specimens may represent undescribed species and are identified with ‘‘sp.’’. Finally, for some specimens their characteristics were close to but did not completely fit the diagnosis of a described species; these examples are noted using the convention of ‘‘cf’’ (e.g., ‘‘Acheilognathus’’ cf. striatus). Outgroup selection included multiple species and was based on previous phylogenetic hypotheses of Chen and Mayden (2009). Outgroup taxa included Tinca tinca, Gobio gobio, Pelecus cultratus, Zacco sieboldii, and Danio dangila, with the most distant outgroup being Danio dangila. Taxa and sample details are provided in Table 1. 2.2. DNA data collection Genomic DNA was extracted either from fin or muscle preserved in 95% ethanol using the Quick Gene DNA tissue Kit S (Fujifilm, Tokyo, Japan). All primers of the six nuclear markers were from Chen et al. (2003, 2008), and López et al. (2004). Protocols for collecting DNA data from these markers follow those outlined in Chen et al. (2008). New pair-specific primers were designed for amplifying and sequencing Cyt b; these include Cyt b-F (5́-GAY TTG AAG AAC CAT CGT TGT A-3́) and Cyt b-R (5́-CTT CGG ATT ACA AGA CCG ATG C-3́). PCR amplifications of Cyt b were performed in a mixture with a final volume of 25 lL containing 10 ng template DNA, 25 lmol of each pair of primers, 12.5 lL of Fast-Run™ Advanced Taq Master Mix (ProTech, Taipei, Taiwan), and distilled water. Thermal cycling began with one cycle at 94 °C for 4 min; subsequently 35 cycles of denaturation at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min; and finally, a single extension step at 72 °C for 5 min. PCR products were purified using a PCR DNA Fragments Extraction Kit (Geneaid, Taipei, Taiwan). Sequencing was performed using ABI 3730 version 3.2 analyzer (Applied Biosystems), following protocols of ABI PRISM BigDye Sequencing Kit (PE Applied Biosystems, USA) and the same pairs of PCR primers (by Mission Biotech Inc., Taipei, Taiwan). All newly obtained sequences are available on GenBank; see Table 1 for accession numbers. 2.3. Sequence alignment and phylogenetic analysis Genes were aligned manually using MEGA 5 (Tamura et al., 2011) and based on inferred amino acid translations. Sequenced bp for genes were as follows: RAG1, 1302 bp; RH, 801 bp; IRBP2, 807 bp; EGR1, 840 bp; ERG2B, 789 bp; EGR3, 843 bp; and Cyt b, 1140 bp. No indels were observed in the aligned sequences except for EGR1 in which a deletion of three continuous nucleotides coding one amino acid occurred. Phylogenetic analyses involved three operational datasets: (1) only mitochondrial-gene data set; (2) only nuclear-gene data set; and (3) a combined nuclear plus mitochondrial sequences. Analyses were performed using partitioned Maximum Likelihood (ML) and partitioned Bayesian approaches (BA). RAxML 7.0.4 (Stamatakis, 2006) was used for ML analyses (MLA). Partitions were set with respect to gene and codon position; the GTR + G + I model (with four discrete rate categories) was adopted for each partition. The ML tree was obtained by performing 100 different runs using the default algorithm of the program. The best ML tree was chosen from likelihood scores among suboptimal trees from each run. Nodal support for MLA was bootstrap analysis and determined using RAxML (Felsenstein, 1985); non-parametric bootstrap replications were 1000 with the ML criterion. BA, as implemented in MRBAYES 3.1.1 (Huelsenbeck and Ronquist, 2001), was used for the combined data set, involving 21 partitions based on gene and codon position. jModelTest (Posada, 2008) was used to select the best-fit model for each partition. Parameters for performing partitioned BA were as follows: ‘‘lset nst = 6’’ (for GTR modle), ‘‘lset nst = 2’’ (for HKY model), ‘‘lset nst = 1’’ (for F81 model), ‘‘rates = invgamma’’ (G + I), ‘‘rates = propinv’’ (I), or ‘‘rates = gamma’’ (G), ‘‘unlink’’ (unlinking of model parameters across data partitions), and ‘‘prset ratepr = variable’’ (rate multiplier variable across data partitions). Two independent MCMC chains were conducted with 6,000,000 replicates, sampling one tree per 100 replications for each run. The distribution of log likelihood scores was examined to determine both stationarity for each search and the necessity for additional runs to reach convergence in log likelihoods. We discarded the initial trees with nonstationary log likelihood (as burn-in), and combined the remaining trees that resulted in convergent log likelihood scores from both independent searches. These trees were used to construct a 50% majority rule consensus tree. The values represented are a posteriori probabilities (PP) for BA. Nodal support was for BA trees was based on a posteriori probabilities (PP). 3. Results A total of 6522 bp were aligned for the combined dataset of 117 bitterling and 5 outgroup taxa; Table 2 includes sequence length, number of variable sites, and number of parsimony informative sites for each genetic marker. Among sequences from the seven gene markers a deletion of one amino acid in EGR1 was observed in only Acheilognathidae gen, sp., A. typus, A. longipinnis, A. gracilis, A. asmussii, A. macropterus (1) and (3), A. macropterus (5), A. barbatus (2), and A. barbatulus (8). However, with reference to the most likely Table 1 List of bitterling species and their outgroup taxa sequenced for seven molecular markers (Cytochrome b [Cyt b], recombination activation gene 1 [RAG1], Rhodopsin [RH], interphotoreceptor retinoid-binding protein gene 2 [IRBP2], early growth response protein genes [EGR] 1, 2B, and 3) from the study with taxonomic identification, sample donor, location, individual number as utilized in Fig. 1 and GenBank accession numbers. Genus Species (individual no.) Sample donor Sample location GenBank accession number Cyt b RAG1 RH IRBP EGR1 EGR2B EGR3 NC_008648 NC_008662 NC_008663 NC_008653 NC_015525 EU711162 EU292689 EU711144 EU292713 EU292697 FJ197070 FJ197056 FJ197045 FJ197069 EU409660 FJ197121 FJ197107 FJ197095 FJ197120 EU409662 FJ531280 FJ531264 FJ531272 FJ531283 EU409724 FJ531309 FJ531293 FJ531301 FJ531312 EU409756 FJ531338 FJ531322 FJ531330 FJ531341 EU409788 KF410698 KF410707 KF410699 KF410700 KF410701 KF410706 KF410702 KF410703 KF410704 KF410705 KF410711 KF410710 KF410708 KF410709 KF410713 KF410712 KF410714 KF410715 KF410717 KF410716 KF410718 KF410719 KF410720 KF410721 KF410722 KF410723 KF410728 KF410726 KF410727 KF410724 KF410725 KF410730 KF410729 KF410732 KF410731 KF410734 KF410733 KF410738 KF410735 KF410736 KF410737 