Hydrobiologia (2006) 553:255–266 Springer 2006 DOI 10.1007/s10750-005-1301-3

Primay Research Paper The phylogenetic relationships of the (Teleostei: ) inferred from mitochondrial cytochrome b gene sequences

Jinquan Yang1,2, Shunping He1,Jo¨ rg Freyhof3, Kai Witte4 & Huanzhang Liu1,* 1Institute of Hydrobiology, The Chinese Academy of Sciences, 430072, Wuhan, China 2Graduate School of Chinese Academy of Sciences, 100039, Beijing, China 3Department of Biology and Ecology of , Leipniz-Institute of Freshwater Ecology and Inlands Fisheries, Mu¨ggelseedamm 310, 12587, Berlin, 4Max Planck Institute for Developmental Biology, Spemannstr. 35/3, 72076, Tu¨bingen, Germany (*Author for correspondence: Tel.: +86-27-68780776; Fax: +86-27-68780123; E-mail: [email protected])

Received 6 April 2005; accepted 4 June 2005

Key words: phylogeny, monophyly, cytochrome b, diversity, Cyprinidae, Gobioninae

Abstract The Gobioninae are a group of morphologically and ecologically diverse Eurasian freshwater cyprinid fishes. The intergeneric relationships of this group are unresolved and the possible monophyly of this subfamily remains to be established. We used complete mitochondrial cytochrome b gene sequences from most genera within the gobionine group, in addition to a selection of cyprinid outgroups, to investigate the possible monophyly of this group and resolve the interrelationships within the group. Our results support the monophyly of the Gobioninae and identify four monophyletic groups within the subfamily; the group, the group, the group, and the group. The mor- phologically aberrant genera , Xenophysogobio and Gobiocypris are included in the Gobioninae, with the latter a sister group of .

Introduction exhibit greatest diversity and richness in eastern Asia (China, Japan, Korea and Vietnam), The Cyprinidae contains more than 2000 species particularly on the Chinese mainland (Ban a: rescu and is one of the largest teleost families (Nelson, & Nalbant, 1973; Luo et al., 1977; Ban a: rescu, 1994). The phylogenetic structure of this family is 1992; Yue, 1998). still under debate, though several subfamilies are Gobionines are a group of small to medium- widely accepted and appear to form well-sup- sized cyprinids that exhibit great morphological, ported monophyletic clades (Chen et al., 1984; ecological, and behavioural variation (Ban a: rescu Howes, 1991). One of these is the Gobioninae, the & Nalbant, 1973; Ban a: rescu, 1992; Yue, 1998). gudgeons, a speciose subfamily with approxi- Some are benthic and rheophilic, while others are mately 130 species in about 30 genera that are semi-pelagic. Some species only inhabit fast-run- widely distributed throughout northern and east- ning cold headwater streams and others are found ern Eurasia from Spain east to Japan and south to in tropical swamps. Some gudgeons possess an central Vietnam. With the exception of the inferior mouth with 8 barbels while in some species endemic western Palaearctic , the mouth is superior and barbels are absent. and the genus Gobio, which is widespread from Some gobionines exhibit parental care of their Spain to northern China, the gobionine cyprinids eggs, while others lay them on the gills of 256 freshwater mussels, and yet others release their chrome b gene sequences for 49 gobionine species pelagic eggs into stream currents. to obtain a better understanding of their phylo- Because of their morphological and ecological genetic relationships and the position of this group diversity, there is debate about which genera within the cyprinids. The aims of this study were: should be included in the Gobioninae and whether (1) to test the monophyly of Gobioninae, (2) to this subfamily constitutes a monophyletic unit estimate the phylogenetic relationships among within the Cyprinidae (Liu, 1940; Ramaswami, gobionine genera. 1955; Hosoya, 1986). Liu (1940) proposed that the morphologically highly derived genera Gobiobotia and Xenophysogobio should to be excluded from Materials and methods the Gobioninae, forming a distinct subfamily the Gobiobotinae. Hosoya (1986) divided the Gobi- Samples collection oninae into two branches, which comprised two monophyletic units, which may constitute two Here we adopt the classification of the Gobioninae distinct subfamilies. sensu Yue (1998). Specimens used in this study were The position of the Gobioninae within the collected from major rivers in China and Europe Cyprinidae is poorly understood. Most authors during 2000–2004. Due to the difficulties of obtain- using morphological data associate these fishes ing samples, one gobionine genus from China, with the large cyprinid subfamily, the Cyprininae Ladislavia, one endemic to Vietnam, Parasqualidus, (also sometimes called the Barbinae) (Mori, 1935; and two endemic to Korea, and Berg, 1940; Luo et al.; 1977; Arai, 1982; Hosoya, , were not included in our analysis. All 1986; Howes, 1991). In contrast, more recent au- specimens for sequencing were preserved in 95% thors, such as Cavender & Coburn (1992) and ethanol. Specimens of all the species used in this Chen et al. (1984), have grouped the Gobioninae study are deposited in the collection of the Institute within the large cyprinid subfamily the Leucisci- of Hydrobiology, Chinese Academy of Sciences nae, with the cyprinid subfamily the Acheilogna- and in the Collection of Jo¨ rg Freyhof (FSJF, thinae as its sister-group. Berlin) and K.-E. Witte (KEW, Tu¨ bingen), Recent molecular data appear to show that the respectively. DNA sequences for some species were Gobioninae may be a monophyletic group (Zar- downloaded from GenBank for comparison. To doya & Doadrio, 1999; Zardoya et al., 1999; Liu & test the systematic position of the Gobioninae Chen, 2003; He et al., 2004), though a contrary within the family Cyprinidae, some species from result was also obtained by Cunha et al. (2002). different subfamilies of the Cyprinidae were also Molecular data also support the view that the included for analysis. Species names, river drain- gobionines should be grouped more closely within ages and GenBank accession numbers are shown in the cyprinids to the subfamily Leuciscinae rather Table 1. than to the Cyprininae (Briolay et al., 1998; Zar- doya et al., 1999; Zardoya & Doadrio, 1999; Gilles mtDNA extraction, amplification and sequencing et al., 2001; Liu & Chen, 2003; He et al., 2004). However, with the exception of the study by Liu & Genomic DNA was extracted from muscle tissue Chen (2003), all these studies are based on a lim- following standard phenol/chloroform protocols ited number of species. Consequently, the sys- (Kocher et al., 1989). The mitochondrial Cyt b gene tematic position of the Gobioninae within the was amplified by the polymerase chain reaction Cyprinidae, the monophyly of the group, and the (PCR) in 50 ll reactions containing 0.5 ll dNTPs phylogenetic relationships of genera within the (1 mM), 5 ll reaction buffer (200 mM Tris-HCl pH subfamily are equivocal. 8.4, 500 mM KCl, 50 mM MgCl), 2.5 ll of each The mitochondrial cytochrome b gene is widely primer (10 lM), 0.5 ll (2.5 U) of Taq DNA Poly- used for phylogenetic analyses and is considered to merase, 3 ll template DNA and 36 llH2O. The be one of the most reliable mitochondrial markers primer pairs used were L14724 (5¢-GAC TTG AAA for phylogenetic studies (Zardoya & Meyer, 1996). AAC CAC CGT TG-3¢) and H15915 (5¢-CTC CGA Here we use complete mitochondrial DNA cyto- TCT CCG GAT TAC AAG AC-3¢) (Xiao et al., 257

