PROGRESS IN NATURAL SCIENCE V ol .15 , No .3 , M arch 2005

Phylogenetic studies of Chinese labeonine (Teleostei : )based on the mitochondrial 16S rRNA gene＊

LI Junbing1, 2 , WANG Xuzhen1 , HE Shunping1 ＊＊ and CHEN Yiy u1 (1 .Institute of Hydrobiology , Chinese Academy of Sciences, Wuhan 430072 , China;2 .Graduate School of the Chinese Academy of S ciences, Beijing 100039 , C hina)

Received July 15 , 2004 ;revised November 11 , 2004

Abstract The mitochondrial 16S ribosomal RNA gene is sequenced from 24 ingroups taxa, including 18 from grouped in 13 genera .Phylogenetic analyses are subjected to neighbor joining, maximum parsimony , maximum likelihood and Bayesian analyses.Phylogenetic analysis indicates that Labeoninae is basically a monophyletic assem blage and can be divided into 2 major clades:one comprising the genera Cirrhin us, and ;and the other consisting of the genera , , , Pseu- doorossocheilus, , , S emilabeo , Pseudogyricheilus, Rectori and .According to our present analysis, the features such as the presence of the adhesive disc on the chin and the in 2 row s used in the traditional of Labeoninae provide scarce information for phylogeny of labeonine fishes.

Keywords: 16S rRNA, Labeoninae, monophyly , phylogeny , Cyprinidae.

Labeoninae , one of the contained species of labeonine fishes , so they cannot be used as diag- w ithin Cy prinidae , is a huge g roup including more nostic for species otherwise than garrini group[ 3] . than 300 species in approximately 26 genera .Labeo- nine fishes are hig hly adaptive to torrential habitats , Based on osteological synapomorphy , Chen et al . treated Labeoninae as a monophylotic assemblage within and they are mainly distributed in the tropical and [ 5] subtropical regio ns of and [ 1] .The labeo- Cyprinidae , and placed Labeoninae in Barbini Series . nine know n in Africa is a diversified g roup and com- Because of the increase of the new records of species and its important role in economy , Labeonins has received prises about 120 species and subspecies placed in 2 [ 6—8] genera , Labeo and Garra[ 2] .This assemblage in- considerable taxonomical attention .However , all the cludes 20 genera over 60 species in China , w ithin previous phylogenies were focused on a fraction of labeo- w hich there are 8 genera endemic to China[ 1] .In nine species only .Therefore , up to date no subsequent China , labeonine fishes are mainly distributed in the work has been done to provide phylogenetic information w ater systems in south of the Yang tze River .The on the taxa of this group . subfamilies Labeoninae and Barbininae , together with The mitocho ndrial DNA has been w idely used in most of the genera of Danioninae and the Puntio- [ 1] the studies of molecular sy stematics of verte- plites of , constitute the south Asian g roup . brates[ 9, 10] , and the mitochondrial 16S rRNA gene Labeonins had a taxonomic difficulty because of sequence w as used as an effective genetic mark- [ 11 , 12] the lack of appropriate taxonomic features for assigna- er .In this study , we used the sequences of the tion of these included genera to higher taxonomic cat- mitochondrial DNA 16S rRNA fo r 18 labeonine egories.Labeonine fishes were affiliated to the sub- species to test the monophyly of Labeoninae , and we Barbininae[ 3] .Based on mo rphology of barbels also aimed to construct phy logenetic relationships of and the pattern of innervations correlated w ith bar- labeonine fishes at the genus level . bels , How es placed labeonins into the Cyprininae[ 4] .On account of the peculiar morphology 1 Materials and methods of lips and associated structures adapted for torrential 1 .1 Sam ple collection inhabit and the pharyngeal teeth in 2 row s , this as- semblage w as recog nized as Garrinae .These two A total of 27 species , including 18 species in 13 characters are , however , irregularly distributed among the genera from Labeoninae and the others from the sum-