KF410740 KF410739 KF410741 KF410742 KF471756 KF471765 KF471757 KF471758 KF471759 KF471764 KF471760 KF471761 KF471762 KF471763 KF471769 KF471768 KF471766 KF471767 KF471771 KF471770 KF471772 KF471773 KF471775 KF471774 KF471776 KF471777 KF471778 KF471779 KF471780 KF471781 KF471786 KF471784 KF471785 KF471782 KF471783 KF471788 KF471787 KF471790 KF471789 KF471792 KF471791 KF471796 KF471793 KF471794 KF471795 KF471798 KF471797 KF471799 KF471800 KF429359 KF429368 KF429360 KF429361 KF429362 KF429367 KF429363 KF429364 KF429365 KF429366 KF429372 KF429371 KF429369 KF429370 KF429374 KF429373 KF429375 KF429376 KF429378 KF429377 KF429379 KF429380 KF429381 KF429382 KF429383 KF429384 KF429389 KF429387 KF429388 KF429385 KF429386 KF429391 KF429390 KF429393 KF429392 KF429395 KF429394 KF429399 KF429396 KF429397 KF429398 KF429401 KF429400 KF429402 KF429403 KF434637 KF434646 KF434638 KF434639 KF434640 KF434645 KF434641 KF434642 KF434643 KF434644 KF434650 KF434649 KF434647 KF434648 KF434652 KF434651 KF434653 KF434654 KF434656 KF434655 KF434657 KF434658 KF434659 KF434660 KF434661 KF434662 KF434667 KF434665 KF434666 KF434663 KF434664 KF434669 KF434668 KF434671 KF434670 KF434673 KF434672 KF434677 KF434674 KF434675 KF434676 KF434679 KF434678 KF434680 KF434681 KF442283 KF442292 KF442284 KF442285 KF442286 KF442291 KF442287 KF442288 KF442289 KF442290 KF442296 KF442295 KF442293 KF442294 KF442298 KF442297 KF442299 KF442300 KF442302 KF442301 KF442303 KF442304 KF442305 KF442306 KF442307 KF442308 KF442313 KF442311 KF442312 KF442309 KF442310 KF442315 KF442314 KF442317 KF442316 KF442319 KF442318 KF442323 KF442320 KF442321 KF442322 KF442325 KF442324 KF442326 KF442327 KF444559 KF444568 KF444560 KF444561 KF444562 KF444567 KF444563 KF444564 KF444565 KF444566 KF444572 KF444571 KF444569 KF444570 KF444574 KF444573 KF444575 KF444576 KF444578 KF444577 KF444579 KF444580 KF444581 KF444582 KF444583 KF444584 KF444589 KF444587 KF444588 KF444585 KF444586 KF444591 KF444590 KF444593 KF444592 KF444595 KF444594 KF444599 KF444596 KF444597 KF444598 KF444601 KF444600 KF444602 KF444603 KF460155 KF460164 KF460156 KF460157 KF460158 KF460163 KF460159 KF460160 KF460161 KF460162 KF460168 KF460167 KF460165 KF460166 KF460170 KF460169 KF460171 KF460172 KF460174 KF460173 KF460175 KF460176 KF460177 KF460178 KF460179 KF460180 KF460185 KF460183 KF460184 KF460181 KF460182 KF460187 KF460186 KF460189 KF460188 KF460191 KF460190 KF460195 KF460192 KF460193 KF460194 KF460197 KF460196 KF460198 KF460199 Outgroup Tinca tinca Gobio gobio Pelecus cultratus Zacco sieboldii Danio dangila Fan Li Fan Li Fan Li Fan Li Fan Li Fan Li Fan Li Fan Li Fan Li CToLa Fan Li Fan Li Fan Li Fan Li CToLa CToLa Maurice Kottelat Akimitsu Hanado Akimitsu Hanado Fan Li Fan Li Fan Li Jae-Seong Lee Masaki Miya Masaki Miya Feng Chen CToLa Takahiro Morosawa Chia-Hao Chang Jie Li Takahiro Morosawa Takahiro Morosawa Fan Li Fan Li Fan Li Fan Li Takahiro Morosawa Akimitsu Hanado Akimitsu Hanado Akimitsu Hanado Fan Li Fan Li CToLa Takahiro Morosawa Khanka Lake, Amur River basin Wuhan City, China Shanghai City, China Wuhan City, China Wuhan City, China Anhui Province, China Anhui Province, China Anhui Province, China Anhui Province, China Anhui Province, China Unrecorded Anhui Province, China Anhui Province, China Anhui Province, China Anhui Province, China Unrecorded Unrecorded Phongsali Province, Laos Gyeonggi-do, Korea Gyeonggi-do, Korea Anhui Province, China Anhui Province, China Anhui Province, China Korea Japan Japan Hubei Province, China Unrecorded Lake Kasumigaura, Japan Guangdong Province, China Guangdong Province, China Lake Kasumigaura, Japan Lake Kasumigaura, Japan Gvangjsih, China Gvangjsih, China Anhui Province, China Anhui Province, China Lake Kasumigaura, Japan Gyeonggi-do, Korea Gyeonggi-do, Korea Gyeonggi-do, Korea Jiangxi Province, China Jiangxi Province, China Okayama, Japan Lake Kasumigaura, Japan 185 (continued on next page) C.-H. Chang et al. / Molecular Phylogenetics and Evolution 81 (2014) 182–194 Acheilognathidae Acheilognathus asmussii Acheilognathus barbatulus (1) Acheilognathus barbatulus (2) Acheilognathus barbatulus (3) ‘‘Acheilognathus’’ barbatulus (4) Acheilognathus barbatulus (5) Acheilognathus barbatulus (6) Acheilognathus barbatulus (7) Acheilognathus barbatulus (8) Acheilognathus barbatulus (9) Acheilognathus barbatus (1) Acheilognathus barbatus (2) Acheilognathus barbatus (3) Acheilognathus barbatus (4) Acheilognathus chankaensis (1) Acheilognathus chankaensis (2) Acheilognathus cyanostigma Acheilognathus deignani Acheilognathus gracilis (1) Acheilognathus gracilis (2) Acheilognathus imberbis (1) Acheilognathus imberbis (2) Acheilognathus imberbis (3) Acheilognathus intermedia Acheilognathus longipinnis (1) Acheilognathus longipinnis (2) Acheilognathus macropterus (1) Acheilognathus macropterus (2) Acheilognathus macropterus (3) Acheilognathus macropterus (4) Acheilognathus macropterus (5) Acheilognathus melanogaster (1) Acheilognathus melanogaster (2) Acheilognathus meridianus (1) Acheilognathus meridianus (2) ‘‘Acheilognathus’’ sp.(1) ‘‘Acheilognathus’’ sp. (2) Acheilognathus rhombeus (1) Acheilognathus rhombeus (2) Acheilognathus rhombeus (3) Acheilognathus rhombeus (4) ‘‘Acheilognathus’’ cf. striatus (1) ‘‘Acheilognathus’’ cf. striatus (2) Acheilognathus tabira tabira Acheilognathus tabira erythropterus Genus Species (individual no.) Sample donor CToLa CToLa Chia-Hao Chang CToLa Jae-Seong Lee CTOLa Jae-Seong Lee Jae-Seong Lee Jae-Seong Lee Ján Koščo Jiří Musil Alexandre Carpentier Martin Reichard Martin Reichard Martin Reichard Martin Reichard Carl Smith Carl Smith CToLa CToLa Kouichi Kawamura Kouichi Kawamura Carl Smith Carl Smith Fan Li Martin Reichard Martin Reichard Akimitsu Hanado Akimitsu Hanado Akimitsu Hanado Akimitsu Hanado Akimitsu Hanado Akimitsu Hanado Chia-Hao Chang Chia-Hao Chang Jie Zhang Xiu-Fa Hou Fan Li Tomoki Oonaka Fan Li Fan Li Fan Li Fan Li Akimitsu Hanado Akimitsu Hanado Carl Smith Carl Smith Fan Li Fan Li Shun-Ping He Tomoki Oonaka Fan Li Akimitsu Hanado Akimitsu Hanado Sample location Shimane, Japan Japan Aquarium shop, Taiwan Unrecorded Korea Korea Korea Jeollabuk-do, Korea Korea Perin Village, near Kosice, Slovakia River Kyjovka (Danube Basin), Czech Cher, France River Oder, Poland River Oder, Poland River Kyjovka (Danube Basin), Czech River Kyjovka (Danube Basin), Czech Khanka Lake, Amur River basin Khanka Lake, Amur River basin Unrecorded Fukuoka, Japan Okayama, Japan Okayama, Japan River Notanebi, Notabeni, Georgia River Notanebi, Notabeni, Georgia Anhui