Table 1. Samples used in the present study, showing GenBank accession number, river drainage and country from which specimens were collected

Species Accession numbers Drainage Origin

Abbottina rivularis AY953020 Yangtze River China Acanthogobio guitheri AY953003 Yellow River China Belligobio numifer AY952987 Yangtze River China Biwa zezera* AF309507 Biwa Lake Japan guichenoti AY953001 Yangtze River China Coreius heterrodon AY953000 Yangtze River China Gnathopogon imberbis AY952998 Yangtze River China Gnathopogon nicholsi AY952997 Yangtze River China Gobio cynocephalus AY953005 Amur River China AY953007 Rhine River Germany Gobio macrocephalus AY953006 Amur River China Gobio tenuicorpus AY953004 Yellow River China Gobiobotia filifer AY953002 Yangtze River China Gobiobotia meridionalis* AF375867 Pearl River China * AF375865 Yangtze River China Hemibarbus labeo AY952988 Yangtze River China Hemibarbus maculatus AY952990 Yangtze River China Hemibarbus medius AY952989 Yangtze River China chinssuensis AY953016 Yellow River China Mesogobio tumenensis AY953008 Tumenjiang River China fukiensis AY953014 Minjiang River China Microphysogobio tungtingensis AY953013 Yangtze River China Paracanthobrama guichenoti AY952994 Yangtze River China Paraleucogobio strigatus AY952999 Amur River China exiguus AY953015 Pearl River China Platysmacheilus longibarbatus AY953017 Yangtze River China Pseudogobio guilinensis AY953018 Pearl River China Pseudogobio vaillanti AY953019 Yangtze River China elongata AY952996 Yangtze River China Pseudorasbora parva AY952995 Yangtze River China herzi* AF375864 Unkown Japan cylindricus AY952992 Yangtze River China Rhinogobio hunanensis AY952993 Yangtze River China Rhinogobio typus AY952991 Yangtze River China Romanogobio banaticus AY952329 Danube River Romanogobio kesslerii AY952328 Dniester River Romanogobio macropterus AY952332 Arax River Turkey AY952331 Danube River Romania Rostrogobio liaohensis AY953012 Liaohe River China Sarcocheilichthys microoculus* NC004694 Unkown Japan Sarcocheilichthys nigripinnis AY952983 Yangtze River China Sarcocheilichthys kiangsiensis AY952984 Yangtze River China dabryi AY953011 Yangtze River China Saurogobio gymnocheilus AY953009 Yangtze River China Saurogobio immaculatus AY953010 Hainan Island China Continued on p. 258 258

Table 1. (Continued)

Species Accession numbers Drainage Origin

Squalidus gracilis* AF375866 Unkown Japan argentatus AY952985 Yangtze River China Squalidus nitens AY952986 Yangtze River China Xenophysogobio boulengeri* AF375868 Yangtze River China Other cyprinid species Acheilognathus chankaensis* AF051854 Unkown China Aphyocypris chinensis* AF307452 Liaohe River China Barbus barbus* Y10450 Unkown Europe Discogobio tetrabarbatus AY953022 Pearl River China Hemiculter leucisculus* AF051865 Unkown China molitrix* AF051866 Unkown China Leuciscus waleckii AY953021 Amur River China Megalobrama terminalis* AF051872 Unkown China Opsariichthys uncirostris* AF308437 Unkown Japan Rhodeus ocellatus* AF051876 Unkown China Squaliobarbus curriculus* AF051877 Unkown China davidi* AF036195 Unkown China Zacco platypus* AF309085 Unkown China

Asterisks indicate sequences obtained from GenBank.

