MOLECULAR PHYLOGENY OF ONYCHOSTOMA (CYPRINIDAE) BASED ON MITOCHONDRIAL GENOMES

ZHANG Chen， CHENG Qi， GENG Hong， LIN Ai-Hua and WANG Hong-Ying

(Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China， Key Laboratory of State Ethnic Affairs Commission for Biological Technology， College of Life Science， South-Central University for Nationalities，Wuhan 430074， China)

Onychostomais a kind of small freshwater fish that inhabits mountain streams and rivers in the upper reaches of rapids. At present， there are 19 nominal species inOnychostoma[1]. The species of this genus widely distributes in the Yellow River， the Yangtze River， the Pearl River， and the Red River systems.Three species， includingO. lini， O. rara， and O. alticorpus， have been listed as endangered fish because of scarce in number and the narrow distribution area[2]. AsOnychostomagenius in cyprinid， however，the molecular phylogenetic relationship inOnychostomais not clear.

Mitochondrial genome is a closed loop and independent of the nuclear genome. Mitochondrial genome consists of 13 protein-coding genes， two mitochondrial ribosomal rRNA (12SrRNA and 16SrRNA)，22 tRNA， and a non-coding region (D-Loop) containing a signal which controls mtDNA transcription and replication initiation[3]. Animal mitochondria gene structure is conserved. It is maternal genetic， independent in terms of replication， and relatively little reorganization. Therefore， mtDNA has become an effective tool for the study of phylogeny and population genetics at different levels[4]. To understand the genetic relationship inOnychostoma， we examined mitochondrial DNA of 8 species inOnychostomaand analyzed genetic relationship to elucidate the major relationship inOnychostoma. Phylogeny had been used extensively for animal and plant classification.The phylogenetic tree of imperial pigeons (Aves:Columbidae) in the Pacific Ocean using mtDNA and nuclear sequences was reconstructed. The result showed that eastern polynesian endemics were not a monophyletic group[5]. Based on nuclear and mtDNA genes， subgenus Tityus was considered neither sister of the remaining species of the genus， nor closely re-lated to the New World microbuthids with decreasing neobothriotaxy[6]. New insights into the phylogeny of Burasaieae (Menispermaceae) with the recognition of a new genus and emphasis on the southern Taiwan，China and mainland Chinese disjunction: By combining cpDNA and ITS data， species of Tinospora are divided into three clades， including a new genus[7].

1 Material and methods

1.1 Taxon selection

8 species ofOnychostomawhich already have the whole mitochondrial DNA sequences were selected. All the mtDNA sequences were downloaded from GenBank. GenBank accession numbers of each species were listed in Tab. 1 .

1.2 Data analysis

All the whole mtDNA sequences were used as multiple comparison by Clustal W[8]. Coding sequences were translated into amino acids by MEGA5.0[9]. Sequences of genetic distance and the content of each nucleotide variation were also analyzed by MEGA5.0. Genetic distance between two species was calculated with Kimura two-parameter model. Neighbor-Joining method (NJ)， Maximum Likelihood method (ML)， and Bayesian Inference method (BI) were used in establishing phylogenetic trees. ML tree and NJ tree were established by MEGA5.0[9]and BI tree was established by MrbayesV3.2.5[10]. DAMBE was used for sequence substitution analysis. Both in NJ and ML， the number of nonparametric bootstrap replications was set as 1000. The GTR+I+G model was chosen by MODELTEST3.7[11，12]as the best fit model for the BI method. The Markov Chain Monte Carlo (MCMC)process in Mrbayes was conducted for 100 million generations. For every 1000 steps， the initial 250 sampled data were discarded as burin-in. The 4 chains include 3 heating chains (temp 0.5) and 1 cold chain.Posterior probability was used for present repeat credibility of each branch.

Tab. 1 Species and their mtDNA genome sequences used in this study

2 Results

2.1 Sequence features and genetic distance

The alignments analysis showed that the fulllength mitochondrial DNA of these 8 species ranged from 16589 to 16607 bp. The average content proportions of A， T， C， and G were 31.3%， 24.4%， 28.2%，and 16.1% respectively. The sum of the content proportion of A+T (55.7%) was higher than that of C+G(44.3%). This was consistent with range of GC in vertebrates[13]. MtDNA ofOnychostomaappeared strong base composition bias and guanine was lower than other three bases. There were 16655 bp length after alignments.

Based on Kimura two-parameter model， we determined the genetic distance to define genetic differentiation in the taxon. The results showed that the genetic distances in the genus ofOnychostomaranged from 0.004 to 0.118， while the distances betweenOnychostomaand outgroups ranged from 0.140 to 0.282 (Tab. 2 ).

Individual sequence transition and transversion values were shown as a linear relationship and unsaturated (Fig. 1). It also showed phylogenetic significance and could be used for data analysis[14]. At the level of variation， the number of transition was significantly more than that of transversion. The range of the ratio of transition and transversion of the sequences was from 3.075 to 15.205 and the average value was 5.787.