Province, China River Vardar, Greece River Vardar, Greece Gyeonggi-do, Korea Gyeonggi-do, Korea Gyeonggi-do, Korea Gyeonggi-do, Korea Gyeonggi-do, Korea Gyeonggi-do, Korea Taiwan Sichuan Province, China Beijing City, China Guizhou Province, China Shanghai City, China Japan Anhui Province, China Anhui Province, China Anhui Province, China Anhui Province, China Gyeonggi-do, Korea Gyeonggi-do, Korea Lake Kenon, Amur River basin, East Russia Lake Kenon, Amur River basin, East Russia Anhui Province, China Anhui Province, China Wuhan City, China Aquarium shop, Japan Shanghai City, China Gyeonggi-do, Korea Gyeonggi-do, Korea GenBank accession number Cyt b RAG1 RH IRBP EGR1 EGR2B EGR3 KF410743 KF410744 KF410745 KF410746 KF410748 KF410747 KF410811 KF410810 KF410812 KF410751 KF410749 KF410750 KF410752 KF410753 KF410754 KF410755 KF410756 KF410757 KF410758 KF410759 KF410760 KF410761 KF410762 KF410763 KF410764 KF410765 KF410766 KF410772 KF410767 KF410768 KF410769 KF410770 KF410771 KF410781 KF410773 KF410774 KF410775 KF410776 KF410782 KF410780 KF410777 KF410778 KF410779 KF410783 KF410784 KF410785 KF410786 KF410787 KF410788 KF410794 KF410793 KF410789 KF410790 KF410791 KF471801 KF471802 KF471803 KF471804 KF471806 KF471805 KF471869 KF471868 KF471870 KF471809 KF471807 KF471808 KF471810 KF471811 KF471812 KF471813 KF471814 KF471815 KF471816 KF471817 KF471818 KF471819 KF471820 KF471821 KF471822 KF471823 KF471824 KF471830 KF471825 KF471826 KF471827 KF471828 KF471829 KF471839 KF471831 KF471832 KF471833 KF471834 KF471840 KF471838 KF471835 KF471836 KF471837 KF471841 KF471842 KF471843 KF471844 KF471845 KF471846 KF471852 KF471851 KF471847 KF471848 KF471849 KF429404 KF429405 KF429406 KF429407 KF429409 KF429408 KF429472 KF429471 KF429473 KF429412 KF429410 KF429411 KF429413 KF429414 KF429415 KF429416 KF429417 KF429418 KF429419 KF429420 KF429421 KF429422 KF429423 KF429424 KF429425 KF429426 KF429427 KF429433 KF429428 KF429429 KF429430 KF429431 KF429432 KF429442 KF429434 KF429435 KF429436 KF429437 KF429443 KF429441 KF429438 KF429439 KF429440 KF429444 KF429445 KF429446 KF429447 KF429448 KF429449 KF429455 KF429454 KF429450 KF429451 KF429452 KF434682 KF434683 KF434684 KF434685 KF434687 KF434686 KF434750 KF434749 KF434751 KF434690 KF434688 KF434689 KF434691 KF434692 KF434693 KF434694 KF434695 KF434696 KF434697 KF434698 KF434699 KF434700 KF434701 KF434702 KF434703 KF434704 KF434705 KF434711 KF434706 KF434707 KF434708 KF434709 KF434710 KF434720 KF434712 KF434713 KF434714 KF434715 KF434721 KF434719 KF434716 KF434717 KF434718 KF434722 KF434723 KF434724 KF434725 KF434726 KF434727 KF434733 KF434732 KF434728 KF434729 KF434730 KF442328 KF442329 KF442330 KF442331 KF442333 KF442332 KF442396 KF442395 KF442397 KF442336 KF442334 KF442335 KF442337 KF442338 KF442339 KF442340 KF442341 KF442342 KF442343 KF442344 KF442345 KF442346 KF442347 KF442348 KF442349 KF442350 KF442351 KF442357 KF442352 KF442353 KF442354 KF442355 KF442356 KF442366 KF442358 KF442359 KF442360 KF442361 KF442367 KF442365 KF442362 KF442363 KF442364 KF442368 KF442369 KF442370 KF442371 KF442372 KF442373 KF442379 KF442378 KF442374 KF442375 KF442376 KF444604 KF444605 KF444606 KF444607 KF444609 KF444608 KF444672 KF444671 KF444673 KF444612 KF444610 KF444611 KF444613 KF444614 KF444615 KF444616 KF444617 KF444618 KF444619 KF444620 KF444621 KF444622 KF444623 KF444624 KF444625 KF444626 KF444627 KF444633 KF444628 KF444629 KF444630 KF444631 KF444632 KF444642 KF444634 KF444635 KF444636 KF444637 KF444643 KF444641 KF444638 KF444639 KF444640 KF444644 KF444645 KF444646 KF444647 KF444648 KF444649 KF444655 KF444654 KF444650 KF444651 KF444652 KF460200 KF460201 KF460202 KF460203 KF460205 KF460204 KF460268 KF460267 KF460269 KF460208 KF460206 KF460207 KF460209 KF460210 KF460211 KF460212 KF460213 KF460214 KF460215 KF460216 KF460217 KF460218 KF460219 KF460220 KF460221 KF460222 KF460223 KF460229 KF460224 KF460225 KF460226 KF460227 KF460228 KF460238 KF460230 KF460231 KF460232 KF460233 KF460239 KF460237 KF460234 KF460235 KF460236 KF460240 KF460241 KF460242 KF460243 KF460244 KF460245 KF460251 KF460250 KF460246 KF460247 KF460248 C.-H. Chang et al. / Molecular Phylogenetics and Evolution 81 (2014) 182–194 Acheilognathus tabira jordani Acheilognathus tabira namakurae Acheilognathus tonkinensis Acheilognathus typus Acheilognathus yamatsutae (1) Acheilognathus yamatsutae (2) Acheilognathus signifer (1) Acheilognathus signifer (2) Acheilognathus somjinensis Rhodeus amarus (1) Rhodeus amarus (2) Rhodeus amarus (3) Rhodeus amarus (E1) Rhodeus amarus (E2) Rhodeus amarus (W1) Rhodeus amarus (W2) Acheilognathidae gen. sp. (1) Acheilognathidae gen. sp. (2) Rhodeus amurensis Rhodeus atremius atremius Rhodeus atremius suigensis (1) Rhodeus atremius suigensis (2) Rhodeus colchicus (1) Rhodeus colchicus (2) Rhodeus fangi Rhodeus meridionalis (1) Rhodeus meridionalis (2) Rhodeus notatus (1) Rhodeus notatus (2) Rhodeus notatus (3) Rhodeus notatus (4) Rhodeus notatus (5) Rhodeus notatus (6) Rhodeus ocellatus ocellatus (1) Rhodeus ocellatus ocellatus (2) Rhodeus ocellatus ocellatus (3) Rhodeus ocellatus ocellatus (4) Rhodeus ocellatus ocellatus (5) Rhodeus ocellatus kurumeus Rhodeus albomarginatus (1) Rhodeus albomarginatus (2) Rhodeus albomarginatus (3) Rhodeus albomarginatus (4) Rhodeus pseudosericeus (1) Rhodeus pseudosericeus (2) Rhodeus sericeus (1) Rhodeus sericeus (2) Rhodeus shitaiensis (1) Rhodeus shitaiensis (2) Rhodeus sinensis (1) Rhodeus sinensis (2) Rhodeus sinensis (3) Rhodeus sinensis (4) Rhodeus sinensis (5) 186 Table 1 (continued) iLength (bp) 560 369 223 276 215 141 160 Number of variable sites 505 245 143 187 103 68 99 Number of parsimony informative sites 187 Locus 1140 1302 801 807 840 789 843 Tinca tinca Zacco sieboldii Acheilognathus clade (details in Fig. 1b) (details in Fig. 1c) (Acheilognathus) Rhodeus clade (Rhodeus) Unnamed clade Tanakia (Paratanakia) clade III Tanakia clade II (Tanakia) Tanakia (Pseudorhodeus) clade I 100 100 100 100 Gobio gobio Pelecus cultratus Danio dangila 100 100 Acheilognathidae 100 90 99 71 Fig. 1. Phylogenetic relationships of the Acheilognathidae from partitioned maximum likelihood analysis (21 partitions) and partitioned Bayesian analysis (21 partitions) of the combined dataset (7 genes: 6522 bp) (Parts A, B, and C). The topology from Bayesian inference is similar to ML tree; differences exist only on those relationships with weak statistical support. Numbers on branches are ML bootstrap values (those below 70% are not shown) and solid circles on branch nodes indicate statistically robust nodes with posteriori probabilities from partitioned Bayesian analysis P0.95. 