2001). The PCR reaction was optimized at 35 cycles 2002) for all positions. Nucleotide saturation was of 94 C for 40 s (denaturation), 56 C for 45 s analyzed by plotting pairwise inferred substitutions (annealing), 72 C for 1 min (extension), and a final against corrected GTR-distance values. 72 C extension for 8 min. Amplified double- Phylogenetic trees were estimated with both stranded DNA was purified using a BioStar glass- maximum parsimony (MP) and maximum likeli- milk DNA purification kit following the manufac- hood (ML) optimality criteria using the computer ture’s instructions. Purified DNA was sequenced by program PAUP*. MP analysis used tree bisection- a commercial sequencing company with the same reconnection (TBR) branch swapping and a primer set L14724 and H15915. heuristic searches with 20 replicates of random stepwise additions. No topological restraints were Sequence alignment and phylogenetic analysis enforced and all characters were included with equal weighting. Support for each node was eval- Nucleotide sequences were aligned using the Clus- uated using bootstrap values (Felsenstein, 1985) tal X multiple alignments program (Thompson et and Bremer decay indices (Bremer, 1994). Quali- al., 1997), with default gap penalties, and the out- tative support for recovered nodes was assessed put was later improved by eye. No ambiguous using a non-parametric bootstrap analysis with alignments were found, and no gaps postulated. 1000 pseudoreplicates and a heuristic tree search DNA sequences were also translated into amino with 20 additional sequence replicates. Bremer acid sequence in MEGA version 2.1 (Kumar et al., decay indices were calculated using TreeRot v2 2001), producing full-length reads with no internal (Sorenson, 1999). stop codons. The amount of sequence variation The appropriate model of sequence evolution and the Kimura’s two parameter distances were was chosen on the basis of hierarchical likelihood- also analyzed using MEGA 2.1. Base composi- ratio tests (LRTs) as implemented in ModelTest tional bias was calculated across all taxa, and a chi- version 3.06 (Posada & Crandall, 1998). The square (v2) test of base heterogeneity was con- parameters of the models were computed using ducted using PAUP* version 4.0b10 (Swofford, PAUP*. Under the Akaike information criterion 259