＊ Supported National Natural S cience Foundation of China and C hinese Academy of Sciences (Grant Nos:30225008 & kscxz-sw-101B) ＊＊ To w hom correspondence should be addressed .E-mail:clad @ihb .ac.cn 214 w ww .tandf.co .uk/journals Prog ress in Natural Science Vol .15 No .3 2005 family Schizo thoracinae , Barbininae and Cy prininae , tions of origin and other relevant info rmation are giv- w ere used for this study .The data of en in Table 1 .All the tissues used for ex traction of carpio (NC001606 ) and auratus DNA were preserved in 95 % ethanol and most of (NC002079)w ere retrieved from GenBank .My xo- them w ere deposited in the Freshw ater M useum cyprinus asiaticus w as chosen as the outg roup ow ing of Institute of Hydrobiology , Chinese Academy of to its clear relationship w ith family Cy prinidae .Loca- Sciences . Table 1 . Species identifications, locations of origin and the length of analyzed sequence Taxons Species Location Length (bp) Labeoninae Cirrhin us molitorella Tengxian , Guangxi 1208 Osteoch ilus sa lsburyi Rongan , Guangxi 1211 Sinilabeo laticeps Mengla , Yunnan 1176 Tengchong , Yunnan 1215 Labeo yu nnanensis Mengla , Yunnan 1217 Pseudoorossocheilus bamaensis Tiane, Guangxi 1243 Parasin ilabeo assinilabeo Rongan , Guangxi 1182 Ptych idio jord ani Tiane, Guangxi 1144 notabilis Tiane, Guangxi 1121 Pseudogyrincheilus procheilus Luding , Sichuan 1274 posehensis Duan , Guangxi 1258 Discogobio la ticeps Tiane, Guangxi 1149 Discogobio bismargaritus Tiane, Guangxi 1157 Discogobio brachyphysa llidos Jinxiu , Guangxi 1148 Discogobio tetrabarbatus Rongan , Guangxi 1271 Garra pingi hainanensis Changhua, Ledong 1174 Chayu Ⅴ , Tibet 1149 Garra orientails Changhua, Ledong 1229 Barbininae sima Hejiang , Sichuan 1254 holland i Tunxi, Huangshan 1148 Cyp rininae Procyp ris rabaudi Hejiang , Sichuan 1215 Carassius au ratus NC 002079 1839 Cyp rin us carpio NC 001606 1839 Cyp rin us m ultitaen iata Guiping , Guangxi 1234 Schizothoracinae Schizothora x walton i Chayu Ⅳ , Tibet 1341 Schizothora x molesworthi Chayu Ⅴ , Tibet 1247 Family Catostomidae Myxocyp rin us asiaticus Wuhan , Hubei 1193