0.06 nferred phylogeny, the unique deletion appeared to have evolved independently eight times (see below, Fig. 1b). All MLA and BA supported a monophyletic Acheilognathidae (see Fig. 1 and Fig. S1 in supplementary material). Topologies of mitochondrial (Fig. S1a) and nuclear (Fig. S1b) trees were largely congruent; the only observed discordance was in the position of T. koreensis relative to some other species of Tanakia and Acheilognathus. In the mitochondrial gene tree, T. koreensis was sister to a clade with polychotomus relationships; this clade included Acheilognathus signifier, T. limbata and A. somjinensis. In the nuclear gene tree T. koreensis was sister to T. lanceolata plus A. intermedia clade Cyt b RAG1 RH IRBP2 EGR1 EGR2B EGR3 Table 2 Descriptive statistics of sequences for each gene locus used in this study. C.-H. Chang et al. / Molecular Phylogenetics and Evolution 81 (2014) 182–194 CToL: Cypriniformes Tree of Life initiative. a KF460249 KF460252 KF460253 KF460254 KF460255 KF460258 KF460256 KF460257 KF460261 KF460259 KF460260 KF460262 KF460264 KF460263 KF460266 KF460265 KF460270 KF460271 KF444653 KF444656 KF444657 KF444658 KF444659 KF444662 KF444660 KF444661 KF444665 KF444663 KF444664 KF444666 KF444668 KF444667 KF444670 KF444669 KF444674 KF444675 KF442377 KF442380 KF442381 KF442382 KF442383 KF442386 KF442384 KF442385 KF442389 KF442387 KF442388 KF442390 KF442392 KF442391 KF442394 KF442393 KF442398 KF442399 KF434731 KF434734 KF434735 KF434736 KF434737 KF434740 KF434738 KF434739 KF434743 KF434741 KF434742 KF434744 KF434746 KF434745 KF434748 KF434747 KF434752 KF434753 KF429453 KF429456 KF429457 KF429458 KF429459 KF429462 KF429460 KF429461 KF429465 KF429463 KF429464 KF429466 KF429468 KF429467 KF429470 KF429469 KF429474 KF429475 KF471850 KF471853 KF471854 KF471855 KF471856 KF471859 KF471857 KF471858 KF471862 KF471860 KF471861 KF471863 KF471865 KF471864 KF471867 KF471866 KF471871 KF471872 KF410792 KF410795 KF410796 KF410797 KF410798 KF410801 KF410799 KF410800 KF410804 KF410802 KF410803 KF410805 KF410807 KF410806 KF410809 KF410808 KF410813 KF410814 Anhui Province, China Iran Iran China Korea Taiwan Shanghai City, China Zhejiang Province, China Taiwan Taiwan Taiwan Korea Lake Kasumigaura, Japan Gifu Province, Japan Japan Gifu, Japan Japan Japan Fan Li Carl Smith Carl Smith CToLa Jae-Seong Lee Chia-Hao Chang Fan Li Fan Li Chia-Hao Chang Chia-Hao Chang Chia-Hao Chang CToLa Tomoki Oonaka Tomoki Oonaka Tomoki Oonaka Tomoki Oonaka Masaki Miya Masaki Miya Rhodeus sinensis (6) Rhodeus sp. (1) Rhodeus sp. (2) Rhodeus spinalis Rhodeus suigensis Tanakia himantegus chii (1) Tanakia himantegus chii (2) Tanakia himantegus chii (3) Tanakia himantegus himantegus (1) Tanakia himantegus himantegus (2) Tanakia himantegus himantegus (3) Tanakia koreensis Tanakia lanceolata (1) Tanakia lanceolata (2) Tanakia limbata (1) Tanakia limbata (2) Tanakia tanago (1) Tanakia tanago (2) 188 C.-H. Chang et al. / Molecular Phylogenetics and Evolution 81 (2014) 182–194 100 Acheilognathidae gen. sp. (2) 98 Acheilognathidae gen. sp. (1) 80 Acheilognathus chankaensis (1) 99 Acheilognathus chankaensis (2) 100 Acheilognathus gracilis (1) Acheilognathus gracilis (2) 100 Acheilognathus longipinnis (1) 100 100 Acheilognathus longipinnis (2) Acheilognathus typus Acheilognathus macropterus (5) Acheilognathus barbatulus (8) 92 Acheilognathus macropterus (3) Acheilognathus asmussii 100 Acheilognathus macropterus (2) 100 100 Acheilognathus macropterus (1) Acheilognathus tonkinensis Acheilognathus barbatulus (1) 100 Acheilognathus deignani Acheilognathus macropterus (4) Acheilognathus barbatulus (4) Acheilognathus barbatulus (3) Acheilognathus barbatulus (9) Acheilognathus barbatulus (6) 97 Acheilognathus barbatulus (5) Acheilognathus rhombeus (1) 100 100 Acheilognathus barbatulus (2) Acheilognathus rhombeus (4) Acheilognathus rhombeus (2) 77 100 Acheilognathus rhombeus (3) Acheilognathus barbatulus (7) 100 Acheilognathus melanogaster (2) Acheilognathus melanogaster (1) 95 Acheilognathus barbatus (2) Acheilognathus barbatus (3) 100 Acheilognathus barbatus (1) Acheilognathus yamatsutae (1) 100 outgroups Acheilognathus yamatsutae (2) Acheilognathus cyanostigma Acheilognathus tabira erythropterus 100 95 100 100 Acheilognathus tabira jordani Acheilognathus tabira tabira Acheilognathus tabira namakurae 95 // Acheilognathus imberbis (1) 100 100 100 Acheilognathus imberbis (2) Acheilognathus imberbis (3) 100 Acheilognathus meridianus (1) Acheilognathus meridianus (2) 100 Acheilognathus clade (Acheilognathus) details in Fig. 1c 0.06 Fig. 1 (continued) (Fig. S1). Among the resulting phylogenies for any data set and analysis (Fig. 1), only Rhodeus, among the three currently recognized genera, was recovered as monophyletic (BS P 71%; PP P 0.95) (Fig. 1c). The Acheilognathidae was resolved as having two main reciprocally monophyletic groups, each with high support. The Acheilognathus clade contains most of species of Acheilognathus (Fig. 1b). The second clade includes all other species and was further subdivided into five clades: (1) a clade containing the remaining species of ‘‘Acheilognathus’’, except for A. intermedia, A. signifier, and A. somjinensis, (2–4) three separate clades of species that are currently classified as Tanakia (clades I–III), and (5) the Rhodeus clade (Fig. 1c). Tanakia clade I included only T. tanago. Tanakia Clade II included only species from Japan and Korea, except for T. tanago, and three species formerly of Acheilognathus (A. intermedia, A. signifier, and A. somjinensis). Clade III included Tanakia himantegus from Taiwan and China. Within Acheilognathus sensu stricto (Acheilognathus clade), relationships among the various main lineages were not well supported and internal branches are short relative to terminal branches. Such a pattern could reflect a rapid radiation occurring during an early diversification of Acheilognathus (Fig. 1b) or conserved anagenesis for the genes examined. Within the second major acheilognathid group, the sister-group relationships among clades and within clades were generally well resolved and have high nodal support (Fig. 1a and c). The Tanakia clade I (or T. tanago) was the basal-most lineage, sister to the other four clades., The Rhodeus clade, while the more speciose group in this analysis, was deeply nested within acheilognathids (Fig. 1a and c). Among the multiple individuals examined of A. tabira, R. notatus, R. atremius, R. ocellatus, and T. himantegus, the phylogenetic results of gene trees support their validity. However, intraspecific sequence divergence within these species was high, possibly indicating additional species diversity (e.g., cryptic species or 189 C.