(AIC), the general time reversible model with some Both weighted MP analyses yielded two different sites assumed to be invariable and with variable consensus trees with most nodes unresolved. sites assumed to follow a discrete gamma distri- Therefore, the two results are not addressed in the bution (GTR+G+I; Yang, 1994) was consis- discussion. tently selected as the best-fit ML model of nucleotide substitution. The cyt b data sets had the following statistics: estimated nucleotide frequen- Results cies A = 0.3276, C = 0.3333, G = 0.0859, and T = 0.2532; nucleotide substitution rate matrix Sequence variations and mutation analysis A-C = 0.3554, A-G = 8.0173, A-T = 0.2929, C- G = 0.5767, C-T = 6.0624, and G-T = 1.0000; A total of 44 complete nucleotide sequences of the assumed proportion of invariable sites = 0.4745 mitochondrial cytochrome b gene were deter- and the Gamma distribution shape parameter mined, of which 42 were gobionine taxa. These (a)=0.6433. A heuristic tree search strategy with sequences were aligned with the complete cyto- 20 random-addition sequences and TBR branch chrome b sequences of 7 gobionine and 12 other swapping was used with PAUP* to find the best cyprinid species obtained from GenBank. All ML tree. Because bootstrap analysis for node protein encoding 1140 positions were analyzed, of support was too time consuming, Bayesian pos- which 578 (50.7%) were constant sites and 485 terior values were determined for the ML tree (42.5%) were phylogenetically informative sites using the computer program MrBayes version using the parsimony criterion. Pairwise sequence 3.0b4 (Huelsenbeck & Ronquist, 2001), with the divergence among gobionines varied from 0.4 optimal model of sequence evolution determined (between (Sauvage and from the LRTs. Bayesian analyses were initiated Dabry) and Squalidus nitens (Gu¨ nther)) to 23.8% from random starting trees and four Markov chain (between Gobiocypris rarus (Ye and Fu) and Monte Carlo (MCMC) simulations were run (Temminck and Schlegel)), with simultaneously for 2 000 000 generations with tree a mean of 18.8% (±0.7%). This figure is higher sampling every 100 generations. Chain stationarity than that among out-group taxa, which ranged was achieved after only 30 000 generations (‘burn- from 4.8 (between (Bleeker) and in’) and, therefore, 300 trees were subsequently Acheilognathus chankaensis (Dybowski)) to 24.7% discarded. The frequency that a particular clade (between Zacco platypus (Temminck and Schlegel) occurs within the last 19701 trees after the burn-in and Barbus barbus L.), with a mean of 17.2% was interpreted as a measure of node support. (±0.7%) (table of genetic distance not shown). In accordance with Wahlberg et al. (2003), The mean base composition of cytochrome b when discussing our results we refer to support gene sequences was similar to that previously values as either giving weak, moderate, good or reported for cyprinid fishes (Briolay et al., 1998; strong support. We define weak support as Bremer Durand et al., 2002), with low G content (15.2%) support values of 1–2 (bootstrap values 50–63% and almost equal A, T and C content (28.1%, and posterior probabilities 50–69%), moderate 28.9%, 27.7%, respectively). The content of A+T support as values between 3 and 5 (bootstrap (56.3%) was higher than that of C+G (43.7%), values 64–75% and posterior probabilities 70– which fell within the range of the GC content 84%), good support as values between 6 and 10 typical for vertebrates (Nei & Kumar, 2000). Sig- (bootstrap values 76–88% and posterior proba- nificant compositional biases exist at the second bilities 85–94%), and strong support as values >10 and especially the third codon position, where (bootstrap values 89–100% and posterior proba- there was a marked under representation of G bilities 95–100%). (13.3 and 6.7%, respectively). Base frequencies Because transitions (Ts) often accumulate at were homogeneous across all taxa for three posi- rates higher than transversions (Tv), we also con- tions (v2 = 167.91, df = 177, p = 0.676). How- ducted weighted parsimony analyses under two ever, nucleotide composition at the third position Ts/Tv weighting schemes (2.6:1 and 5:1). The Ts/ exhibited significant heterogeneity: first position, Tv ratio of 2.6:1 was derived from ModelTest. v2 = 36.29, df=177, p = 1.0; second position, 260 v2 = 2.51, df=177, p = 1.0; and third position, consensus Bayesian tree differed slightly for ML v2 = 474.07, df=177, p < 0.001. trees. Most nodes of ML trees were supported with Variability among sequences was detected significant ML Bayesian posterior probabilities mainly in third codon positions, of which 98.4% (Fig. 1). The phylogenetic position of the Gobion- were variable. For first and second positions, 37.6 inae closer to the Leuciscine than to the Cyprinine and 13.4% were variable, respectively. Substitu- was recovered with a significant posterior proba- tions showed some level of saturation in third co- bility (PP = 100). The monophyly of the subfamily don positions but not in first and second codon Gobioninae was also strongly supported positions (not shown). (PP = 100), and the gobionine genera were grouped into four discrete assemblages (Fig. 1). Phylogenetic relationships The first assemblage, the Hemibarbus group, has strong support as a monophyletic group Maximum likelihood analysis yielded two trees (PP = 100), comprising the genera Belligobio, with a likelihood score of )ln L=20779.6. The Hemibarbus and Squalidus (Fig. 1). However, the

