Zoological Studies 56: 40 (2017) doi:10.6620/ZS.2017.56-40 Open Access Molecular Phylogeny of the Opsariichthys Group (Teleostei: Cypriniformes) Based On Complete Mitochondrial Genomes Shih-Pin Huang1, Feng-Yu Wang2, and Tzi-Yuan Wang1,* 1Biodiversity Research Center, Academia Sinica, Nankang, Taipei, Taiwan. E-mail: [email protected] 2Taiwan Ocean Research Institute, National Applied Research Laboratories, Kaohsiung, Taiwan. E-mail: [email protected] (Received 13 December 2016; Accepted 6 December 2017; Published 21 December 2017; Communicated by Benny K.K. Chan) Shih-Pin Huang, Feng-Yu Wang, and Tzi-Yuan Wang (2017) The complete mitochondrial genomes of 76 species from 43 genera under Cyprinidae sensu lato were collected to reassess the molecular phylogeny of Opsariichthyinae sensu Liao et al. 2011. The mitogenomes of three species, Candidia barbata, Opsariichthys evolans, and Opsariichthys pachycephalus, were newly sequenced. Phylogenetic trees were reconstructed based on 13 concatenated multiple protein-coding genes with two ribosomal RNA genes. The concatenated dataset provided a new perspective on systematics and relationships. Tree topologies show that a monophyletic group containing Parazacco, Candidia, Nipponocypris, Zacco, and Opsariichthys should belong to the Opsariichthys group. In addition, the present results also strongly support that Candidia and Nipponocypris should be regarded as distinct genera within the Opsariichthys group. Aphyocypris, Yaoshanicus, Nicholsicypris, and Pararasbora form a monophyletic group within Xenocyprididae, distinct from the Opsariichthys group. Furthermore, Hemigrammocypris is nested with four species of Metzia, a genus of ex-Cultrinae in Xenocyprididae. In addition, two major types of distinct stripes - longitudinal and vertical - were observed among species of the Opsariichthys group and were highly correlated with molecular phylogenetic relationships. Such types of vertical and longitudinal stripes presented in the Opsariichthys group might have originated in an ancestor species, after which distinct vertical stripes might have been lost among these cyprinids but retained in the Opsariichthys group. Key words: Molecular phylogeny, Mitochondrial genome, Freshwater fish, Cyprinidae, Opsariichthyinae. BACKGROUND et al. 2010 2013; Stout et al. 2016). Some were subsequently renamed in an attempt to reflect their Cyprinidae sensu lato (originally called family new taxonomic placements (Liao et al. 2011c). Cyprinidae) is the largest family of teleosts in the Among these were Opsariichthyinae, a group of world, containing 3090 valid species (Eschmeyer et minnows in Cyprinidae sensu lato occurring widely al. 2017). Several recent studies have been carried in East Asia that contains the genera Aphyocypris, out to assess the phylogeny and systematics of this Candidia, Hemigrammocypris, Nipponocypris, group and/or the rest of Cypriniformes based on Opsariichthys, Parachela, Parazacco, Yaoshanicus molecular evidence (Tang et al. 2010 2013; Stout and Zacco (Liao et al. 2011c) (Fig. 1). Most et al. 2016). of these genera were previously assigned to The taxonomic placement of several major Danioninae. However, their taxonomic assignments subfamilies belonging in Cyprinidae sensu lato has have been continuously changed over recent undergone a large change. Several subfamilies, years (Mayden et al. 2009; Tang et al. 2010 2013; especially Danioninae and Cultrinae, were Liao et al. 2011a; Stout et al. 2016). Among these reported to be paraphyletic or polyphyletic (Tang common minnows, Yaoshanicus, Nicholsicypris, *Correspondence: E-mail: [email protected] © 2017 Academia Sinica, Taiwan 1 Zoological Studies 56: 40 (2017) page 2 of 13 and Pararasbora were considered junior synonyms The subfamilies Cultrinae, Xenocyprinae, of Aphyocypris (Liao et al. 2011b), although Huynh Squaliobarbinae, Alburninae, and Opsariichthyinae and Chen (2013) still considered Nicholsicypris were formerly in Cyprinidae sensu lato but to be a valid genus. Fang et al. (2009) defined considered a monophyletic group by Stout ex-Rasborinae, which included Candidia, et al. (2016) and therefore reassigned to the Nipponocypris, Opsariichthys, Parazacco, and family Xenocyprididae. However, the taxonomic Zacco. Subsequently, Liao et al. (2011c) renamed placements and relatedness of subfamilies ex-Rasborinae as Opsariichthyinae to include under Xenocyprididae remained ambiguous Aphyocypris, Candidia, Hemigrammocypris, because there was an insufficient number of taxa Nipponocypris, Opsariichthys, Parachela, and none of the taxonomic assignments were Parazacco, Yaoshanicus and Zacco (Fig. 1). included. For example, two species from former Recently, these genera along with several other subfamily Cultrinae (Chanodichthys