-H. Chang et al. / Molecular Phylogenetics and Evolution 81 (2014) 182–194 outgroups Acheilognathus clade details in Fig. 1b 100 100 100 Tanakia tanago (2) Tanakia tanago (1) Tanakia clade I (Pseudorhodeus) Acheilognathus intermedia Tanakia lanceolata (1) 91 Tanakia lanceolata (2) Tanakia koreensis Acheilognathus signifer (2) 100 Acheilognathus signifer (1) Acheilognathus somjinensis 100 Tanakia limbata (2) 92 Tanakia limbata (1) 100 Tanakia himantegus himantegus (1) Tanakia himantegus himantegus (3) 100 100 Tanakia himantegus himantegus (2) Tanakia himantegus chii (2) Tanakia himantegus chii (1) Tanakia himantegus chii (3) 100 100 100 99 100 90 Tanakia clade II (Tanakia) Tanakia clade III (Paratanakia) “Acheilognathus” barbatus (4) 100 “Acheilognathus” cf. striatus (1) “Acheilognathus” cf. striatus (2) “Acheilognathus” sp. (1) 100 100 “Acheilognathus” sp. (2) Unnamed clade Rhodeus spinalis Rhodeus ocellatus ocellatus (2) 96 Rhodeus ocellatus ocellatus (4) 100 Rhodeus ocellatus kurumeus Rhodeus ocellatus ocellatus (1) Rhodeus ocellatus ocellatus (5) 77 Rhodeus ocellatus ocellatus (3) 100 Rhodeus shitaiensis (1) Rhodeus shitaiensis (2) 71 100 Rhodeus albomarginatus (1) Rhodeus albomarginatus (3) 98 Rhodeus albomarginatus (4) 76 Rhodeus albomarginatus (2) 100 Rhodeus pseudosericeus (1) Rhodeus pseudosericeus (2) 100 100 Rhodeus sericeus (1) Rhodeus sericeus (2) 99 Rhodeus sp. (1) 100 Rhodeus sp. (2) 100 86 99 Rhodeus amarus (1) Rhodeus amarus (2) Rhodeus amarus (W2) Rhodeus meridionalis (2) Rhodeus meridionalis (1) 91 100 100 Rhodeus colchicus (2) Rhodeus colchicus (1) 100 European bitterlings Rhodeus amarus (E2) Rhodeus amarus (E1) Rhodeus amarus (W1) Rhodeus amarus (3) Rhodeus sinensis (1) Rhodeus sinensis (3) 100 Rhodeus sinensis (6) Rhodeus sinensis (2) Rhodeus amurensis Rhodeus sinensis (5) 93 83 Rhodeus sinensis (4) Rhodeus fangi 74 R. atremius atremius 100 94 99 100 R. atremius suigensis (1) 100 R. atremius suigensis (2) Rhodeus suigensis Rhodeus notatus (3) Rhodeus notatus (2) Rhodeus notatus (5) Rhodeus notatus (4) Rhodeus notatus (1) 81 Rhodeus notatus (6) Rhodeus smithii complex 100 0.06 96 Rhodeus clade (Rhodeus) Fig. 1 (continued) species complexes). Seven other species, representing about 18% of the species sampled in the study, where more than one specimen was examined (usually from different locations) did not group as single gene-tree lineages by species. Gene lineages of Acheilognathus chankaensis, A. rhombeus, R. sinensis, and R. amarus were paraphyletic with respect to Acheilognathid gen. sp., A. barbatulus, 190 C.-H. Chang et al. / Molecular Phylogenetics and Evolution 81 (2014) 182–194 R. amurensis, and R. meridionalis plus R. colchicus, respectively. Gene lineages of A. macropterus, A. barbatulus, and A. barbatus were resolved as polyphyletic groupings. Finally, ‘‘Acheilognathus’’ sp. and Rhodeus sp. are herein regarded as putatively undescribed species as they are morphologically distinct from other known species and both display unique phylogenetic positions. 4. Discussion 4.1. Phylogeny of the Acheilognathidae Reciprocal monophyly of the three traditionally recognized acheilognathid genera has never been examined with large taxon and character sampling. Previous analyses had limitations in taxon and character sampling (Chen and Mayden, 2009; Fujiwara et al., 2009; Tang et al., 2011). The phylogeny of the Acheilognathidae based on mitochondrial 12S rRNA sequences, morphological, and karyological characters in earlier studies resolved Tanakia (inferred as monophyletic) as the sister group to Acheilognathus and Rhodeus (Arai, 1988; Arai and Kato, 2003). This result led Arai and Kato (2003) to hypothesize that Tanakia was the ‘‘ancestral’’ group, and both Acheilognathus and Rhodeus evolved from Tanakia. However, this hypothesis is inconsistent with results from two recent molecular studies based on either mitochondrial cytochrome b sequence data (Kawamura et al., 2014) or the combined data set with 12S rRNA sequences (Cheng et al., 2014). In these studies two reciprocal clades are resolved in the Acheilognathidae (Acheilognathus and Tanakia-Rhodeus). Species relationships in the current study with a substantial increase in both taxa and characters, received high support. The resulting relationships are not consistent with the early hypothesis but are consistent with hypotheses of the two later studies (Cheng et al., 2014; Kawamura et al., 2014). The present analysis does not support the monophyly of Tanakia or Acheilognathus. While Acheilognathus was shown to be monophyletic in all previous molecular phylogenetic analyses (Cheng et al., 2014; Fujiwara et al., 2009; Kawamura et al., 2014; Okazaki et al., 2001; Yang, Q. et al., 2011; Zhu and Liu, 2006) the genus is not recovered as monophyletic. The Acheilognathus clade (or Acheilognathus sensu stricto) forms the sister group to remaining acheilognathid species (Fig. 1a). The well-corroborated and consistent phylogenies resolved herein, based on two independent data sets, and the strong nodal support suggests that taxonomic revisions are warranted for the Acheilognathidae. Furthermore, given these relationships, diagnostic characters identified by Arai and Akai (1988) will have to be reexamined. Moreover, as the diploid chromosome numbers of the out-group taxa Tinca tinca and Zacco are 48 (Okazaki et al., 2001; Yu et al., 1987) this number used to group Tanakia (2n = 48) and Rhodeus (2n = 48 or 46) is not diagnostic. In comparison with previous studies (Cheng et al., 2014; Kawamura et al., 2014; Okazaki et al., 2001), this investigation is the first to provide evidence, with high statistical support, for the monophyly of Rhodeus, and the first to reveal the paraphyly of Tanakia (Fig. 1c). Given that the evolutionary history of a species is directly tied to its traits, previous studies drawing from earlier phylogenetic hypotheses as a framework for behavioral and biological evolution within acheilognathids should be reevaluated. Moreover, the present phylogenetic hypothesis also highlights several immediate taxonomic implications in need of attention, as well as highlighting opportunities to further test hypotheses regarding the evolution of bitterling species and their biology. 