Figure 1. Tree resulting from maximum likelihood analysis of the complete cyt b gene dataset under the GTR+G+I model. Max- imum-likelihood score=)20779.58, a=0.64, and I=0.47. Numbers at nodes represent Bayesian posterior probabilities (given for posterior probabilities greater than 50%). 261 monophyly of the second assemblage, the Sar- MP trees weakly supported the integrity of the cocheilichthys group, is not supported by ML Pseudogobio group. Moderate decay scores sup- Bayesian posterior probability values greater than ported the formation of sister groups between 50%. In addition, interrelationships within the Gobiobotia and Xenophysogobio as well as Sarcocheilichthys group are not well resolved. Abbottnia and Saurogobio with the other genera of Within this group, Coreius is at the basal position the Pseudogobio group, but were not supported in in the Bayesian tree with a posterior probability bootstrap analysis. Like our results with ML trees, lower than 50% and forming an independent when , Gobiobotia, Sauragobio and group in the ML tree. Sarcocheilichthys and the Xenophysogobio were excluded, the monophyly of remaining genera form a monophyletic group with the remaining genera with strongly papillated lips moderate ML Bayesian posterior probability val- received moderate support by bootstrap analysis ues (PP=81) (Fig. 1) and Gobiocypris appears in and good support from Bremer decay indices this group as a sister genus of Gnathopogon with (Fig. 2). strong support. The genus Pseudorasbora is not monophyletic in the ML analyses, with Pseudo- Tests of alternative topologies rasbora elongata (Temminck & Schlegel) the sister taxon of (Herzenstein). To further test the monophyly of the Pseudogobio The monophyly of the third assemblage, the and Sarcocheilichthys groups, we constrained these Gobio group including Acanthogobio and Me- groups as monophyletic and performed further sogobio tumensis (Chang), is strongly supported heuristic searches. The resulting trees did not differ (PP = 100). A sister-group relationship between significantly from those obtained without con- the genera Romanogobio and Gobio is strongly straint enforcement in Kishino–Hasegawa (KH) supported (PP = 100) (Fig. 1). (Kishino & Hasegawa, 1989) and two-tailed Wil- There is weak support for monophyly in the coxon signed-ranks tests (Templeton, 1983) fourth assemblage, the Pseudogobio group (p > 0.05 in each case) (Table 2). (PP = 56). Within this group, Gobiobotia and Xenophysogobio are situated basally; Abottina and Saurogobio form a sister-group, being the sister- Discussion group to several genera characterized by strongly papillated lips (PP = 59) (Fig. 1). Monophyly and systematic position of Gobioninae The two most parsimonious trees were recov- ered in maximum parsimony analysis using uni- Our results concur with those of Ban a: rescu & form weights for all changes. Most of the MP trees Nalbant (1965, 1973) and Luo et al. (1977), sup- are congruent with the ML tree in topology (Fig. 2). porting the view that the Gobioninae is a mono- The monophyly of the Gobioninae is weakly sup- phyletic subfamiliy within Cyprinidae. Our results ported by bootstrap analysis but well supported by contradict the polyphyletic origin of this group Bremer decay indices (BP = 56 and decay proposed by Hosoya (1986) and Howes (1991). score = 7). Only two of the four monophyletic Previous molecular work has demonstrated the assemblages recovered in ML analyses are sup- monophyly of the European and North American ported with moderate bootstrap values and good subfamily the Leuciscinae, the African, Palearctic decay scores (BP = 64 and decay score of 6 in the and East Asian subfamily the Cyprininae and the Hemibarbus group, BP = 69 and decay score of 6 East Asian subfamily the Cultrinae, including the in the Gobio group). The monophyly of the Sar- former subfamily, the Xenocyprininae (Zardoya & cocheilichthys group is recovered with neither high Doadrio, 1999; Zardoya et al., 1999; Gilles et al., bootstrap values nor high decay scores. When the 2001; Cunha et al., 2002; Liu & Chen, 2003; He et genus Coreius is excluded, the remaining genera al., 2004). These studies indirectly demonstrate the formed a monophyletic unit with a moderate decay gobionines to be monphyletic, since if these groups score but with bootstrap values not greater than are all monophyletic within the cyprinid family, 50%. Within this group the monophyly of the genus the gobionines cannot be members of these groups Pseudorasbora is also not supported. and vice versa. Within the Cyprinidae, the 262