1 .2 DNA ex traction , PCR amplification and se- ing agarose gels , recovered from the gels and purified quencing using Biostar Glassmilk DNA purification Kit follow- ing the manufacture' s instructions .The purified frag- Total DNA w as extracted from muscle tissues o r ments were directly sequenced in both forw ard and fins follow ing Ausubel' s method[ 13] .The follow ing reverse directions by Shang hai Biostar Genechip Inc . primers , 16S L (5′- CTT ACA CCG AGA ARA CAT 1 .3 Sequence analy sis C -3′)and 16SH (5′- CTT AAG C TC CAA AGG GTC -3′), were used fo r amplification and sequenc- Multiple alignments of sequences were performed ing .Amplification reactions w ere performed in a using Clustal W[ 14] with a gap-opening penalty of 20 60 μL volume containing approximately 100 ng of and a gap-extension penalty of 20 and Seaview[ 15] template DNA , 0 .75 μL dNTP (2 .5 mmol/L of alignment edito r and verified by eye .Measures of nu- each), 1 .5 μL of each primer , 5 μL of 10 X reaction cleotide composition were carried out by MEGA2 .1 buffer and 3 units of Taq DNA polymerase software[ 16] .The mutational saturation for nucleotide (Biostar).The thermocycling conditions included an substitutions w as examined by plotting transversions initial denaturation at 95 ℃ for 3 min , followed by 30 and transitions against Kimura two-parameter dis- cycles of 94 ℃ for 30 s , 60 ℃ for 30 s , and 72 ℃ fo r tance using DAM BE4 .1[ 17] ;saturation w as estimated 1 min and were completed w ith a final extension at by the extent to which the slope of a linear regression 72 ℃ for 5 min .The amplified fragments were frac- departs from a value of 1 .Aligned sequences w ere an- tionated by electrophoresis through 0 .8 % low-melt- alyzed using maximum parsimony (M P), neighbor Prog ress in Natural Science Vol.15 No .3 2005 w ww .tandf.co .uk/journals 215 joining (NJ), m aximum likelihood (M L ) and age proportions of the different nucleotide was T = Bayesian methods .All phylogenetic analyses includ- 0 .201 , C =0 .23 , A =0 .38 , G =0 .19 .These data ing maximum parsimony , neighbo r joining and maxi- clearly show that the base composition is somew hat mum likelihood were performed w ith PAUP A-rich and the base pair composition is rich in A and 4 .0b10[ 18] except for Bayesian method , the latter w as T bases (58 .1 %).An overall transitions/transver- carried out with M rBayes3 .0b4[ 19] . sions ratio observed w as 2 .1 for these data sets .The saturation of transition and transversion changes was M aximum parsimony search w as conducted using checked by plotting the percentage of sequence diver- heuristic search methods w ith tree bisection-reconnec- gence against the pairwise number of inferred substi- tion (TBR)branch sw apping , and 10 random se- tutions .The transitions/ transversions ratio did not quence addition replicates .Non-parametric bootstrap exhibit any plateau and indicated a non-mutational analy ses w ith 1000 pseudoreplicates and 10 random saturation (data no t show n).The uncorrected p-dis- sequence additions w ere conducted on the equally tance matrix observed from the analysis of the align- w eighted data .In to determine w hich model is ment of all sequences is also not show n here , and the the best-fit , w e selected models using Modeltest value of pairw ise distance among labeonine fishes [ 20] 3 .06 .Finally , the GTR model w hich incorporates ranged from 0 .1 % to 16 %, and from 14 .8 % to rate variation (Γ)along w ith the number of invariable 20 .4 % betw een ingroups and outg roup . sites (I)w as utilized for neig hbor joining and maxi- mum likelihood analy ses based on a hierarchical hy- 2 .2 Phylogenetic relationships po thesis test of alternative models implemented with Bayesian analy sis resulted in a consensus tree modeltest 3 .06 .The Ti/ Tv ratio , gamma shape pa- w ith g reat posterio r probabilities at most nodes (Fig . rameter and proportion of non-variant sites w ere esti- 1).Within the Barbini species studied , tw o major mated by maximum likelihood from a maximum par- clades could be recog nized with great posterior proba- simony tree .After the best-fit model w as found , we bilities (PP =98):one is composed of the subfamily perfo rmed a heuristic search using the same branch Barbininae (Onychostoma sima and S pinibarbus hol- sw apping as described previously for NJ and M L .In landi), Schizothoracinae ( waltoni and M L analysis , starting tree w as obtained via neighbo r Schizothorax moleworthi)and Cyprininae (Cy pri- joining , then the bootstrap tests w ere performed a- nus m ultitaeniate , Cyprinus carpio , Carassius au- g ain , 1000 for NJ analy sis and 100 for M L analy sis . ratus and rabandi)(PP =79).While the Bayesian analy sis using Markov Chain M onte other comprises labeonine fishes with great posterior Carlo (MCMC)w as performed using MrBayes 3 .0b4 . probabilities (PP =100).The Labeoninae species is To decrease the chance of reaching apparent station- also related to two major g roups:one includes genera ary on local optima , tw o separate analyses w ere con- , Crossocheilus and Garra , and the o ther ducted , each analysis consisted of four chains , ran- accommodates genera Labeo , Sinilabeo , Os- dom starting trees and the appropriate model (GTR + teochilus , Pseudocrossocheilus , Parasinilabeo , Pty- I +G)determined by Modeltest version 3 .06 .The chiodio , Semilabeo , Pseudogyrinocheilus , Rectoris chains w ere run for 2 ×105 generations w ith trees be- and Discogobio .In this latter association , the genera ing sampled every 100 generations, stationary w as Pseudogyrinocheilus , Rectoris and Ptychiodio are determined visually , burn-in trees discarded and the closely related , and Pseudocrossocheilus , Parasinil- remaining trees used to estimate Bayesian posterio r abeo and Semilabeo closely related .They are related probabilities . as the sister g roup of the genus Discogobio .Trees constructed using M L and Bayesian methods are very 2 Results similar , m aximum likelihood analy sis yielded one tree w ith likelihood ln L =-6167 .42083 (not show n). 2 .1 Sequences and variations of the mitochondrial 16S rRNA gene Maximum parsimony analysis yielded tw o most- parsimonious trees w ith a tree length =1039 , CI = In this study , the leng th of the mitochondrial 0 .5600 and RI =0 .5733 (Fig .2).In the M P tree , 16S rRNA gene ranged from 1144 bp to 1341 bp in 25 the genera Sinilabeo , Osteochilus , Cirrhinus , taxa .Of the 1148 alig ned sequences , 402 were vari- Crossocheilus and Garra fo rm one clade ;the genera able and 242 were parsimony informative .The aver- Pseudocrossocheilus , Parasinilabeo , Ptychiodio , 216 w ww .tandf.co .uk/journals Prog ress in Natural Science Vol .15 No .3 2005