4.2. Taxonomic implications and revised classification The Acheilognathidae includes six lineages, each being referred to separate genera. Only species of Rhodeus form a monophyletic group, and species of ‘‘Acheilognathus’’ and ‘‘Tanakia’’ must be reallocated to newly proposed genera. The Acheilognathus clade, as recognized herein, retains its generic allocation given that it includes the type species, A. melanogaster (Kottelat, 2013). Arai and Akai (1988) indicate that this genus can be diagnosed morphologically from other acheilognathids, except for species of ‘‘Acheilognathus.’’ Species of Acheilognathus are diagnosed as having two transverse rows of white spots, equal in size, on the dorsal fin rays, with the upper row being narrower than the lower, a transverse row of black spots on the dorsal fin membranes, and serrations on pharyngeal teeth (versus teeth with vestigial serrations or less developed). Among the lineages of ‘‘Tanakia,’’ the genus name Tanakia applies to Tanakia Clade II, as it includes the type species, Tanakia limbata Jordan and Seale, 1906. Species of Tanakia Clade I and Tanakia Clade III can be diagnosed from species of Tanakia spp. of Clade II using the combination of morphological, genetic, and karyological characters. The details of the six genera including one retained, two revised, two new, and one undescribed genus are as follows: Genus Rhodeus Type species: Rhodeus amarus (Bloch, 1782) Diagnosis: Diagnosed by Arai and Akai (1988) and retained herein. Species possessing a well-developed wing-like yolk sac projection, weakly developed and minute tubercles, and a diploid chromosome number 46. Comments. While Rhodeus smithii was not included in this analysis, the other four species of this group form a monophyletic group, and we propose that the diagnostic characters of the Rhodeus smithii complex are synapomorphic. Genus Acheilognathus Synonyms: Acanthorhodeus, Paracheilognathus, and Rhodeops (Eschmeyer, 2014) Type species: Acheilognathus melanogaster Bleeker, 1860 Diagnosis: Acheilognathus corresponds to our Acheilognathus clade (Fig. 1b). The diagnosis established by Arai and Akai (1988) is insufficient in distinguishing species of bitterlings in this clade from those in the unnamed clade. However, genetic data reveal that some molecular characters, in combination with characters provided by Arai and Akai (1988), can serve to diagnose this lineage. Additional morphological examination of species in this lineage is warranted to more fully develop a diagnosis of the lineage. Comments: Whether the reduced diploid chromosome number of Acheilognathus (2n = 44 or 42) is shared with ‘‘Acheilognathus’’ from the unnamed clade is unknown and requires further karyologidal investigations of species of the latter lineage. Genus Tanakia Type species: Tanakia limbata (Temminck and Schlegel, 1846) Diagnosis: Includes only species of Tanakia clade II (Fig. 1c). Diagnosis for the newly revised Tanakia, is derived from a modified diagnosis by Arai and Akai (1988). Lateral line complete, diploid chromosome number 48, and absence of 8 M + 20SM + 18ST + 2A chromosomal constitution (Ojima et al., 1973; Sola et al., 2003; Ueda et al., 2001, 2006). Comments: The three recently revised species of ‘‘Acheilognathus’’: A. intermedia, A, signifier, and A. somjinensis (Yang, Q. et al., 2011) should be placed in Tanakia rather than Acheilognathus. Our findings confirm’s the taxonomic proposition by Arai and Akai (1988) to consider these three species as Tanakia. Thus, these species are transferred to Tanakia. Genus Pseudorhodeus gen. nov. Chang, Chen, and Mayden Type species: Rhodeus tanago Tanaka, 1909 Diagnosis: This new name applies to Tanakia clade I (Fig. 1c). Based on the description of Pseudorhodeus tanago (Tanaka, 1909) and karyological studies, this monotypic genus can be diagnosed from other ‘‘Tanakia’’ (Arai and Akai, 1988) in having an incomplete lateral line, a diploid chromosome number of 48, and a chromosomal constitution of 8M + 20SM + 20ST (Ojima et al., 1973). C.-H. Chang et al. / Molecular Phylogenetics and Evolution 81 (2014) 182–194 Etymology: Pseudorhodeus. A noun in apposition. Latin pseudo, meaning false, and similarity to species of Rhodeus. Tanaka (1909) originally identified Pseudorhodeus tanago as a species of Rhodeus. The species was later transferred to Tanakia by Okada (1961). Most recently, it has been recognized in Tanakia (Fricke, 2014). Given that P. tanago was once viewed as Rhodeus tanago, and P. tanago and species of Rhodeus both possess an incomplete lateral line, Pseudorhodeus refers to the phenetic similarity of P. tanago being similar but not closely related to Rhodeus. Genus Paratanakia gen. nov. Chang, Chen, and Mayden Type species: Achilognathus himantegus Günther, 1868 Diagnosis: This new name applies to Tanakia Clade III (Fig. 1c). Lineage distinguished from other species of ‘‘Tanakia’’ (including Pseudorhodeus tanago) using morphological and chromosomal characters identified in Arai and Akai (1988), Chen et al. (1998) and Ueda et al. (1997, 2006). Lateral line complete, diploid chromosome number 48, and chromosomal constitution 8M + 20SM + 18ST + 2A. Molecular bp characters also serve as synapomorphies for this genus at this time; nucleotide at the position 707 of the RAG1 is A for this genus but is T for other acheilognathids, and bp composition at position 66 of the EGR1 is C for this genus and A for other acheilognathids. Etymology: Paratanakia. A noun in apposition. Para, from Greek word parilis meaning beside, near or by, and the genus name Tanakia, referring to the similarity to species of Tanakia "himantegós" is composed from himás and himántos. It’s a noun and the feminine form is equal. Paratanakia himantegus was placed in Paracheilognathus by Günther (1868), and then reclassified as Tanakia by Arai and Akai (1988). Currently, it is considered a member of Tanakia (Eschmeyer, 2014). Since Paracheilognathus is synonymous to Acheilognathus (Arai and Akai, 1988), Paracheilognathus is unavailable. Paratanakia derives from Paratanakia himantegus being morphologically more similar to the herein described monophyletic Tanakia (Tanakia Clade II) than to Pseudorhodeus tanago by having a complete lateral line. The unnamed clade in (Fig. 1c) currently contains taxa inhabiting the Yangtze River basin. Species of ‘‘Acheilognathus’’ in the unnamed lineage (identified as unnamed clade, Fig. 1c) can only be diagnosed from Acheilognathus at this time using genetic