Figure 2. Strict consensus of two equally parsimonious trees from the equally weighted cyt b gene dataset. The tree length is 5083 steps, CI is 0.192 and RI is 0.808. Numbers given above branches are bootstrap values and numbers below the branch are Bremer support values (given for bootstrap values greater than 50%).

Table 2. Summary of the two-tailed Wilcoxon signed-ranks tests and Kishino–Hasegawa (KH) test of alternative topologies

Topology Wilcoxon signed-ranks test Kishino–Hasegawa test Maximum parsimony Maximum likelihood

Tree length nz p Ln L Ln L diff. p

MP 5099 25 )1.4000 0.16 )25464.76 16.4401 0.144 Pseudogobionini monophyletica 5092 Best )25448.32 Best Sarcocheilichthyini monophyletica 5096 49 )0.6676 0.50 )25456.52 8.2031 0.623 aMaximum parsimony tree recovered from constraint search.

Gobioninae seem to be more closely associated et al. (2001), Liu & Chen’s (2003) and He et al. with the Leuciscinae than the Cyprinine, as sus- (2004), contradicting the view of Luo et al. (1977) pected by Arai (1982), Chen et al. (1984), Gilles and Hosoya (1986). However, our results do not 263 support the idea that the Gobioninae are the sister genera of our Sarcocheilichthys group are not group to the Acheilognathinae (the bitterlings) as certain in the present study. Rhinogobio appears to proposed by Chen et al. (1984). be a sister group of Gobiocypris or Gnathopogon, although it had neither bootstrap values greater Major groups within Gobioninae than 50% nor significant posterior probabilities, it did show a high decay score in MP analyses. Four major evolutionary lineages within the Assignment to the genus Pseudorasbora is mainly Gobioninae are postulated in the present study, based on characters related to the position of the which partly confirms the division of the gobio- mouth, with Pseudorasbora and Pungtungia mor- nines proposed in previous studies (Liu, 1940; phologically similar. Further studies may show Ramaswami, 1955; Luo et al., 1977; Kim, 1984; Pungtungia to be a synonym of Pseudorasbora, Hosoya, 1986; Rainboth, 1991; Ban a: rescu, 1992; alternatively Pseudorasbora elongata (Wu) may be Naseka, 1996; Yu & Yue, 1996; Yue, 1998). transferred to the genus Pungtungia. Gobiocypris Ban a: rescu (1992) suspected that Hemibarbus was formerly thought to be allied to Aphyocypris and Belligobio might represent the ancestral group and was treated as a member of the cyprinid of gobionine cyprinids. Hemibarbus has distinct subfamily the Danioninae (Ye & Fu, 1983). morphological characters considered to be primi- However, it has six branched anal fin rays, which is tive; the last simple dorsal ray is strongly ossified, a diagnostic morphological character for the the vent is positioned immediately in front of the Gobioninae. He et al. (2004) and results from the anus, and the pharyngeal teeth are arranged in present study support the assignment of Gobiocy- three rows. The main morphological difference pris to the Gobioninae, with a relationship to between Hemibarbus and Belligobio is that the Gnathopogon. Additional molecular data are former has a strongly ossified last unbranched needed to better resolve the relationships among dorsal ray. Consequently, Ban a: rescu & Nalbant the genera of the Sarcocheilichthys group. (1973) assigned Belligobio as a subgenus of In the present study the position of the genus Hemibarbus. Because the type species of Belligobio, Coreius appears ambiguous. This genus shows B. eristigma (Jordan & Hubbs), is not included in molariform teeth in one row, large body size, the present analysis, further studies on the genus small eyes, and long barbels and tongue, which Belligobio and Hemibarbus may demonstrate that made it inconsistent with members of other Belligobio should be included within the genus groups. It was treated as an isolated aberrant Hemibarbus. The close relationship of Squalidus to group by Ban a: rescu (1992), a conclusion that is Hemibarbus and Belligobio is also supported by supported by our ML analysis. However, it sequence data for the mitochondrial control region affiliates with the Sarcocheilichthys group in our (Yang et al. unpublished data), whereas, mor- Bayesian and MP analyses, so we tentatively phological data indicate that Squalidus may be group it with the Sarcocheilichthys group. Further close to Gobio (Ban a: rescu & Nalbant, 1965, 1973; molecular data are needed to confirm the taxo- Hosoya, 1986; Ban a: rescu, 1992) or Gnathopogon nomic position of this genus. (Luo et al., 1977; Yue, 1995). The monophyletic Gobio group comprises the The monophyly of the Sarcocheilichthys group genera Gobio, Romanogobio, Acanthogobio and was supported by both ML and MP results, Mesogobio tumensis. Our data support the view though bootstrap support, posterior probabilities that Acanthogobio and M. tumensis should be in- and decay scores for this conclusion are not high. cluded in the genus Gobio. Ban a: rescu & Nalbant Our analysis showed that our Sarcocheilichthys (1973) highlighted that Acanthogobio seem to be a group comprised most of the genera that were morphologically derived species of Gobio. We excluded from the Gobioninae by