Semilabe , Pseudogyrinocheilus , Rectoris and Disco- ported value of BP =80 .The genus Labeo is basal in are related as monophy letic w ith a strong ly sup- the labeonine fishes .

Fig .1 . Consensus tree obtained from Bayesian methods.Numbers at the nodes represen t the posterior probabilities of the phylogeny , w hich w as determined from the follow ing 1001 trees.FM , the first morphotype ;SM , the second morphotype ;TM , the third morphotype . Prog ress in Natural Science Vol.15 No .3 2005 w ww .tandf.co .uk/journals 217

Fig.2 . Consensus tree of the tw o equiparsimonious trees inferred from maximum parsimony methods based on the mitochondrial 16S rRNA gene sequences.Length of each tree is 1039 steps and C I =0.5600 , RI =0 .5733 , numbers at the nodes refer to the bootstrap .

The NJ tree is of the same topology w ith the M P abeo , Pseudocrossocheilus , Parasinilabeo and Disco- tree fo r the labeonine species except for the position of gobio . the genus Labeo .In the NJ tree , Labeo is related as the sister group w ith the group including the genera Overall, trees constructed using NJ , MP , ML Pseudogyrinocheilus , Rectoris , Ptychiodio , Sem il- and Bayesian methods show that labeonine fishes are 218 w ww .tandf.co .uk/journals Prog ress in Natural Science Vol .15 No .3 2005 related closely as a monophyly . nocheilus , Rectoris and Ptychiodio , there is a strong support for a sister group relationship between the 3 Discussions genera Pseudogyrinocheilus and Rectoris (PP =94); the second clade includes the genera Pseu- 3 .1 Monophyly of the subfamily Labeoninae docrossocheilus , Parasinilabeo and Sem ilabeo w ith low bootstrap support . The genus Pseu- On the basis of several morphological synapo- docrossocheilus endemic to China had been regarded as morphies , the labeonine fishes were defined as mono- [ 3 , 22] a sy nonym of the genus Crossocheilus .Zhang phyletic group[ 5, 21] .When My xocyprinus asiaticus made a taxonomical revision of the Chinese cyprinid w as assigned as the outgroup , our present study sug- genus Crossocheilus and considered the genus Pseu- gested that Labeoninae is basically a monophyletic docrossocheilus as a valid genus[ 6] .In this study , the g roup and related as sister group with non-labeonine genus Pseudocrossocheilus is not clustered w ith the fishes in Barbininae , Cyprininae and Schizothoracinae . genus Crossocheilus , how ever , it has a closer rela- 3 .2 Phy logenetic relationships tionship w ith parasinilabeo-like fishes .