erythropterus subfamilies (Cultrinae, Hypophthalmichthyinae, and Parabramis pekinensis) were nested with Squaliobarbinae, Xenocyprindinae, parts of Hypophthalmichthys molitrix, Ctenopharyngodon Alburninae, and Danioninae) were reclassified into idella, Elopichthys bambusa, and Squaliobarbus Oxygastrinae (Tang et al. 2013). Subsequently, curriculus, which were part of the former Kottelat (2013) proposed that Oxygastrinae was subfamilies Leuciscinae and Squaliobarbinae not available, and instead Hypophthalmichthyinae (Stout et al. 2016). This classification was and Xenocypridinae were the earliest available inconsistent with another study (Tang et al. names. Therefore, the genera and subfamilies 2013). In addition, the taxonomic status of were assigned to Xenocyprididae (Stout et Hemigrammocypris remained controversial. Liao al. 2016). These taxonomic placements and et al. (2011c) proposed that it should be assigned assignments will be used and discussed in this to Opsariichthyinae. However, Tang et al. (2013) study. and Stout et al. (2016) proposed that it was Fig. 1. Systematic positions of the Opsariichthys group and related genera from this and other studies. © 2017 Academia Sinica, Taiwan Zoological Studies 56: 40 (2017) page 3 of 13 closest to Metzia, a genus of ex-Cultrinae (Fig. molecular evidence (Liao et al. 2011c; Tang et 1). The systematic positions of these genera are al. 2013; Huynh and Chen 2013), but several summarized in figure 1. contrary findings have been reported in recent The complete mitochondrial genome could years. For example, a phylogenetic tree of twelve be regarded as an alternative molecular marker Opsariichthyines species was reconstructed based for processing at a higher level of phylogenetic on mitochondrial Cyt b and COI and nuclear analysis (Saitoh et al. 2006; Mayden et al. 2009; RAG1 and Rh1 (Tang et al. 2010). Although they Huang et al. 2016). In order to verify the systematic all belonged to a monophyletic group, Candidia, positions of Opsariichthyinae, a reassessment of Nipponocypris, and Parazacco were nested molecular phylogenetic analysis was performed. together and therefore formed an indeterminate We expected that mitogenomes would be useful lineage. Yin et al. (2015) revealed the relationship for resolving these ambiguous relationships in of Candidia, Nipponocypris, Opsariichthys, Opsariichthyinae and Xenocyprididae. Fortunately, Parazacco, and Zacco based on complete complete mitochondrial genomes of many species mitochondrial genomes. Remarkably, Candidia under Opsariichthyinae and Xenocyprididae have was only nested within Nipponocypris, which is been sequenced (Jang-Liaw et al. 2013a b; Chang consistent with Tang et al. (2010). In contrast, two et al. 2016; Chen et al. 2016a b). However, none of Japanese species of Nipponocypris were assigned these studies analyzed the complete mitochondrial to Candidia (Nakabo 2013). Thus, this study also genome. Recently, mitochondrial DNA has been used further molecular research to reassess these frequently used for resolving the taxonomic and inconsistencies and the validity of Nipponocypris. phylogenetic problems in East Asian cyprinids (Tsao et al. 2016; Huang et al. 2017). In order to verify taxonomic placement and assignment and MATERIALS AND METHODS attempt to provide a new molecular perspective on different genetic marks, this study analyzed Whole mitogenome collection more species and genera from Opsariichthyinae and family Xenocyprididae based on complete Seventy-six species from 43 genera of mitochondrial genomes. Opsariichthyinae, Xenocyprididae, and related Among East Asian common minnows, families were used to reassess their molecular there are three major stripe patterns that can be phylogenetic analysis (Table 1). Among the East roughly grouped. One is an indistinct stripe or Asian common minnows, three species, Candidia band on the side of the body and the remaining barbata, Opsariichthys evolans, and Opsariichthys two are a distinct vertical or longitudinal stripe pachycephalus, were sequenced for the first time. or band on the side of the body. These minnows Mitogenomes for the remaining nine species occur in five genera in East Asia: Candidia, (Aphyocypris chinensis, Hemigrammocypris Nipponocypris, Opsariichthys, Parazacco, and rasborella, Nicholsicypris normalis, Nipponocypris Zacco. Among these, Candidia is endemic to temminckii, Opsariichthys uncirostris, Pararasbora Taiwan, Nipponocypris is restricted to Japan moltrechti, Parazacco spilurus, Yaoshanicus and Korea, Opsariichthys is widely distributed arcus, and Zacco platypus), each of which is the in East Asia, Parazacco is restricted to southern type species of its genus, were retrieved from China, and Zacco is distributed in northern China, GenBank