Hosoya (1986), cannot exclude the possibility that Acanthogobio with the exception of Rhinogobio and Paracant- and M. tumensis might by introgressed with hobrama. Paracanthobrama was formerly classified mtDNA from Gobio species. Introgressive as a subgenus of Hemibarbus by Ban a: rescu & hybridization is not rare between closely related Nalbant (1973) and Ban a: rescu (1992). Relation- species (Avise, 2000; Doiron et al., 2002; Rognon ships between Paracanthobrama and the remaining & Guyomard, 2003) and is well known from 264 cyprinid fishes (Gilles et al., 1998; Durand et al., group. On the basis of our data, interrelationships 2000; Tsigenopoulos et al., 2002). Resolution of among genera within the Sarcocheilichthys and the position of Acanthogobio and Mesogobio will Pseudogobio groups were not well resolved. Fur- come with further examination of the type species ther molecular work examining the interrelation- of Mesogobio, M. lachneri (Ban a: rescu & Nalbant) ships of the Gobioninae should include additional and the use of further molecular markers. mitochondrial or nuclear genes. The monophyly of the Pseudogobio group was not well supported in our study by the uniform weighted MP method. However, the monophyletic Acknowledgements constrained tree represented the best tree with our data and was not rejected by the Templeton and We thank Dr. C. Smith for English correction and KH tests. This group comprises the genera suggestion. Our thanks also to Q. Tang, Y. Zhu, Gobiobotia, Sauragobio, Abbottina, Pseudogobio, X. Xia, L. Zhang and Y. Zeng for their help in Platysmcheilus, Huigobio, Microphysogobio, collecting samples and laboratory work. We also Rostrogobio, and Xenophysogobio. Several would like to extend our thanks to D. He and morphological studies already support the view Z. Peng for discussion and information about the that these genera form a monophyletic unit manuscript. Funding supports are from NSFC (Hosoya, 1986; Ban a: rescu, 1992), though some 40432003, CAS KZCX-SW-126, and Cyprini- authors excluded Gobiobotia and Xenophysogobio formes TOL. from the group (Luo et al., 1977; Yu & Yue 1996; Yue, 1998). Gobiobotia and Xenophysogobio have had inconsistent systematic positions because of References the curious bony cups and four pairs of barbels (contrasting with one pair in other genera). Most Arai, R. M., 1982. A chromosome study on two cyprinid fishes, authors concur that Gobiobotia and Xenophy- Acrossocheilus labiatus and pumila, sogobio belong to the Gobioninae (Taranetz, with notes on Eurasian cyprinids and their karyotypes. 1938; Ramaswami, 1955; Ban arescu & Nalbant, Bulletin of the National Science Museum, Tokyo (A) 8: 131– : 152. 1973; Hosoya, 1986; Ban a: rescu, 1992; Naseka, Avise, J. C., 2000. Phylogeography. The History and Forma- 1996), while Luo et al. (1977), Kim (1984) and Yue tion of Species. MA: Harvard University Press, Cambridge. (1998) ascribed it to an independently subfamily. Ban arescu, P., 1992. A critical updated checklist of Gobioninae Hosoya (1986) believed Gobiobotia and Xenophy- (Pisces, Cyprinidae). Travaux Du Muse´um D’histoire Nat- sogobio should be placed in the Gobioninae since urelle ‘‘Grigore Antipa’’ 32: 303–330. Ban a: rescu, P. & T. T. Nalbant, 1965. Studies on the systematics they share synapomorphies in the lower jaw and in of Gobioninae (Pisces, Cyprinidae). Revue Roumaine de the caudal base with other genera of the Gobion- Biologie. Serie Biologie Animale 10: 219–229. inae (Pseudogobio, Abbottina, Saurogobio, Micro- Ban arescu, P. & T. T. Nalbant, 1973. Pisces, Teleostei, Cyp- physogobio and Biwia). Our molecular results rinidae (Gobioninae). Das Tierreich, Lieferung 93. Walter de support the view that Gobiobotia and Xenophy- Guryter, Berlin. Berg, L. S., 1940. Classification of fishes, both recent and fossil. sogobio belong to the Pseudogobio group. With Transactions of Institute of Zoology, Academy of Sciences, the exclusion of Saurogobio, Gobiobotia, Abbottina USSR 5: 87–345. and Xenophysogobio, all the genera exhibit Bremer, K., 1994. Branch support and tree stability. Cladistics strongly papillose lips with one or two smooth or 10: 295–304. papillose pads and a reduced air bladder with an Briolay, J., N. Galtier, M.R. Brito & Y. Bouvet, 1998. Molec- ular phylogeny of Cyprinidae inferred from cytochrome b encapsulated anterior chamber. DNA sequences. Molecular Phylogenetics and Evolution 9: In conclusion, our phylogenetic analysis using 100–108. complete mitochondrial cytochrome b gene se- Cavendar, T. M. & M. Coburn, 1992. Phylogenetic relationships quences suggests that the subfamily Gobioninae is of North American Cyprinidae. In: Mayden. R. (ed.), Sys- a monophyletic group. In addition, the subfamily tematics, historical ecology, and North American freshwater fishes. Stanford University Press, Stanford, CA, pp. 293–327. can be divided into a further four monophyletic Chen, X., P. Yue & R. Lin, 1984. Major groups within the groups: the Hemibarbus group, the Sarcocheilich- family Cyprinidae and their phylogenetic relationships. Acta thys group, the Gobio group, and the Pseudogobio Zootaxonomica Sinica 9: 424–440. 265