characters. As the taxonomic sampling in our study included only 50% of the recognized species of Acheilognathus we refrain from naming this lineage at this time. 4.3. Species diversity and cryptic species Taxonomy plays a crucial role in modern evolutionary biology (Padial et al., 2009; Smith et al., 2013; Winsor, 2009) with its primary focus being descriptions of new taxa, resolution of names that are valid, available, synonyms, and/or homonyms. Clarity in the classification of life with proper names applied to taxa is fundamental to studies of life forms (Imamura and Nagao, 2011; Iwatsuki and Heemstra, 2010; Nielsen, 2011). Acheilognathidae currently includes about 74 valid species, yet around 120 species names are available and have been used (Eschmeyer, 2014). Species of this family continue to be discovered and described. Cryptic species are likely to exist within identified complexes and, based on gene trees, not all populations of currently recognized species are most closely related to one another. For example, gene trees of A. rhombeus, T. koreensis and R. spinalis are paraphyletic sensu Kawamura et al. (2014) and may indicate that additional species diversity exists within such groups. The phylogenetic framework established herein represents an initial step in the systematic revision of Acheilognathidae. Among the species in the study from multiple geographic samples, gene trees suggest that seven do not group as would be expected of descendants from a most recent common ancestor. 191 This indicates that it is possible, given gene tree resolutions, multiple species exist within these seven species. These species include A. chankaensis, A. rhombeus, R. sinensis, R. amarus, A. macropterus, A. barbatulus, and A. barbatus (Fig. 1b and c). One of the most interesting instances is A. macropterus. Among the five individuals examined (from four different localities in China and Japan) they have resolved, with strong nodal support, into four independent lineages nested within the Acheilognathus (Fig. 1b). This may indicate that other species are currently masquerading under the name A. macropterus. Acheilognathus asmussii (Lake Khanka,Amur River basin) is resolved as closely related to A. macropterus (3) from Lake Kasumigaura, Japan. This lineage is sister to another lineage including A. macropterus (1) and A. macropterus (2). Acheilognathus macropterus (4) from Guangdong Province in southern China is sister to A. deignani from the Mekong River in northern Laos (Fig. 1b). Finally, the phylogenetic position of A. macropterus (5), also collected from Guangdong Province in southern China, remains unresolved. Thus, the widely distributed species A. macropterus and A. asmussii may consist of at least four separate, geographically independent lineages, and possibly new species. Species delimitation requires additional sampling in other geographic areas and an examination of diagnostic traits. Acheilognathus chankaensis groups with acheilognathidid gen. sp. with high statistical support, possibly indicating that the latter specimen is A. chankaensis or a close relative. Currently available information indicates that the four specimens of A. barbatus, examined are nearly identical morphologically. However, these specimens group into two separate and distantly related lineages within the Acheilognathus clade (Fig. 1b) and unnamed clade (Fig. 1c). Cryptic species within A. barbatus may explain these divergent lineages under one name. Gene trees of A. rhombeus resolve the species as an artificial grouping. Acheilognathus rhombeus (1) is nested within A. barbatulus (Fig. 1b), a result also revealed in analysis by Kawamura et al. (2014). Moreover, gene trees of A. barbatulus (7) resolve this specimen as an independent lineage relative to other A. barbatulus genetic lineages and A. rhombeus (Fig. 1b). Comparisons of nuclear and mitochondrial gene trees reveal no discordance in these relationships (Fig. S1). Thus, the possibility of mitochondrial introgression or inter-species hybridization is unlikely and additional species, yet to be discovered may exist within A. rhombeus and A. barbatulus. Within Rhodeus, R. sinensis and R. amurensis are closely related. Rhodeus sinensis (1) is the most genetically divergent specimen found among samples (Fig. 1c). Rhodeus amurensis (Amur River drainage) is the sister-taxon to R. sinensis (4) and (5) from Korea. Notably, gene trees for specimens of R. sinensis (1, 3, 6), all sampled from the Yangtze River basin, revealed a paraphyletic grouping with respect to R. amurensis and the Korean sample of R. sinensis. A detailed morphological examination and taxonomic revision for these two species of Rhodeus is warranted. The most recently described species of Rhodeus, R. albomarginatus (Li and Arai, 2014), is morphologically most similar to R. ocellatus, but gene trees resolve the species as sister to R. shitaiensis, an example illustrating that phenetic similarity does not necessarily reflect a genealogical relationship (Fig. 1c). Incongruence between the consistently resolved nuclear and mitochondrial gene trees and morphological similarity of samples of a particular species, may explain the above examples wherein gene trees do not support some specimens from the same or different populations of a species as sharing a common lineage. The phylogenetic results presented here, based on multiple nuclear and mitochondrial gene sequences, indicate that some genetic groups of the individual species A. chankaensis, A. macropterus, A. barbatulus, A. barbatus, and R. sinensis (Fig. 1b and c), should 192 C.-H. Chang et al. / Molecular Phylogenetics and Evolution 81 (2014) 182–194 be carefully reexamined morphologically to investigate the existence of cryptic species and to better resolve the taxonomy of these species. Moreover, our phylogenetic results revealed that some taxa (‘‘Acheilognathus’’ sp. and Rhodeus sp.) that appear as independent lineages are likely new species. Acheilognathus tabira (endemic to Japan) is represented by five subspecies based on differentiation in morphological characters, coloration, and their geographic distributions. Discrimination among these subspecies was also observed in genetic data (Arai et al., 2007; Kitamura et al., 2012). In our analysis of samples of four of the five subspecies (A. t. tohokuensis not sampled) gene trees corroborated their sharing of a well-supported common lineage. The resulting phylogeny presented herein is identical to that of Kitamura et al. (2012) based on Cyt b, Glyt, Myh6, RAG1, and Ryr3 data. It is interesting that there is a substantial degree of genetic divergence among these subspecies derived from these two molecular studies. This finding implies a high nucleotide substitution rate for the gene markers used and, with further investigation, the diversification of possibly multiple new species (via taxonomic elevation from subspecies) currently within A. tabira. 