The morphology of lips and associated structures The third group , defined by the presence of the have been used to analy ze the phy logeny of Labeoni- adhesive disc on the chin and phary ngeal teeth in 2 nae[ 22] , and the subfamily Labeoninae is divided into rows includes the genera Garra , Discogobio , Pla- three major groups[ 1] .The first group found is that cochilus and Discocheilus .Wu et al .considered that the upper lip is present and rostral cap plus lower lip the garra-like group , composed of the genera Garra , D iscogobio and Placochilus , might be the most de- can no t form a prebuccal cavity .It consists of the [ 22] genera Sinilabeo , Henicorhynochus , Lobocheilus , rived g roup .The studies on development , func- Labeo , Cirrhinus , and Osteocheilus . tion and surface ultrastructure of the adhesive disc al- In this group , it w as thought that the genera Sinil- so supported that the genus Garra had a close rela- [ 25] abeo and Lobocheilus were the sister group w hich w as tionship with genus Discogobio .However , our closely related w ith Labeo and Cirrhinus[ 22] .How- phylogenetic analysis indicates that Garra has a close ever , the phy logeny based on the mitochondrial 16S relationship w ith Corrocheilus and Cirrocheilus , rRNA gene show s that the genus S inilabeo is not w hile it is distantly related w ith the genus D iscogob- clustered w ith the genera Lobocheilus , Labeo and io .The garra-like g roup might be a paraphy letic or Cirrhinus , w hereas it is closely related with the gen- polyphyletic group , because the genus Garra is clus- era Parasinilabeo , Discogobio and Rectoris .Surpris- tered with the first g roup , w hile the genus D iscogob- ing ly , the positioning of the genera Labeo and Os- io has a close relationship with the second group .The teocheilus was problematic and constitutes the m ain adhesive disc on the chin is only a feature adapted to source of differences between the M P tree presented torrential environment and of little significance in the [ 26, 27] in Fig .2 and trees obtained from the NJ , M L and phylogeny of the subfamily Labeoninae . Bayesian methods with the low boo tstrap support . In Labeoninae , modifications of the lips and The second g roup , characterized by a prebuccal associated structures are an indication for adaptive cavity , has been considered including the genera evolution to lotic systems .These characters, as Crossocheilus , , Rectoris , Pseu- mentioned above , w ere used as traditional techniques docrossocheilus , Parasinilabeo , Semilabeo , Pseudo- to elucidate the relationships of labeonine fishes. gy rinocheilus , Ptychiodio and [ 23] , In the present paper we present an inconsistent phy- and Parasinilabeo-like fishes are a g roup endemic to logeny based on the analysis of sequence data of the China and composed of the genera Parasinilabeo , mitochondrial 16S rRNA gene .Our phylogenetic Semilabeo and Pseudogyrinocheilus .Zhang defined analysis indicates that Labeoninae may be divided into parasinilabeo-like fishes as a monophyletic group fo r 2 major clades :one comprising the genera Cirrhi- the possession of four synapomorphies[ 24] .However , nus , Crossocheilus and Garra , and the other consist- our study show s that parasinilabeo-like g roup is ing of the genera Labeo , Sinilabeo , Osteochilus , maybe a paraphyletic o r poly phy letic g roup .Within Pseudocrossocheilus , Parasinilabeo , Ptychiodio , this group , two w ell-supported clades are presented . Sem ilabeo , Pseudogyrinocheilus , Rectori and Disco- The first clade contains the genera Pseudogy ri- gobio . Prog ress in Natural Science Vol.15 No .3 2005 w ww .tandf.co .uk/journals 219

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