Cunha, C., N. Mesquita, T. E. Dowling, A. Gilles & M. M. Luo, Y., P. Yue & Y. Chen, 1977. Gobioninae. In Wu, X. W. Coelho, 2002. Phylogenetic relationships of Eurasian and (ed.), Cyprinid Fishes of China, II. People’s Press, Shanghai: American cyprinids using cytochrome b sequences. Journal 439–549. of Fish Biology 61: 929–944. Mori, T., 1935. Description of two new genera and seven new Doiron, S., L. Bernatchez & P. Blier, 2002. A comparative species of Cyprinidae from chosen. Annotationes of Zoo- mitogenomic analysis of the potential adaptive value of logicae Japonenses 15: 161–181, pls. 11–13. arctic charr mtDNA introgression in brook charr popula- Naseka, A. M., 1996. Comparative study on the vertebral col- tions. Molecular Biology and Evolution 19: 1902–1909. umn in the Gobioninae (Cyprinidae, Pisces) with special Durand, D. J., S. C. Tsigenopoulos, E. U¨ nlu¨ & P. Berrebi, 2002. reference to its systematics. Publicaciones Especiales. Insti- Phylogeny and biogeography of the family Cyprinidae in the tuto Espanol de Oceanografia 21: 149–167. Middle East inferred from Cytochrome b DNA – evolu- Nei, M. & S. Kumar, 2000. Molecular Evolution and Phylog- tionary significance of this region. Molecular Phylogenetics enetics. Oxford University Press, New York. and Evolution 22: 91–100. Nelson, J. S., 1994. Fishes of the World (3rd edn.). John Wiley Felsenstein, J., 1985. Confidence limits on phylogenies: an ap- and Sons, New York. proach using the bootstrap. Evolution 39: 783–791. Posada, D. & K. A. Crandall, 1998. MODELTEST: testing the Gilles, A., G. Lecointre, E. Faure, R. Chappaz & G. Brun, model of DNA substitution. Bioinformatics 14: 817–818. 1998. Mitochondrial phylogeny of European cyprinids: Rainboth, W. J., 1991. Cyprinids of South-East Asia. In Win- implications for their systematics, reticulate evolution, col- field, I. J. & J. S. Nelson (eds), Cyprinid fishes. Systematics, onization time. Molecular Phylogenetics and Evolution 10: Biology and Exploitation. Chapman and Hall, New York: 132–143. 156–211. Gilles, A., G. Lecointre, A. Miquelis, M. Loerstcher, R. Chappaz Ramaswami, L. S., 1955. Skeleton of cyprinoid fishes in relation & G. Brun, 2001. Partial combination applied to phylogeny of to phylogenetic studies. VI. The skull and Weberain appa- European cyprinids using the mitochondrial control region. ratus in the subfamily Gobioninae (Cyprinidae). Acta Zoo- Molecular Phylogenetics and Evolution 19: 22–33. logica 36: 127–158. He, S., H. Liu, Y. Chen, M. Kuwahara & T. Nakajima, 2004. Rognon, X. & R. Guyomard, 2003. Large extent of mito- Phylogenetic analysis of cyprinid fishes based on mitochon- chondrial DNA transfer from Oreochromis aureus to O drial cytochrome b gene sequences. Science in China (C) 34: niloticus in West Africa. Molecular Ecology 12: 435–445. 96–104. Sorenson, M. D., 1999. TreeRot (version 2). MA: Boston Hosoya K., 1986. Interrelationships of the Gobioninae (Cyprini- University, Boston. dae) In Uyeno T. et al. (ed.), Indo-Pacific Fish Biology: pro- Swofford, D. L., 2002. PAUP*: Phylogenetic Analysis Using ceeding of the Second International Conference on Indo-Pacific Parsimony (* and other Methods) (version 4.0). MA: Sina- Fishes Ichthyological Society of Japan, Tokyo: 484–501. uer Associates, Sunderland. Howes, G., 1991. Systematics and biogeography: an overview. Taranetz, A., 1938. On the relationship and origin of gudgeons In Winfield, I. J. & J. S. Nelson (eds), Cyprinid fishes. Sys- of the basin of the Amur River. Moskva: Zoologicheskij tematics, Biology and Exploitation. Chapman and Hall, Zhurnal 17: 453–471. New York: 1–33. Templeton, A. R., 1983. Phylogenetic inference from restriction Huelsenback, J. P. & F. Ronquist, 2001. MrBayes: Bayesian endonuclease cleavage site maps with particular reference to inference of phylogeny. Bioinformatics 17: 754–755. the evolution of human and apes. Evolution 37: 221–244. Kim, I. S., 1984. The taxonomic study of gudgeons of the Thompson, J. D., T. J. Gibson, F. Plewwniak, F. Jeanmougin & subfamily Gobioninae (Cyprinidae) in Korea. Bulletin of D. G. Higgins, 1997. The Clustal X windows interface: flex- Korean Fishery Society 17: 436–448. ible strategies for multiple sequence alignment aided by Kishino, H. & M. Hasegawa, 1989. Evaluation of the maximum quality analysis tools. Nucleic Acids Research 25: 4876–4882. likelihood estimate of the evolutionary tree topologies from Tsigenopoulos, C. S., P. Ra´b, D. Naran & P. Berrebi, 2002. DNA sequence data, and the branching order in Hominoi- Multiple origins of polyploidy in the phylogeny of southern dea. Journal of Molecular Evolution 29: 170–179. African barbs (Cyprinidae) as inferred from mtDNA Kocher, T. D., W. K. Thommas, A. Meyer, S. V. Edwards, markers. Heredity 88: 466–473. F. X. Pallablanca & A. C. Wilson, 1989. Dynamics of Wahlberg, N., E. Weingartner & S. Nylin, 2003. Towards a mitochondrial DNA evolution in : amplification and better understanding of the higher systematics of Nymp- sequencing with conserved primers. Proceedings of the Na- halidae (Lepidoptera: Papilionoidea). Molecular Phyloge- tional Academy of Sciences of the United States of America netics and Evolution 28: 473–484. 86: 6196–6200. Xiao, W., Y. Zhang & H. Liu, 2001. Molecular systematics of Kumar, S., K. Tamura, I. B. Jakobsen & M. Nei, 2001. MEGA: (Teleostei: Cyprinidae): , biogeo- Molecular Evolutionary Genetics Analysis. Ver 2.1. Bioin- graphy, and coevolution of a special group restricted in East formatics 17: 1244–1245. Asia. Molecular Phylogenetics and Evolution 18: 163–173. Liu, C. K., 1940. Preliminary studies on the air-bladder and its Yang, Z., 1994. Estimating the pattern of nucleotide substitu- adjacent structures in Gobioninae. Sinensia 11 (1/2): 77–104. tion. Journal of Molecular Evolution 39: 105–111. Liu, H. & Y. Chen, 2003. Phylogeny of the East Asian cyprinids Ye, M. & T. Fu, 1983. Description of a new genus and species inferred from sequences of the mitochondrial DNA control of Danioninae from China (: Cyprinidae). region. Canadian Journal of Zoology 81: 1938–1946. Acta Zootaxonomica Sinica 8: 434–437. 266

Yu, L. & P. Yue, 1996. Studies on Phylogeny of the Pseu- pean cyprinids. Journal of Molecular Evolution 49: 227– dogobionini fishes (Cypriniformes: Cyprinidae). Acta Zoo- 237. taxonomica Sinica 21: 244–255. Zardoya, R., P. S. Economidis & I. Doadrio, 1999. Phyloge- Yue, P., 1995. Revision of the classification on the cyprinid netic relationships of Greek Cyprinidae: Molecular evidence fishes of the genus Squalidus from China. Acta Hydrobio- for at least two origins of the Greek cyprinid fauna. logica Sinica 19: 89–91. Molecular Phylogenetics and Evolution 13: 122–131. Yue, P., 1998. Gobioninae. In Chen, Y. et al. (eds), Fauna Zardoya, R. & A. Meyer, 1996. Phylogenetic performance of Sinica. Osteichthyes. Cypriniformes II Science Press, Beijing: mitochondrial protein-coding genes in resolving relation- 232–389. ships among vertebrates. Molecular Biology and Evolution Zardoya, R. & I. Doadrio, 1999. Molecular evidence on 13: 933–942. the evolutionary and biogeographical patterns of Euro-