4.4. Origin of European bitterling species In the discovery and description of Rhodeus colchicus (a morphologically distinct European species) from the western Caucasus, Bogutskaya and Komlev (2001) evoked the assumption of putative reciprocal faunal exchange of bitterling species between Europe and East Asia. This conclusion was inferred from the fact that European R. amarus and East Asian R. sericeus have high-levels of morphological similarity (Bogutskaya and Komlev, 2001). This similarity led the authors to suggest two alternative colonization scenarios for Eurasian bitterlings; (1) speciation of R. colchicus and R. sericeus in East Asia and a subsequent parallel colonization of Europe by both lineages with subsequent extinction of the ancestral lineage of R. colchicus and (2) colonization of Europe by a common ancestor of R. colchicus and R. sericeus with subsequent split into R. colchicus and R. sericeus and a subsequent recolonization of eastern Asia by R. sericeus. Bohlen et al. (2006) examined these hypotheses. They proposed species diversification and colonization of the Euro-Mediterranean area (R. amarus, R. colchicus, and R. meridionalis) occurred following their divergence of their most recent common ancestor (MRCA) from R. sericeus (East Asia), and a single dispersal event of the MRCA from East Asia to Europe. Results herein, incorporating R. amarus from the western and eastern lineages, sensu Bohlen et al. (2006), R. colchicus, R. meridionalis, and an undescribed Iranian species of Rhodeus (Rhodeus sp.) supports the hypothesis of Bohlen et al. (2006). The Iranian Rhodeus (not in Bohlen et al., 2006) is the sister-group to the monophyletic Euro-Mediterranean bitterling species, suggesting a possible broader and more continuous distribution of the MRCA of the Euro-Mediterranean and Iranian Rhodeus, extending from Europe through western Eurasia. The Korean species Rhodeus pseudosericeus, another species morphologically similar to R. sericeus, is the sister of the lineage including R. sericeus, Rhodeus from Iran, and the Euro-Mediterranean bitterlings. These relationships further corroborate the hypothesis that Euro-Mediterranean bitterlings are more recently evolved from Rhodeus and refute the hypothesis that R. sericeus (East Asia) is closely related to the morphologically similar R. amarus (European), providing another example of similarities in a simple context not reflecting genealogical relationships. The phylogenetic relationships among the Euro-Mediterranean species are not well resolved, possibly the result of relatively rapid speciation, incomplete lineage sorting and/or the gene flow among populations/species (Bryja et al., 2010). We have no conclusive evidence to identify which, if any, of these possible processes is the most. 5. Conclusions Species of Acheilognathidae have emerged as a valuable model group in behavioral and evolutionary studies over the preceding two decades. We present a strongly supported phylogeny of this diversity in this family, including multiple species. This analysis represents the most comprehensive phylogenetic investigation of the family to date. Relationships are derived from 117 individuals from at least 41 species (some cryptic or undescribed species likely exist), exploiting nuclear and mitochondrial gene sequences, and evaluated using model-based analytical methods of evolutionary inference. Sister-group relationships in all trees generated are essentially identical and serve to reject the current classification for the Acheilognathidae, and provides evidence for several areas for further taxonomic investigation. This study also reveals additional species currently listed within a single species, which warrants additional investigation and possible reevaluation of the classification by Arai and Akai (1988). Rhodeus is monophyletic and its status is maintained. However, Acheilognathus and Tanakia are not monophyletic but represent multiple lineages in paraphyly. For phylogenetic and taxonomic consistency it is necessary to describe two new monotypic genera for two of the lineages— Pseudorhodeus and Paratanakia. An unnamed clade is recognized and discovery of this clade highlights the need for more species sampling and additional tests of its monophyly. Further taxonomic studies are recommended to aid in the identification of morphological or other traits useful in diagnosing lineages. Finally, the phylogenetic results strongly suggest that species diversity within the Acheilognathidae has been underestimated and warrants comprehensive revision. Acknowledgments The authors thank Dr. Kouichi Kawamura, Dr. Jiang Zhou, Dr. Shen-Horn Yen, Mr. Xiu-Fa Hou, Mr. Tomoki Oonaka, Mr. Akimitsu Hanado, Mr. Masumi Ozaki, Dr. Jano Kosco, Dr. Meta Povz, and Dr. Maurice Kottelat for providing bitterling specimens. This research was supported by USA NSF Grant (DEB-1021840 to RLM) and by Taiwanese MOST Grants (MOST 99-2611-M-002-001-MY2 and MOST 101-2611-M-002-016-MY3 to W.J.C.). MM acknowledges the Japan Science for the Promotion of Science (Grant No. 17207007). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2014. 08.026. References Agbali, M., Reichard, M., Bryjová, A., Bryja, J., Smith, C., 2011. Mate choice for nonadditive genetic benefits correlate with MHC dissimilarity in the rose bitterling (Rhodeus ocellatus). Evolution 64, 1683–1696. Arai, R., 1988. Fish Systematics and Cladistics. Asakura Shoten, Tokyo, Japan. 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Phylogenetic relationships of Acheilognathidae (Cypriniformes: Cyprinoidea) as revealed from evidence of both nuclear and mitochondrial gene sequence variation: evidence for necessary taxonomic revision in the family and the identification of cryptic species

Phylogenetic relationships of Acheilognathidae (Cypriniformes: Cyprinoidea) as revealed from evidence of both nuclear and mitochondrial gene sequence variation: evidence for necessary taxonomic revision in the family and the identification of cryptic species

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