2.2 Molecular phylogenetic trees

NJ， ML， and BI methods were used in phylogenetic analysis of mtDNA sequence of 8Onychostoma.Danio rerioandSpinibarbus denticulatuswere used as outgroups. The trees from three methods were similar whileO. Barbatulumwas identical in NJ and ML trees but it was different in BI tree (Figs. 2—4).In NJ and ML method，O. raraandO. alticorpuswere sister groups at the base， suggesting they were relatively primitive in evolution. Meanwhile，O. simumandO. gerlachiwere in the sister groups whileO.lini，O. barbatum，O. macrolepis， andO. barbatulumwere in monophyletic groups (with a bootstrap value 99 in ML and 86 in NJ). According to the NJ and ML tree，Onychostomawas divided into 3 taxa:O.rara+O. alticorpus，O. simum+O. gerlachiandO.lini+O. barbatum+O .macrolepis+O. barbatulum(Fig. 2 and 3).

The topologies of the NJ tree and the ML tree were similar with only difference in bootstrap and Bayesian posterior probability (BPP) value. The posi-tion ofO. barbatulumvaried in different methods(Fig. 2—Fig. 4). In BI tree，O. raraandO. alticorpuswere in sister groups at the basal with a high BPP value 100， which was similar in NJ and ML tree. Unlike the NJ tree and the ML tree， in the BI tree，O.gerlachiandO. simumwere in sister groups (with a BPP value of 100)， but they were sister groups toO.barbatulum(with a BPP value of 100).O. lini，O.barbatum， andO. macrolepiswere in the same taxa which is identical in NJ and ML tree (with a BPP value of 100).

Tab. 2 Genetic distance between two species based on Kimura two-parameter model

Fig. 1 Saturation processes of whole mitochondrial DNA sequence. Transitions (squares) and Transversions (triangles) were plotted against sequences

Fig. 2 The maximum likelihood tree was based on mitochondrial DNA sequence of 8 Onychostoma. S. denticulatus and D. rerio were used as outgroups

Fig. 3 The neighbor-joining tree was based on mitochondrial DNA sequence of 8 Onychostoma. S. denticulatus and D. rerio were used as outgroups

Fig. 4 The Bayesian inference tree was based on mitochondrial DNA sequence of 8 Onychostoma. S. denticulatus and D. rerio were used as outgroups

3 Discussion

3.1 Phylogenetic relationships within Onychostoma

Recent phylogenetic analysis ofOnychostomafrom RAG2 gene and mitochondrial 16s rRNA indicated thatOnychostomawas not a monophyletic group[15，16]. The result from the whole mtDNA ge-nome supported this view as well. Both the phylogenetic analysis from molecular data were in agreement with previous morphological classification[17]. Accordingly， theOnychostomawas divided into 3 groups. The first group containsO. barbatum，O. lini，andO. rara. The second group containedO. macrolepisandO. simum. The last one containedO. alticorpusandO. barbatulum. However， Xin suggested this classification might result in some deviations due to the limitation of samples quantity[18]. Xin dividedOnychostomainto 3 groups:O. barbatumandO. lini；O. macrolepis，O. simum，O. rara， andO. barbatulum； andO. alticorpusandO. barbatulum. From the NJ and ML trees of whole mtDNA sequences，Onychostomawas divided into 3 groups:O. raraandO.alticorpusform group 1，O. simumandO. gerlachiform group 2， andO. macrolepis，O. lini，O. barbatum， andO. barbatulumform the last group. In BI tree，O. gerlachi，O. simum， andO. barbatulumconsist of a monophyletic group， and the rest was similar as NJ and BI tree.

Tab. 3 Geographical distribution of 8 Onychostoma

Based on the significant difference between phylogenetic tree from mtDNA and morphological classification by Chen and Xin， the only consistent classfication result was thatO. simumandO. gerlachibelonged to the same group， while other results were inconsistent[17，18]. The phylogenetic tree tended to classifyO. macrolepis，O. lini，O. barbatum， andO. barbatulumto Clade 1，O. gerlachiandO. simumto Clade 2， andO. raraandO. alticorpusto Clade 3.Both the limited collection quantity of the specimens and the variable perspectives from scholars bring about this gap， although it was not sufficient to divideOnychostomasolely. Therefore， the classification method only by the phylogenetic trees based on mtDNA genome sequences was considered to be objective within the genus.

3.2 Biogeography of Onychostoma

According to the phylogenetic tree，Onychostomacan be divided into three groups. It is clear that Clade 1 mainly distributes in the Yellow River，Yangtze River， and Pearl River. Clade 2 mainly distributes in the Yangtze River and the Pearl River.Clade 3 mainly distributes in the Yuan River， Xijiang，and Taiwan， China (Tab. 3 ).

Due to the lack of reliable fossil records and insufficient geological isolation time， the evolution and biogeography distribution ofOnychostomacannot be well interpreted via the fossil data and geological events. By comparing the presence molecular clock limit ML tree level log-likelihood values， molecular clock was accepted (–lnL nonclock=57111.229271，–lnL clock=57111.22919，P＜1). We used cyprinidaeCytb0.76% Mya to estimate the divergence time[19]among which，O. raraandO. alticorpuswere at the base of the tree. The estimated divergence time between them and otherOnychostomawas 7.74—6.25 Mya. The estimated divergence time between Clade 1 and Clade 2 was 7.44—6.73 Mya and Clade 1 and Clade 3 was 6.55—6.23 Mya.

In this study， we established molecular phylogenetic tree based on analysis of 8Onychostomafulllength mitochondrial DNA sequences. The result showed thatOnychostomawas not a monophyletic group. Further testing of this hypothesis using additional taxa from this clade and additional genes is expected to lead to a better understanding of the endemic clade ofOnychostoma.

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